WO2012145269A1 - Yield and stress tolerance in transgenic plants - Google Patents

Abstract

Polynucleotides and polypeptides incorporated into expression vectors have been introduced into plants and were ectopically expressed. The polypeptides of the instant description have been shown to confer at least one regulatory activity and confer increased heat tolerance, increased yield, greater vigor, greater biomass as compared to the control plant as compared to a control plant.

Description

YIELD AND STRESS TOLERANCE IN TRANSGENIC PLANTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/476,608, filed 18 April 2011 , which is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to plant genomics and plant improvement. BACKGROUND OF THE INVENTION

Yield of commercially valuable species in the natural environment may be suboptimal as plants often grow under unfavorable conditions, including at an inappropriate temperatures or with a limited supply of soil nutrients, light, or water availability.

Temperature has a profound impact on plant development and survival. Low temperature or chilling conditions are detrimental to many plants of tropics and subtropics which cannot acclimatize to cold. Such effects may result from, for example, loss of function of biomembranes connected with a decrease of their fluidity and an inactivation of or at least a reduction in activity of the membrane-bound ion pumps. Light energy which is absorbed independently of the temperature, produces oxidative stress, such that metabolism cannot keep pace with the energization of the photo synthetic membranes (Beck et al. 2004).

On the other hand, high temperatures can accelerate the onset of reproductive development, which shortens the time for photosynthesis to overall vegetative growth or to contribute to fruit or seed production, resulting substantially reduced biomass or total fruit or grain yield. In some cases, heat can even inhibit reproductive development of many crop species such that they produce no flowers or if they produce flowers, those flowers have reduced fertility such that they set few, if any, fruit or seeds. In addition, heat can damage cellular structures, including organelles and cytoskeleton, and impairs membrane function effects.

Germination of many crops is also very sensitive to temperature. Factors that would enhance germination in hot conditions would be useful for crops that are planted late in the season or in hot climates. Seedlings and mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs, including leaves and particularly fruit, when transpiration is insufficient to overcome heat stress.

A plant's phenotypic characteristics that enhance tolerance to heat stress or yield may be controlled through a number of cellular processes. One important way to manipulate that control is through regulatory proteins known as transcription factors; proteins that influence the expression of a particular gene or sets of genes. Transformed and transgenic plants that comprise cells having altered levels of at least one selected transcription factor, for example, often possess advantageous or desirable traits. Strategies for manipulating traits by altering a plant cell's transcription factor content or expression level can therefore result in plants and crops with commercially valuable properties.

SUMMARY OF THE INVENTION

An object of this description is to provide plants which can express genes to increase plant' s tolerance to heat, and/or increase the yield of commercially significant plants.

The present description thus pertains to novel methods for producing transgenic plants that have greater heat tolerance, greater biomass, size, or vigor and/or as compared to a control plant by introducing recombinant polynucleotides or expression vectors comprising polynucleotides into target plants or plant cells, and selecting from the transformed plants a transgenic plant line that have greater heat tolerance relative to a control plant.

The recombinant polynucleotides that can be used as disclosed or claimed may include any of the following sequences:

(a) polypeptide sequences that are at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 90%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, or at least 99%, or about 100% identical in their amino acid sequence to the entire length of any of SEQ ID NOs: 2n, where n=l-32;

(b) nucleotide sequences that are at least 40% identical to any of the nucleotide sequences that encode the polypeptide sequences of (a);

(c) nucleotide sequence that hybridize to any of the nucleotide sequences of (b) under stringent conditions, which may include, for example, hybridization with wash steps of 6x SSC and 65° C for ten to thirty minutes per step; and

(d) polypeptides, and the nucleotide sequences that encode them, having a conserved AP2 domain required for the function of regulating transcription and altering a trait in a transgenic plant, the conserved domain being at least 59% to about 100% identical in its amino acid residue sequence to the conserved AP2 domains of SEQ ID NO: 2n, where n=l-32 (i.e., a polypeptide listed in the sequence listing, or encoded by any of the above nucleotide sequences, the conserved domains being represented by those set forth as SEQ ID NOs: 65-96, respectively). The conserved domains listed in Table 4 comprise a domain required for the function of regulating transcription and altering a trait in a transgenic plant, said trait selected from the group consisting of increasing yield, increasing tolerance to heat, as compared to the control plant.

(e) polypeptides, and nucleotide sequences that encode them, sharing at least 30%, 37%, 40%, 45%, 47%, 48%, 50%, 60%, 65%, 66%, 68%, 50%, 68%, 80%, 85%, 90%, or 95% identical in their amino acid sequence to the entire length any of SEQ ID NOs: 2n, where n=l-32 and having a conserved

AP2 domain required for the function of regulating transcription and altering a trait (for example, increasing heat tolerance) in a transgenic plant, the conserved domain being at least about 59% to about 100% identical in its amino acid residue sequence to the conserved AP2 domains of SEQ ID NO: 2n, where n=l-32.

The method of this description includes exposing a plant or plants containing the one or more plant cells that overexpress at least one of the polynucleotides above to a heat stress, and selecting from the plant or plants a transgenic plant that expresses the polypeptide which, when expressed in the transformed plant, confers greater heat stress tolerance, or greater biomass, vigor, or size relative to a control plant that does not contain the recombinant polynucleotide.

The expression vectors, and hence the transgenic plants, of the instant description, comprise putative transcription factor polynucleotides sequences and, in particular, the conserved AP2 domain. When any of the claimed polypeptides are overexpressed in a plant, the polypeptide confers at least one regulatory activity to the plant, which in turn is manifested in a trait selected from the group consisting of increased yield and increased tolerance to heat, increased biomass, increased vigor, increased weight, increased diameter, increased size, and having darker green leaves as compared to the control plant.

Brief Description of the Sequence Listing and Drawings

The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the instant description. The traits associated with the use of the sequences are included in the Examples.

Incorporation of the Sequence Listing. The Sequence Listing provides exemplary polynucleotide and polypeptide sequences. The copy of the Sequence Listing, being submitted electronically with this patent application, provided under 37 CFR § 1.821-1.825, is a read-only memory computer-readable file in ASCII text format. The Sequence Listing is named "MBI-0130_ST25.txt", the electronic file of the Sequence Listing was created on April 18, 2011, and is 152,464 bytes in size (148 kilobytes in size as measured in MS-WINDOWS). The Sequence Listing is herein incorporated by reference in its entirety.

Figure 1 shows a conservative estimate of phylogenetic relationships among the orders of flowering plants (modified from Soltis et al. (1997)). Those plants with a single cotyledon (monocots) are a monophyletic clade nested within at least two major lineages of dicots; the eudicots are further divided into rosids and asterids. Arabidopsis is a rosid eudicot classified within the order Brassicales; rice is a member of the monocot order Poales. Figure 1 was adapted from Daly et al. (2001).

Figure 2 shows a phylogenic dendogram depicting phylogenetic relationships of higher plant taxa, including clades containing tomato and Arabidopsis; adapted from Ku et al. (2000); and Chase et al. (1993).

In Figure 3, a phylogenetic tree of the CBF superclade members, including the CBF clade members, and related full length proteins were constructed using ClustalW (CLUSTAL W Multiple Sequence Alignment Program version 1.83, 2003). The CBF superclade members appear in the large box with the dashed-line boundary. ClustalW multiple alignment parameters were:

Multiple matrix :Gonnet Multiple Open Gap Penalty : 10.000

Multiple Extended Gap Penalty :0.05

Delay Divergent: 30 Gap Separation Distance: 8

End Gap Separation: false

Residue Specific Penalties: false

Hydrophilic Penalties: false

Hydrophilic Residues: GPSNDQEKR

SEQ ID NOs: appear in parentheses after each Gene IDentifier (GID).

A FastA formatted alignment was then used to generate a phylogenetic tree in Accelrys Gene v2.5 software (accelrys.com/) using the neighbor joining algorithm and an absolute (#differences) distance model. A test of phylogeny was done via bootstrap with 1000 replications. Closely-related homologs of CBF superclade members are considered as being those proteins within the node of the tree below with a bootstrap value of 1000, bounded by G3440 and G3373 (indicated by the box around these sequences). The ancestral sequence is represented by the node of the tree indicated by the arrow "(a)" in Figure 3 having a bootstrap value of 1000. Closely-related CBF clade members are considered as being those proteins within the node of the tree below with a bootstrap value of 1000, bound by G42 and G912. The ancestral sequence of the CBF clade is represented by the node of the tree indicated by the arrow "(b)" in Figure 3.

Figure 4A-4G shows a Clustal W alignment of the CBF superclade and related proteins, including sequences in the CBF superclade, which appear in the large boxes having dashed-line boundaries. SEQ ID NOs: appear in parentheses after each Gene Identifier (GID). The highly conserved AP2 domain is boxed, the conserved CBF superclade box 1 and 2 are noted by the double arrows below the alignment. The conserved residues are identified by arrows above the alignment.

Figure 5 shows the sequence of the G912 polypeptide (SEQ ID NO: 8). The AP2 domain and the C-terminal half of the G912 protein which harbors the activation domain are noted in boxes. The amino acid residue coordinates of these domains are noted above the boxes. The conserved CBF boxes are noted by the double arrows above the sequence.

Figure 6 shows effects of heat stress on Arabidopsis plants, including greater heat stress-induced anthocyanin accumulation (shown by arrows) in the wild type plants (Ws2_Wt) than in plants overexpressing either CBF2 (lines "E2-3" and "E8-1-4") or CBF3 (lines "A28-3" and lines A30-3"). In addition, heat stress caused a higher percentage of tissue bleaching in the wild type plants (shown in ovals) than in the overexpressors.

Figure 7 shows that heat stress induced greater anthocyanin accumulation (shown by arrows) in wild type plants (Ws2_Wt) than in plants overexpressing CBF1 (line "G6-7"). Slight tissue bleaching was also observed in CBF1 overexpressors (circled by the open oval). Note that CBF1 transgenic seeds and control seeds were germinated at 22 °C for 11 days as described in Example IV before they were subjected to heat stress (34 °C for five days).

DETAILED DESCRIPTION The present description relates to polynucleotides and polypeptides for modifying phenotypes of plants, particularly those associated with increased abiotic stress tolerance and increased yield with respect to a control plant (for example, a wild-type plant). Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and World Wide Web browser-inactive page addresses. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of "incorporation by reference" is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the instant description.

As used herein and in the appended claims, the singular forms "a", "an", and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "a stress" is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.

DEFINITIONS

"Polynucleotide" is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides. A polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single- stranded or double-stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). The polynucleotide can comprise a sequence in either sense or antisense orientations. "Oligonucleotide" is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single-stranded.

A "recombinant polynucleotide" is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.

"Gene" or "gene sequence" refers to the partial or complete coding sequence of a gene, its complement, and its 5' or 3' untranslated regions. A gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter may be subjected to subsequent processing such as chemical modification or folding to obtain a functional protein or polypeptide. A gene may be isolated, partially isolated, or found with an organism's genome. By way of example, a transcription factor gene encodes a transcription factor polypeptide, which may be functional or require processing to function as an initiator of transcription.

Operationally, genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and that may be used to determine the limits of the genetically active unit (Rieger et al. (1976)). A gene generally includes regions preceding ("leaders"; upstream) and following ("trailers"; downstream) the coding region. A gene may also include intervening, non-coding sequences, referred to as "introns", located between individual coding segments, referred to as "exons". Most genes have an associated promoter region, a regulatory sequence 5' of the transcription initiation codon (there are some genes that do not have an identifiable promoter). The function of a gene may also be regulated by enhancers, operators, and other regulatory elements.

A "polypeptide" is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise: (i) a localization domain; (ii) an activation domain; (iii) a repression domain; (iv) an oligomerization domain; (v) a protein-protein interaction domain; (vi) a DNA-binding domain; or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.

"Protein" refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.

"Portion", as used herein, refers to any part of a protein used for any purpose, but especially for the screening of a library of molecules which specifically bind to that portion or for the production of antibodies.

A "recombinant polypeptide" is a polypeptide produced by translation of a recombinant polynucleotide. A "synthetic polypeptide" is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art. An "isolated polypeptide," whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein. "Homology" refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence.

"Identity" or "similarity" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases "percent identity" and "% identity" refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. "Sequence similarity" refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical, matching or corresponding nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at corresponding positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared by the polypeptide sequences.

"Alignment" refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues at corresponding positions) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. Alignments such as those of Figures 4A-4G may be used to identify conserved domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art, such as MACVECTOR software (1999) (Accelrys, Inc., San Diego, CA).

A "conserved domain" or "conserved region" as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. An AP2 domain is an example of a conserved domain. With respect to polynucleotides encoding presently disclosed polypeptides, a conserved domain is preferably at least nine base pairs (bp) in length. A conserved domain with respect to presently disclosed polypeptides refers to a domain within a polypeptide family that exhibits a higher degree of sequence homology, such as at least about 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 90%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, or at least 99%, or about 100% identity to a conserved domain of a polypeptide of the Sequence Listing (e.g., any of SEQ ID NOs: 65-96) or listed in Table 4. Sequences that possess or encode for conserved domains that meet these criteria of percentage identity, and that have comparable biological and regulatory activity to the present polypeptide sequences, thus being members of the CBF clade polypeptides or sequences in the CBF superclade, are described. A fragment or domain can be referred to as outside a conserved domain, outside a consensus sequence, or outside a consensus DNA-binding site that is known to exist or that exists for a particular polypeptide class, family, or sub-family. In this case, the fragment or domain will not include the exact amino acids of a consensus sequence or consensus DNA-binding site of a transcription factor class, family or sub-family, or the exact amino acids of a particular transcription factor consensus sequence or consensus DNA- binding site. Furthermore, a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide, can be "outside a conserved domain" if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.

As one of ordinary skill in the art recognizes, conserved domains may be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al. (2000a,

2000b)). Thus, by using alignment methods well known in the art, the conserved domains of the plant polypeptides, for example, for the AP2 domain proteins (Riechmann and Meyerowitz, 1998), may be determined.

An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved consecutive amino acid residues, PKK/RPAGRxKFxETRHP (SEQ ID NO: 100) and DSAWR (SEQ ID NO: 101), which bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. (Jaglo et al.

(2001)). A broader clade of sequences ("the CBF superclade" includes a number of sequences descended from a common ancestral sequence, as shown in the phylogenetic tree seen in Figure 3 at the node with a bootstrap value of 1000, bounded by G3440 and G3373. In addition to the conserved AP2 domain, the presently disclosed CBF superclade polypeptides also contain the CBF superclade boxes 1 and 2, i.e., SEQ ID NO: 115 and 99, respectively. The CBF superclade sequences thus may be defined as having a highly conserved AP2 domain, at least 59% identical in its amino acid sequence to the conserved AP2 domain of SEQ ID NO: 8, and comprise SEQ ID NO: 115 and SEQ ID NO: 99. A subgroup of CBF superclade, i.e., the CBF clade sequences, may be defined as having a highly conserved AP2 domain, at least 70% identical in its amino acid sequence to the conserved domain of SEQ ID NO; 8, and comprise the CBF box 1 (SEQ ID NO: 100) and CBF box 2 (SEQ ID NO: 101).

The polypeptides of Table 4 have conserved domains specifically indicated by amino acid coordinate start and stop sites. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example, Reeves and Nissen (1990, 1995)) to identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure.

The presently disclosed CBF superclade polypeptides are "functionally-related and/or closely- related" by having descended from a common ancestral sequence (see the node in Fig. 3 with the bootstrap value of "100"), and/or by being sufficiently similar to the sequences and domains listed in Table 4 that they confer the same function to plants of increased heat tolerance and associated improved plant vigor, quality, yield, size, and/or biomass. "Functionally-related and/or closely-related" polypeptides may thus be created artificially, semi-synthetically, or may occur naturally by having descended from the same ancestral sequence as the disclosed CBF-related sequences, where the polypeptides have the function of conferring increased heat tolerance to plants. These "functionally- related and/or closely-related" CBF superclade polypeptides generally contain the consensus sequence SEQ ID NO: 114, which comprises smaller highly conserved subsequences: K-X-X-A-G-R-X-X-F-X-E- T-R-H-P-V/I-Y/F-R -G-V/I-R-X-R (SEQ ID NO: 97, where X represents any amino acid; seen in

Figure 4B) and L-N-F-X-X-S-X-X-X-L/M (SEQ ID NO: 98, where X represents any amino acid; seen in Figure 4C). The CBF superclade sequences also contain the CBF superclade box 1 : P/K-K-X-X-A-G-R- X-X-F-X-E-T-R-H-P-V I (SEQ ID NO: 115; where X represents any amino acid), which comprises the CBF box 1 (SEQ ID NO: 100); and the CBF superclade box 2: L-N-F-X-X-S-X-X-X-L (SEQ ID NO: 99; where X represents any amino acid), which comprises the CBF box2 (SEQ ID NO: 101). The presence of one or more of these consensus sequences is correlated with the conferring of improved or increased heat tolerance to a plant when the expression level of the polypeptide is altered in a plant by being reduced, knocked-out, or overexpressed. A CBF superclade polypeptide sequence that is "functionally-related and/or closely-related" to the listed full length protein sequences or domains provided in Table 4 may also have at least 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 90%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, or at least 99%, or about 100% identical in their amino acid sequence to the entire length of a listed sequence, and having an AP2 domain that is at least 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 90%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% or 96%, 97%, 98%, or at least 99%, or about 100% identical in its amino acid sequence to the conserved AP2 domain of SEQ ID NO: 8, where the polypeptide sequence comprises one or more consensus sequences: SEQ ID NO: 97 -101, or 114-115, and the presence of the domain in the polypeptide sequence is correlated with the conferring of improved or increased heat tolerance to a plant when the expression level of the polypeptide is altered in a plant by being reduced, knocked-out, or overexpressed. All of the sequences that adhere to these functional and sequential relationships are herein referred to as "CBF superclade polypeptides", or which fall within the "CBF superclade" exemplified in the tree in Fig. 3 as those polypeptides within the node with a bootstrap value of "1000", bounded by G3440 and G3373.

A transgenic plant is expected to have improved or increased heat tolerance relative to a control plant when the transgenic plant is transformed with a recombinant polynucleotide encoding any of the listed sequences or another CBF superclade sequence defined in this paragraph, or when the transgenic plant contain or expresses a CBF superclade sequence.

The terms "highly stringent" or "highly stringent condition" refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs. Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present description may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are disclosed in detail by Kashima et al. (1985), Sambrook et al. (1989), and by Haymes et al. (1985), which references are incorporated herein by reference.

In general, stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure (for a more detailed description of establishing and determining stringency, see the section "Identifying Polynucleotides or Nucleic Acids by Hybridization", below). The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity. Thus, similar nucleic acid sequences from a variety of sources, such as within a plant's genome (as in the case of paralogs) or from another plant (as in the case of orthologs) that may perform similar functions can be isolated on the basis of their ability to hybridize with known related polynucleotide sequences. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate related polynucleotide sequences having similarity to sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed polynucleotide sequences, such as, for example, encoded transcription factors having 56% or greater identity with the conserved domains of disclosed sequences (see, for example, Table 4, showing CBF superclade polypeptides having at least 56%, 63%, 73%, 76%, 79%, 81%, 83%, 91%, or 93% amino acid identity with the conserved domains of disclosed sequences).

The terms "paralog" and "ortholog" are defined below in the section entitled "Orthologs and Paralogs". In brief, orthologs and paralogs are evolutionarily related genes that have similar sequences and functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.

In general, the term "variant" refers to molecules with some differences, generated synthetically or naturally, in their base or amino acid sequences as compared to a reference (native) polynucleotide or polypeptide, respectively. These differences include substitutions, insertions, deletions or any desired combinations of such changes in a native polynucleotide of amino acid sequence.

With regard to polynucleotide variants, differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences may be silent (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide. Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations may result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.

Also within the scope of the instant description is a variant of a nucleic acid listed in the Sequence Listing, that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code. Included within this definition are polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding polypeptide.

"Allelic variant" or "polynucleotide allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations may be "silent" or may encode polypeptides having altered amino acid sequence. "Allelic variant" and "polypeptide allelic variant" may also be used with respect to polypeptides, and in this case the terms refer to a polypeptide encoded by an allelic variant of a gene.

"Splice variant" or "polynucleotide splice variant" as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. Thus, splice variants may encode polypeptides having different amino acid sequences, which may or may not have similar functions in the organism. "Splice variant" or "polypeptide splice variant" may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.

As used herein, "polynucleotide variants" may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences. "Polypeptide variants" may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.

Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in a functionally equivalent polypeptides. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the polypeptides and homolog polypeptides of the instant description. A polypeptide sequence variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties.

Conservative substitutions include substitutions in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 1 when it is desired to maintain the activity of the protein. Table 1 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.

Table 1. Possible conservative amino acid substitutions

The polypeptides provided in the Sequence Listing have a novel activity, such as, for example, regulatory activity. Although all conservative amino acid substitutions (for example, one basic amino acid substituted for another basic amino acid) in a polypeptide will not necessarily result in the polypeptide retaining its activity, it is expected that many of these conservative mutations would result in the polypeptide retaining its activity. Most mutations, conservative or non-conservative, made to a protein but outside of a conserved domain required for function and protein activity will not affect the activity of the protein to any great extent.

Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as a significant amount of the functional or biological activity of the polypeptide is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine. More rarely, a variant may have "non-conservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example,

DNASTAR software (see USPN 5,840,544).

"Fragment", with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A "polynucleotide fragment" refers to any subsequence of a polynucleotide, typically, of at least about nine consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein. Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the polynucleotides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a region that encodes an conserved domain of a polypeptide. Exemplary fragments also include fragments that comprise a conserved domain of a polypeptide. Exemplary fragments include fragments that comprise an conserved domain of a polypeptide, for example, amino acid residues 54-112 of G912 (SEQ ID NO: 8), amino acid residues 48-106 of G40 (SEQ ID NO: 4) or amino acid residues 42-100 of G41 (SEQ ID NO: 2).

Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential. In some cases, the fragment or domain is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription.

Fragments can vary in size from as few as three amino acid residues to the full length of the intact polypeptide, but are preferably at least about 30 amino acid residues in length and more preferably at least about 60 amino acid residues in length.

The instant description also provides production of DNA sequences that encode polypeptides and derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding polypeptides or any fragment thereof. "Derivative" refers to the chemical modification of a nucleic acid molecule or amino acid sequence. Chemical modifications can include replacement of hydrogen by an alkyl, acyl, or amino group or glycosylation, pegylation, or any similar process that retains or enhances biological activity or lifespan of the molecule or sequence.

The term "plant" includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and cells (for example, guard cells, egg cells, and the like), and progeny of same. The class of the plants that can be transformed using the methods provided of the instant description is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae (see for example, Figure 1, adapted from Daly et al. (2001), Figure 2, adapted from Ku et al. (2000); and see also Tudge (2000).

A "control plant" as used in the present description refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transgenic or genetically modified plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present description that is expressed in the transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.

A "transgenic plant" refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.

A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the controlled expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell. A transgenic line or transgenic plant line refers to the progeny plant or plants deriving from the stable integration of the transgene(s), carried within the T-DNA borders contained within a

transformation construct, into a specific location or locations within the genome of the original transformed cell.

"Wild type" or "wild-type", as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which a polypeptide's expression is altered, e.g., in that it has been knocked out, overexpressed, or ectopically expressed.

A "trait" refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as hyperosmotic stress tolerance or yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.

"Trait modification" refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present description relative to a plant not doing so, such as a wild-type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease, or an even greater difference, in an observed trait as compared with a control or wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution and magnitude of the trait in the plants as compared to control or wild-type plants.

"Modulates" refers to a change in activity (biological, chemical, or immunological) or lifespan resulting from specific binding between a molecule and either a nucleic acid molecule or a protein.

The term "transcript profile" refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state. For example, the transcript profile of a particular polypeptide in a suspension cell is the expression levels of a set of genes in a cell knocking out or overexpressing that polypeptide compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that polypeptide. The transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods.

With regard to gene knockouts as used herein, the term "knockout" refers to a plant or plant cell having a disruption in at least one gene in the plant or cell, where the disruption results in a reduced expression or activity of the polypeptide encoded by that gene compared to a control cell. The knockout can be the result of, for example, genomic disruptions, including transposons, tilling, and homologous recombination, antisense constructs, sense constructs, RNA silencing constructs, or RNA interference. A T-DNA insertion within a gene is an example of a genotypic alteration that may abolish expression of that gene.

"Ectopic expression" or "altered expression" in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term "ectopic expression or altered expression" further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.

The term "overexpression" as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more polypeptides are under the control of a strong promoter (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may also under the control of an inducible or tissue specific promoter. Thus, overexpression may occur throughout a plant, in specific tissues of the plant, or in the presence or absence of particular environmental signals, depending on the promoter used.

Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present polypeptides. Overexpression may also occur in plant cells where endogenous expression of the present polypeptides or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or "overproduction" of the polypeptide in the plant, cell or tissue. "High temperature" or "heat" with regard to plant species has been defined by others (see U.S. patent application US20060150285). Plant species vary in their capacity to tolerate high temperatures. Very few plant species can survive temperatures higher than 45 °C. The effects of high temperatures on plants, however, can begin at lower temperatures depending on the species and other environmental conditions such as humidity and soil moisture. "High temperature" can be defined as the temperature at which a given plant species will be adversely affected as evidenced by symptoms such as decreased photosynthesis. Since plant species vary in their capacity to tolerate high temperature, the precise environmental conditions that cause high temperature stress can not be generalized. However, high temperature tolerant plants are characterized by their ability to retain their normal appearance or recover quickly from high temperature conditions. Such high temperature tolerant plants produce higher biomass and yield than plants that are not high temperature tolerant. Differences in physical appearance, recovery and yield can be quantified and statistically analyzed using well known measurement and analysis methods (U.S. patent application US20060150285).

"Yield" or "plant yield" refers to increased plant growth, increased crop growth, increased biomass, and/or increased plant product production (including grain), and is dependent to some extent on temperature, plant size, organ size, planting density, light, water and nutrient availability, and how the plant copes with various stresses, such as through temperature acclimation and water or nutrient use efficiency. Increased or improved yield may be measured as increased seed yield, increased plant product yield (plant products include, for example, plant tissue, including ground plant tissue, and products derived from one or more types of plant tissue), or increased vegetative yield.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Transcription Factors Modify Expression of Endogenous Genes

A transcription factor may include, but is not limited to, any polypeptide that can activate or repress transcription of a single gene or a number of genes. As one of ordinary skill in the art recognizes, transcription factors can be identified by the presence of a region or domain of structural similarity or identity to a specific consensus sequence or the presence of a specific consensus DNA-binding motif (see, for example, Riechmann et al. (2000a)). The plant transcription factors of the instant description belong to the AP2 family (Riechmann and Meyerowitz, 1998) and are putative transcription factors.

Generally, transcription factors are involved in cell differentiation and proliferation and the regulation of growth. Accordingly, one skilled in the art would recognize that by expressing the present sequences in a plant, one may change the expression of autologous genes or induce the expression of introduced genes. By affecting the expression of similar autologous sequences in a plant that have the biological activity of the present sequences, or by introducing the present sequences into a plant, one may alter a plant's phenotype to one with improved traits related to osmotic stresses. The sequences of the instant description may also be used to transform a plant and introduce desirable traits not found in the wild-type cultivar or strain. Plants may then be selected for those that produce the most desirable degree of over- or under-expression of target genes of interest and coincident trait improvement. The sequences of the present description may be from any species, particularly plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant. The sequences of the instant description may also include fragments of the present amino acid sequences. Where "amino acid sequence" is recited to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

In addition to methods for modifying a plant phenotype by employing one or more

polynucleotides and polypeptides of the instant description described herein, the polynucleotides and polypeptides of the instant description have a variety of additional uses. These uses include their use in the recombinant production (i.e., expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, e.g., mutation reactions, PCR reactions, or the like; as substrates for cloning e.g., including digestion or ligation reactions; and for identifying exogenous or endogenous modulators of the transcription factors. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can comprise a sequence in either sense or antisense orientations.

Expression of genes that encode polypeptides that modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997) and Peng et al. (1999). In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al. (2001); Nandi et al. (2000); Coupland (1995); and Weigel and Nilsson (1995)).

In another example, Mandel et al. (1992b), and Suzuki et al. (2001), teach that a transcription factor expressed in another plant species elicits the same or very similar phenotypic response of the endogenous sequence, as often predicted in earlier studies of Arabidopsis transcription factors in Arabidopsis (see Mandel et al. (1992a); Suzuki et al. (2001)). Other examples include Miiller et al. (2001); Kim et al. (2001); Kyozuka and Shimamoto (2002); Boss and Thomas (2002); He et al. (2000); and Robson et al. (2001).

In yet another example, Gilmour et al. (1998) teach an Arabidopsis AP2 transcription factor, CBF1, which, when overexpressed in transgenic plants, increases plant freezing tolerance. Jaglo et al. (2001) further identified sequences in Brassica napus which encode CBF-like genes and that transcripts for these genes accumulated rapidly in response to low temperature. Transcripts encoding CBF proteins were also found to accumulate rapidly in response to low temperature in wheat, as well as in tomato. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved consecutive amino acid residues, PKK/RPAGRxKFxETRHP (SEQ ID NO: 100, where X represents any amino acid) and DSAWR (SEQ ID NO: 101), which bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2 EREBP protein family. (Jaglo et al. (2001).

Transcription factors mediate cellular responses and control traits through altered expression of genes containing cis-acting nucleotide sequences that are targets of the introduced transcription factor. It is well appreciated in the art that the effect of a transcription factor on cellular responses or a cellular trait is determined by the particular genes whose expression is either directly or indirectly (e.g., by a cascade of transcription factor binding events and transcriptional changes) altered by transcription factor binding. In a global analysis of transcription comparing a standard condition with one in which a transcription factor is overexpressed, the resulting transcript profile associated with transcription factor overexpression is related to the trait or cellular process controlled by that transcription factor. For example, the PAP2 gene and other genes in the MYB family have been shown to control anthocyanin biosynthesis through regulation of the expression of genes known to be involved in the anthocyanin biosynthetic pathway (Bruce et al. (2000); and Borevitz et al. (2000)). Further, global transcript profiles have been used successfully as diagnostic tools for specific cellular states (e.g., cancerous vs. non-cancerous;

Bhattacharjee et al. (2001); and Xu et al. (2001)). Consequently, it is evident to one skilled in the art that similarity of transcript profile upon overexpression of different transcription factors would indicate similarity of transcription factor function.

Polypeptides and Polynucleotides of the Present Description

The present description includes putative transcription factors (TFs), and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or polynucleotides encoding novel variants of polypeptides derived from the specific sequences provided in the Sequence Listing; the recombinant polynucleotides of the instant description may be incorporated in expression vectors for the purpose of producing transformed plants. Also provided are methods for modifying yield from a plant by modifying the mass, size or number of plant organs or seed of a plant by controlling a number of cellular processes, and for increasing a plant's resistance to abiotic stresses. These methods are based on the ability to alter the expression of critical regulatory molecules that may be conserved between diverse plant species. Related conserved regulatory molecules may be originally discovered in a model system such as Arabidopsis and homologous, functional molecules then discovered in other plant species. The latter may then be used to confer increased yield or abiotic stress tolerance in diverse plant species.

Exemplary polynucleotides encoding the polypeptides of the instant description were identified in the Arabidopsis thaliana GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known polypeptides. In addition, further exemplary polynucleotides encoding the polypeptides of the instant description were identified in the plant GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known polypeptides.

Additional polynucleotides of the instant description were identified by screening Arabidopsis thaliana and/or other plant cDNA libraries with probes corresponding to known polypeptides under low stringency hybridization conditions. Additional sequences, including full length coding sequences, were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure using a commercially available kit according to the manufacturer' s instructions. Where necessary, multiple rounds of RACE are performed to isolate 5' and 3' ends. The full-length cDNA was then recovered by a routine end-to-end polymerase chain reaction (PCR) using primers specific to the isolated 5' and 3' ends. Exemplary sequences are provided in the Sequence Listing.

Many of the sequences in the Sequence Listing, derived from diverse plant species, have been ectopically expressed in overexpressor plants. The changes in the characteristic(s) or trait(s) of the plants were then observed and found to confer increased yield and/or increased abiotic stress tolerance.

Therefore, the polynucleotides and polypeptides can be used to improve desirable characteristics of plants.

The polynucleotides of the instant description were also ectopically expressed in overexpressor plant cells and the changes in the expression levels of a number of genes, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be used to change expression levels of genes, polynucleotides, and/or proteins of plants or plant cells.

The data presented herein represent the results obtained in experiments with polynucleotides and polypeptides that may be expressed in plants for the purpose of reducing yield losses that arise from biotic and abiotic stress.

Background Information for G912, the CBF clade, and the CBF superclade.

G912 (CBF4) belongs to the AP2 ERF family of transcription factors. This large gene family includes approximately 145 transcription factors in the model plant Arabidopsis and can be further divided in several subgroups

The APETALA2 class is characterized by the presence of two AP2 DNA binding domains (DBDs), and contains 14 genes.

The RAV subgroup includes six genes which all have a B3 DBD in addition to the AP2 DBD.

The AP2/ERF is the largest subfamily, and includes 125 genes that are characterized by a single AP2 DBD. Sakuma et al. (2002) have further categorized this class into three subgroups: DREB related (56 genes), ERF related (65 genes), and others (four genes; these lack the conserved WLG-motif in the middle of the AP2 DBD). The DREB and ERF subgroup were distinguished by the amino acids present at position 14 and 19 of the AP2 DBD. While DREBs are characterized by Val-14 and Glu-19, ERFs typically have Ala-14 and Asp-19. The involvement of the DREB subfamily in the plant response to dehydration stress has been shown for members of the subgroup A-1 to A-3 (CBFl-3: e.g. Gilmour et al., 1998, Liu et al., 1998, CBF4: Haake et al., 2002; DREB2: Nakashima et al., 2000; ABI4: Finkelstein et al., 1998; DDF1 and DDF2; Magome et al., 2004). Subgroups A-4 to A-6 are only poorly characterized in the public domain. However, data from the few genes with published function indicate that the ERF subfamily is involved in ethylene signaling, and, in particular, in the response to biotic stress.

Table 2. Overview of the DREB subfamily transcription factors.

The different subgroups of DREB subfamily have distinct structure features. The polypeptides used in the instant description belong to the A-l subgroup of the DREB subfamily, a.k.a. CBF clade polypeptides and CBF superclade polypeptides. Thus, the exemplary polypeptides of this clade include, but not limited to, the sequences listed in Table 4. CBFl-4 are highly related paralogs and have been extensively characterized (see e.g. Haake et al., 2002; Thomashow, 2001, Liu et al., 1998, Gilmour et al., 2000, Shinozaki and Yamaguchi-Shinozaki, 2000). Note that CBF1 a.k.a., DREB IB or G40 (SEQ ID NO: 4); CBF2, a.k.a., DREB1C or G41 (SEQ ID NO: 2); CBF3, a.k.a., DREB 1 A, or G42 (SEQ ID NO: 6); CBF4, a.k.a., G912 (SEQ ID NO: 8).

The CBF superclade polypeptides all contain an AP2 DBD which is localized in the N-terminal region of the protein, an acidic activation domain is found at the C-terminus, and two characteristic motifs: CBF superclade box-1 (SEQ ID NO: 115) and CBF superclade box-2 (SEQ ID NO: 99) flanking the AP2 domain. Box-1 is rich in basic amino acids (Arg, Lys), a property typical for nuclear localization signals as well as for DNA-binding domains (Liu et al., 1999). Box-2 is a shorter motif of unknown function. The CBF clade polypeptides, which belong to the CBF superclade, all share two highly conserved regions, i.e., the CBF box-1 (SEQ ID NO: 100) and the CBF box-2 (SEQ ID NO: 101) (Jaglo et al., 2001). Figure 4 shows the conserved regions of some CBF clade and CBF superclade polypeptides.

In vitro, the AP2 domain of CBF1 is sufficient to confer binding to the CCGAC core of the CRT/DRE element (Kanaya et al., 1999; Hao et al., 2002; in both studies, the protein fragments used for the analysis contained the complete CBF box-2 and parts of box-1). In vitro binding studies have shown that Arabidopsis CBF3 and barley HvCBFl bind with high affinity to the pentamer (C/t)CG(A/n)C (Sakuma et al., 2002; Xue, 2002) illustrating that the protein specificity is highly conserved between monocots and dicots. Regions flanking this motif contribute to binding specificity at the target promoters (Xue, 2002). Characterization of the protein structure of CBF1 has revealed the presence of three β- sheets and one a-helix in the AP2 domain (Kanaya et al., 1999). Binding of the AP2 domain to the major groove of the DNA occurs via the three β-sheets, which are stabilized by the a-helix (Allen et al., 1998). Four arginine, two tryptophan and one glutamic acid residue have been directly implicated in the interaction of the AP2 DBD with the target DNA (indicated by red diamonds in Figure 4).

Transcriptional activation by CBF proteins involves interaction with the histone acetyltransferase (HAT) complex (Stockinger et al. 2001). CBF1 directly interacts with the AtHAT protein, GCN5, and the ADA2 adapter protein. HAT complexes are recruited to promoters by transcription factors, and are thought to remodel chromatin structure by histone acetylation thus making certain promoters more accessible to the transcriptional apparatus. A putative nuclear localization signal is located in the CBF box-1 in the N-terminal region of G912 (Figure 5). The C -terminal half of the G912 protein harbors the activation domain which is rich in acidic amino acids. This region is sufficient for transcriptional activation as evidenced by domain swap analysis (Haake et al., unpublished results).

Genes encoding proteins from the CBF superclade have reportedly been used to engineer low temperature tolerance, osmotic stress tolerance or drought tolerance in a number of different species, such as canola, tomato, strawberry, maize, wheat, rice, and cucumber. See, for example, Jaglo et al., 2001, Hieh et al., 2002a, Zhang et al., 2004; Owen et al., 2002, Chiappetta, 2002 (Thesis); Pellegrineschi et al., 2002; Pellegrineschi et al., 2004; Kim et al., 2002 (Abstract); Tawfik and Grumet, 2003.

Overexpression of the CBF genes results in dark green, dwarfed plants with higher levels of soluble sugars, proline and COR gene transcript levels (Gilmour et al., 2000). Published metabolic profiling experiments indicated that out of 325 metabolites which increase in Arabidopsis (ecotype Ws-2) in response to cold temperatures, 256 (79%) of these metabolites increase in non-acclimated 35S: :CBF3 plants (Cook et al., 2004). CBF genes represent a critical component in the signal transduction of cold acclimation via changes in the transcriptome and metabolome.

DREB A-2 subgroup proteins, for example, DREB2A, a.k.a., G38 (SEQ ID NO: 102) and DREB2B, a.k.a., G1141 (SEQ ID NO: 103) are readily distinguishable from the A-l group of polypeptide in that they do not have the conserved CBF boxes flanking the AP2 domains. They also lack the consensus sequences shared by the CBF superclade transcription factors, i.e., SEQ ID NO: 115 and SEQ ID NO: 99. In addition, both DREB2 genes are known to be induced by drought and salt stress, whereas DREB A-l subgroup genes are known to be induced by cold, but not by drought or salt stress, has reported that DREB2A, which belongs to the A-2 subgroup of the DREB subfamily, functions to improve plant's heat tolerance through the regulation of the expression of HSPs (heat shock proteins) when overexpressed (Sakuma et al., 2006, and Schramm et al., 2008). However, despite of the extensive studies on the DREB A-l group, no role of CBF proteins in heat tolerance has been reported up to date.

Different species of plants often have different temperature requirements for optimal growth. Table 3 shows a number of cool-season annuals and warm-season annuals. Distinct mechanisms are involved for plants to adapt to cold or warm conditions, for example, in contrast to cold-stress response, the acquisition of heat tolerance is correlated to the induction of heat shock proteins (HSPs), which act as molecular chaperons to maintain homeostasis of protein folding and thus help to maintain metabolic and structural integrity of cells. CBF superclade polypeptides have been extensively characterized and many were known to function in cold responsive gene expression, but none had been previously known to have any implications in how plants respond to heat stress. Heat stress is a complex function of intensity (temperature degrees), duration and rate of increase in temperature. The magnitude of heat stress rapidly increases as temperature increases above a threshold level. A plant is under a heat stress when it is subjected to a temperature that is higher than the optimal growth temperature for a wild type plant of the same species for an extended period that is sufficient to cause undesirable changes to the wild type plant. For plants that have similar climate requirements to Arabidopsis, such a temperature could be any that is greater than 25°C, greater than 28°C, greater than 32°C, greater than 39°C, greater than 40°C, or greater than 50 °C. The heat stress duration can range from 1 hour to multiple days. The higher the temperature, the shorter the duration is needed to constitute a heat stress effect on plants. Increasing a plant's tolerance to heat will help minimize the damaging effects of heat stress and thus have a positive impact in plant yield, plant biomass, growth vigor or size. Applicants were surprised to discover that a representative number of the CBF clade and superclade sequences were able to confer greater tolerance to heat stress compared to the control plants in their experiments. The experimental results are described in the Examples herein.

Table 3. Annual cro s ecies ada ted to cool and warm seasons (Hall 2001).

A number of phylogenetically-related sequences have been found in other plant species. Table 4 shows a number of CBF clade and superclade sequences and includes the SEQ ID NO: (Column 1), the species from which the sequence was derived and the Gene Identifier ("GID"; Column 2), the percent identity of the polypeptide in Column 1 to the full length G912 polypeptide, SEQ ID NO: 8, as determined by a BLASTp analysis with a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix Henikoff & Henikoff (1989, 1991) (Column 3), the amino acid residue coordinates for the conserved AP2 domains, in amino acid coordinates beginning at the n-terminus, of each of the sequences (Column 4) , the conserved AP2 domain sequences of the respective polypeptides (Column 5); the SEQ ID NO: of each of the AP2 domains (Column 6), and the percentage identity of the conserved domain in Column 5 to the conserved domain of the Arabidopsis G912 sequence, SEQ ID NO: 68 (Column 7).

Table 4. Conserved domains of G912 and closely related sequences

Column 1 Column 2 Column 3 Column 4 Column 5 Column Column 7

0

Polypeptide Species/ Percent AP2 AP2 domain Percent

SEQ ID GID No. identity of domain in SEQ ID identity of polypeptide amino acid NO: of AP2 in Column coordinates AP2 domain in 1 to G912 domain Column 5 to conserved domain of G912

YRGVRQRNSGKW

VCEVREPNKKSRI

At/G912 100% 54-112 WLGTFPTVEMAAR 68 100%

AHDVAALALRGRS

ACLNFADS

YRGVRQRNSGKW

VCELREPNKKTRI

At/G41 68% 42-100 WLGTFQTAEMAAR 65 91 %

AHDVAAIALRGRS

ACLNFADS

YRGVRQRNSGKW

VSEVREPNKKTRI

At/G40 71% 48-106 WLGTFQTAEMAA 66 93%

RAHDVAALALRG

RSACLNFADS

YRGVRRRNSGKW VCEVREPNKKTRI

At/G42 65% 51-109 WLGTFQTAEMAA 67 93%

RAHDVAALALRG RSACLNFADS

YRGVRKRNLDKW

VCEMREPNMKTRI

Ms/G3498 72% 54-112 WLGTFPTADMAAR 69 81 %

AHDVAAKALRGRY

ACLNFAYS

YKGVRSRNPGRW

VCEVREPHGKQRI

Os/G3375 50% 53-111 WLGTFETAEMAAR 70 81 %

AHDVAAMALRGR

AACLNFADS

FKGVRRRNPGRW

VCEVREPHGKQRI

Os/G3376 50% 55-113 WLGTFETAEMAAR 71 81 %

AHDVAALALRGR

AACLNFADS

YRGVRRRNNNKW

VCEVRVPNDKSTRI

Ms/G3499 66% 50-109 WLGTYPTPEMAAR 72 83%

AHDVAALALRGKS

ACLNFANS

YRGVRRRNKNKW

VCEMRVPNNNSRI

Gm/G3467 48% 63-121 WLGTYPTPEMAAR 73 83%

AHDVAALALRGKS

ACLNFADS

FRGVRRRGRAGRW

Os/G3377 37% 45-105 VCEVRVPGSRGDRL 74 63%

WVGTFDTAEEAAR AHDAAMLALCGAS

ASLNFAD

YRGVRRRDGDKW

VCEVREPIHQRRV

22 At/G2107 47% 30-88 WLGTYPTADMAAR 75 76%

AHDVAVLALRGRS

ACLNFSDS

YRGIRRRNGDKW

VCEVREPTHQRRI

24 At/G2513 47% 30-88 WLGTYPTADMAAR 76 79%

AHDVAVLALRGRS

ACLNFADS

YRGVRRRNNNKW

VCEVRVPNDKSTRI

26 Gm/G3468 48% 67-126 WLGTYPTPEMAAR 77 83%

AHDVAALSLRGKS

ACLNFADS

YRGVRARAGGSRW

VCEVREPQAQARI

28 Os/G3373 47% 47-107 WLGTYPTPEMAAR 78 73%

AHDVAAIALRGER

GAELNFPD

FRGVRRRGAAGRW

VCEVRVPGRRGARL

30 Zm/G3442 40% 64-126 WLGTYLAAEAAAR 79 59%

AHDAAILALQGRGA

GRLNFPDS

Species abbreviations for Table 4: At - Arabidopsis thaliana; Gm - Glycine max; Ms: Medicago sativa; Os - Oryza sativa; Zm - Zea mays

Orthologs and Paralogs

Homologous sequences as described above can comprise orthologous or paralogous sequences. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. General methods for identifying orthologs and paralogs, including phylogenetic methods, sequence similarity and hybridization methods, are described herein; an ortholog or paralog, including equivalogs, may be identified by one or more of the methods described below.

As described by Eisen (1998) Genome Res. 8: 163-167, evolutionary information may be used to predict gene function. It is common for groups of genes that are homologous in sequence to have diverse, although usually related, functions. However, in many cases, the identification of homologs is not sufficient to make specific predictions because not all homologs have the same function. Thus, an initial analysis of functional relatedness based on sequence similarity alone may not provide one with a means to determine where similarity ends and functional relatedness begins. Fortunately, it is well known in the art that protein function can be classified using phylogenetic analysis of gene trees combined with the corresponding species. Functional predictions can be greatly improved by focusing on how the genes became similar in sequence (i.e., by evolutionary processes) rather than on the sequence similarity itself (Eisen, supra). In fact, many specific examples exist in which gene function has been shown to correlate well with gene phylogeny (Eisen, supra). Thus, "[t]he first step in making functional predictions is the generation of a phylogenetic tree representing the evolutionary history of the gene of interest and its homologs. Such trees are distinct from clusters and other means of characterizing sequence similarity because they are inferred by techniques that help convert patterns of similarity into evolutionary relationships .... After the gene tree is inferred, biologically determined functions of the various homologs are overlaid onto the tree. Finally, the structure of the tree and the relative phylogenetic positions of genes of different functions are used to trace the history of functional changes, which is then used to predict functions of [as yet] uncharacterized genes" (Eisen, supra).

Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994); Higgins et al. (1996)). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987)). For example, a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001)), and a group of very similar AP2 domain transcription factors from Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998)). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount (2001))

Transcription factor gene sequences are conserved across diverse eukaryotic species lines (Goodrich et al. (1993); Lin et al. (1991); Sadowski et al. (1988)). Plants are no exception to this observation; diverse plant species possess transcription factors that have similar sequences and functions. Speciation, the production of new species from a parental species, gives rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al. (1994); Higgins et al. (1996)) potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.

By using a phylogenetic analysis, one skilled in the art would recognize that the ability to deduce similar functions conferred by closely-related polypeptides is predictable. This predictability has been confirmed by our own many studies in which we have found that a wide variety of polypeptides have orthologous or closely-related homologous sequences that function as does the first, closely-related reference sequence. For example, distinct transcription factors, including:

(i) AP2 family Arabidopsis G47 (found in US patent 7,135,616), a phylogenetically-related sequence from soybean, and two phylogenetically-related homologs from rice all can confer greater tolerance to drought, hyperosmotic stress, or delayed flowering as compared to control plants;

(ii) CAAT family Arabidopsis G481 (found in PCT patent publication WO2004076638), and numerous phylogenetically-related sequences from eudicots and monocots can confer greater tolerance to drought-related stress as compared to control plants;

(iii) Myb-related Arabidopsis G682 (found in US Patents 7,223,904 and 7,193, 129) and numerous phylogenetically-related sequences from eudicots and monocots can confer greater tolerance to heat, drought-related stress, cold, and salt as compared to control plants;

(iv) WRKY family Arabidopsis G1274 (found in US patent 7,196,245) and numerous closely- related sequences from eudicots and monocots have been shown to confer increased water deprivation tolerance, and

(v) AT-hook family soy sequence G3456 (found in US patent publication 20040128712A1) and numerous phylogenetically-related sequences from eudicots and monocots, increased biomass compared to control plants when these sequences are overexpressed in plants.

The polypeptides sequences belong to distinct clades of polypeptides that include members from diverse species. In each case, most or all of the clade member sequences derived from both eudicots and monocots have been shown to confer increased yield or tolerance to one or more abiotic stresses when the sequences were overexpressed. These studies each demonstrate that evolutionarily conserved genes from diverse species are likely to function similarly (i.e., by regulating similar target sequences and controlling the same traits), and that polynucleotides from one species may be transformed into closely- related or distantly-related plant species to confer or improve traits.

Conserved domains have been used as building blocks in molecular evolution and recombined in various arrangements to make proteins of different protein families with different functions. Conserved domains often correspond to the 3-dimensional (3D) domains of proteins and contain conserved sequence patterns or motifs, which allow for their detection in polypeptide sequences with, for example, the use of a Conserved Domain Database. With such a database a query sequence may provide a good

correspondence between structural units (3D domains), identified by purely geometric criteria, and units asserted to be evolutionary conserved (domain families). Conserved domain models are based on multiple sequence alignments of related proteins spanning a variety of organisms to reveal sequence regions containing the same, or similar, patterns of amino acids. Multiple sequence alignments, three- dimensional structure and three-dimensional structure superposition of conserved domains can be used to infer sequence/structure/function relationships (Conserved Domain Database:

www.ncbi.nlm.nih.gov/Structure/cdd/cdd help.shtml#CDWhat). Since the presence of a particular conserved domain within a polypeptide is highly correlated with an evolutionarily conserved function, a conserved domain database may be used to identify the amino acids in a protein sequence that are putatively involved in functions such as binding or catalysis, as mapped from conserved domain annotations to the query sequence. For example, the presence in a protein of an AP2 DNA-binding domain that is structurally and phylogenetically similar to one or more domains shown in Table 4 would be a strong indicator of a related function in plants (e.g., the function of regulating heat tolerance, yield, size, biomass, and/or vigor; i.e., a polypeptide with such a domain is expected to confer altered heat tolerance, yield, size, biomass, and/or vigor when its expression level is altered). Sequences that are herein referred to as functionally-related and/or closely-related to the sequences or domains listed in Table 4 include polypeptides that are closely related to the polypeptides of the instant description may have conserved domains that share at least about 59% to about 100% amino acid sequence identity to the sequences provided in the Sequence Listing or in Table 4, as indicated above, and have similar functions in that the polypeptides of the instant description may, when their expression level is altered by underexpression, knocking out, or overexpression, confer at least one regulatory activity selected from the group consisting of increased heat tolerance, greater yield, greater size, greater biomass, and/or greater vigor as compared to a control plant.

At the nucleotide level, the claimed sequences will typically share at least about 30% or 40% nucleotide sequence identity, preferably at least about 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to one or more of the listed full-length sequences, or to a listed sequence but excluding or outside of the region(s) encoding a known consensus sequence or consensus DNA-binding site, or outside of the region(s) encoding one or all conserved domains. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.

Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method (see, for example, Higgins and Sharp (1988). The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including Accelrys Gene, FASTA, BLAST, or ENTREZ, FASTA and BLAST, and which may be used to calculate percent similarity. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, WI), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology

Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1 , e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see USPN 6,262,333).

Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (see internet website at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1990); Altschul et al. (1993)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989, 1991)). Unless otherwise indicated for comparisons of predicted polynucleotides, "sequence identity" refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter "off (see, for example, internet website at www.ncbi.nlm.nih.gov/).

Other techniques for alignment are described by Doolittle (1996). Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments (see Shpaer (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.

The percentage similarity between two polypeptide sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method (see, for example, Hein (1990)) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see US Patent Application No. 20010010913).

The percent identity between two polypeptide sequences can also be determined using Accelrys Gene v2.5 (2006) with default parameters: Pairwise Matrix: GONNET; Align Speed: Slow; Open Gap Penalty: 10.000; Extended Gap Penalty: .100; Multiple Matrix: GONNET; Multiple Open Gap Penalty: 10.000; Multiple Extended Gap Penalty: .05; Delay Divergent: 30; Gap Separation Distance: 8; End Gap Separation: false; Residue Specific Penalties: false; Hydrophilic Penalties: false; Hydrophilic Residues: GPSNDQEKR. The default parameters for determining percent identity between two polynucleotide sequences using Accelrys Gene are: Align Speed: Slow; Open Gap Penalty: 10.000; Extended Gap Penalty: 5.000; Multiple Open Gap Penalty: 10.000; Multiple Extended Gap Penalty: 5.000; Delay Divergent: 40; Transition: Weighted.

Thus, the instant description provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.

In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997)), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992)) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1990);

Altschul et al. (1993)), BLOCKS (Henikoff and Henikoff (1991)), Hidden Markov Models (HMM; Eddy (1996); Sonnhammer et al. (1997)), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997), and in Meyers (1995).

A further method for identifying or confirming that specific homologous sequences control the same function is by comparison of the transcript profile(s) obtained upon overexpression or knockout of two or more related polypeptides. Since transcript profiles are diagnostic for specific cellular states, one skilled in the art will appreciate that genes that have a highly similar transcript profile (e.g., with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or with greater than 90% regulated transcripts in common) will have highly similar functions. Fowler and Thomashow (2002), have shown that three paralogous AP2 family genes (CBF1, CBF2 and CBF3) are induced upon cold treatment, and each of which can condition improved freezing tolerance, and all have highly similar transcript profiles. Once a polypeptide has been shown to provide a specific function, its transcript profile becomes a diagnostic tool to determine whether paralogs or orthologs have the same function.

Furthermore, methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and AP2 domains. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function and a polypeptide sequence encoded by a polynucleotide sequence that has a function not yet determined. Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline -rich regions, cysteine repeat motifs, and the like.

Orthologs and paralogs of presently disclosed polypeptides may be cloned using compositions provided by the present description according to methods well known in the art. cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present sequences. Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue. Polypeptide-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.

Examples of orthologs of the Arabidopsis polypeptide sequences and their functionally similar orthologs are listed in Table 4 and the Sequence Listing. In addition to the sequences in Table 4 and the Sequence Listing, the claims include isolated nucleotide sequences that are phylogenetically and structurally similar to sequences listed in the Sequence Listing) and can function in a plant by increasing heat tolerance and/or and increasing yield, vigor, or biomass when ectopically expressed, or

overexpressed, in a plant.

Since a significant number of these sequences are phylogenetically and sequentially related to each other and have been shown to increase yield from a plant and/or heat stress tolerance, one skilled in the art would predict that other similar, phylogenetically related sequences falling within the present clades of polypeptides, including CBF clade and superclade polypeptide sequences, would also perform similar functions when ectopically expressed.

Identifying Polynucleotides or Nucleic Acids by Hybridization

Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof), as described in more detail in the references cited below (e.g., Sambrook et al. (1989); Berger and Kimmel (1987); and Anderson and Young (1985)).

Also provided in the instant description are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger (1987); and Kimmel (1987)). In addition to the nucleotide sequences listed in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.

With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al. (1989); Berger (1987) , pages 467-469; and Anderson and Young (1985).

Stability of DNA duplexes is affected by such factors as base composition, length, and degree of base pair mismatch. Hybridization conditions may be adjusted to allow DNAs of different sequence relatedness to hybridize. The melting temperature (T_m) is defined as the temperature when 50% of the duplex molecules have dissociated into their constituent single strands. The melting temperature of a perfectly matched duplex, where the hybridization buffer contains formamide as a denaturing agent, may be estimated by the following equations:

(I) DNA-DNA:

T_m(° C)=81.5+16.6(log [Na+])+0.41(% G+C)- 0.62(% formamide)-500/L

(II) DNA-RNA:

T_m(° C)=79.8+18.5(log [Na+])+0.58(% G+Q+ 0.12(%G+C)²- 0.5(% formamide) - 820/L

(III) RNA-RNA:

T_m(° C)=79.8+18.5(log [Na+])+0.58(% G+Q+ 0.12(%G+C)²- 0.35(% formamide) - 820/L where L is the length of the duplex formed, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, and % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, approximately 1° C is required to reduce the melting temperature for each 1 % mismatch.

Hybridization experiments are generally conducted in a buffer with a pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson and Young (1985)). Γη addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinylpyrrolidone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.

Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency (as described by the formula above). As a general guidelines high stringency is typically performed at T_m-5° C to T_m-20° C, moderate stringency at T_m-20° C to T_m-35° C and low stringency at T_m-35° C to T_m-50° C for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50° C below T_m), followed by post-hybridization washes at increasing stringencies.

Maximum rates of hybridization in solution are determined empirically to occur at T_m-25° C for DNA- DNA duplex and T_m-15° C for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.

High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter- based method such as a Southern or Northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5°C to 20°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50° C and about 70° C. More preferably, high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50° C. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, e.g., to a unique subsequence, of the DNA.

Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whereas high stringency hybridization may be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C with formamide present. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.

The washing steps that follow hybridization may also vary in stringency; the post-hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.

Thus, hybridization and wash conditions that may be used to bind and remove polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present polypeptides include, for example:

6X SSC at 65° C;

50% formamide, 4X SSC at 42° C; or

0.5X SSC, 0.1 % SDS at 65° C;

with, for example, two wash steps of 10 - 30 minutes each. Useful variations on these conditions will be readily apparent to those skilled in the art.

A person of skill in the art would not expect substantial variation among polynucleotide species provided with the present description because the highly stringent conditions set forth in the above formulae yield structurally similar polynucleotides.

If desired, one may employ wash steps of even greater stringency, including about 0.2x SSC,

0.1 % SDS at 65° C and washing twice, each wash step being about 30 minutes, or about 0.1 x SSC, 0.1 % SDS at 65° C and washing twice for 30 minutes. The temperature for the wash solutions will ordinarily be at least about 25° C, and for greater stringency at least about 42° C. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3° C to about 5° C, and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6° C to about 9° C. For identification of less closely related homologs, wash steps may be performed at a lower temperature, e.g., 50° C.

An example of a low stringency wash step employs a solution and conditions of at least 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1 % SDS over 30 minutes. Greater stringency may be obtained at 42° C in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1 % SDS over 30 minutes. Even higher stringency wash conditions are obtained at 65° C -68° C in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 % SDS. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, US Patent Application No. 20010010913).

Stringency conditions can be selected such that an oligonucleotide that is perfectly

complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10x higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a polypeptide known as of the filing date of the application. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15x or more, is obtained. Accordingly, a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2x or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

The present description also provides polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the polynucleotides within the Sequence Listing, and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger (1987), pages 399-407; and Kimmel (1987)). In addition to the nucleotide sequences in the Sequence Listing, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries, orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.

EXAMPLES

It is to be understood that this description is not limited to the particular devices, machines, materials and methods described. Although particular embodiments are described, equivalent embodiments may be used to practice the claims.

The specification, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present description and are not intended to limit the claims or description. It will be recognized by one of skill in the art that a polypeptide that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the first trait.

Example I. Project Types and Vector and Cloning Information

A variety of constructs were used to modulate the activity of lead transcription factors, and to test the activity of orthologs and paralogs in transgenic plant material. This platform provided the material for all subsequent analysis. Independent transgenic lines from each particular transformation "project" were examined for morphological and physiological phenotypes. Each individual "line" (also sometimes known as an "event") refers to the progeny plant or plants deriving from the stable integration of the transgene(s), carried within the T-DNA borders contained within a transformation construct, into a specific location or locations within the genome of the original transformed cell. It is well known in the art that different lines deriving from transformation with a given transgene may exhibit different levels of expression of that transgene due to so called "position effects" of the surrounding chromatin at the locus of integration in the genome. For this reason, multiple lines were usually examined for each transformation project. An individual project was referred to the analysis of lines for a particular construct or knockout (for example this might be 35S lines for a given gene sequence (GID, Gene Identifier) being tested, 35S lines for a paralog or ortholog of that gene sequence, lines for an RNAi construct, lines for a GAL4 fusion construct, or lines in which expression of the gene sequence is driven from a particular promoter that enhances expression in particular cell, tissue or condition.)

(1) Overexpression/tissue-specific/conditional expression

Expression of a given TF from a particular promoter was achieved either by a direct-promoter fusion construct in which that TF was cloned directly behind the promoter of interest or by a two component system. A list of all constructs used in these analyses (PIDs), including compilations of the sequences of promoter fragments and the expressed transgene sequences within the PIDs, are provided in the Sequence Listing.

The two-component expression system

For the two-component system, two separate constructs were used: Promoter:: Lex A-GAL4TA and opLexA: :TF. The first of these (Promoter: :LexA-GAL4TA) comprised a desired promoter cloned in front of a LexA DNA binding domain fused to a GAL4 activation domain. The construct vector backbone (pMEN48, also known as P5375) also carried a kanamycin resistance marker, along with an opLexA: :GFP (green fluorescent protein) reporter. Transgenic lines were obtained containing this first component, and a line was selected that showed reproducible expression of the reporter gene in the desired pattern through a number of generations. A homozygous population was established for that line, and the population was supertransformed with the second construct (opLexA: :TF) carrying the TF of interest cloned behind a LexA operator site. This second construct vector backbone (pMEN53, also known as P5381) also contained a sulfonamide resistance marker.

Each of the above methods offers a number of pros and cons. A direct fusion approach allows for much simpler genetic analysis if a given promoter-TF line is to be crossed into different genetic backgrounds at a later date. The two-component method, on the other hand, potentially allows for stronger expression to be obtained via an amplification of transcription.

In general, Arabidopsis TFs from different study groups were expressed from a range of different promoters, often with a two component method. (A study group here refers to a set of related sequences that were chosen for experimental comparison to an initial "lead" sequence of interest. For example, in this work, the sequences studied were part of the G912 Study Group.) Arabidopsis paralogs were also sometimes analyzed by the two-component method, but were typically analyzed using the only 35S promoter. An alternative promoter was sometimes used for paralogs when there was a specific indication that a different promoter might afford a more useful approach (such as when use of the 35 S promoter was expected to generate deleterious effects). Putative orthologs from other species were usually analyzed by overexpression from a 35S CaMV promoter via a direct promoter-fusion construct. The vector backbone for most of the direct promoter-fusion overexpression constructs was pMEN65, but pMEN1963 and pMEN20 were sometimes used.

Conditional expression:

Various promoters can be used to over-express of the disclosed polypeptides in plants to confer heat tolerance. However, in some cases, there may be limitations in the use of various proteins that confer stress tolerance when the proteins are overexpressed. Negative side effects associated with constitutive overexpression such as small size, delayed growth, increased disease sensitivity, and development and alteration in flowering time are common. A number of stress inducible promoters can be used promote protein expression during the periods of stress, and therefore may be used to induce overexpression of polypeptides that can confer improved tolerance when they are needed without the adverse

developmental or morphological effects that may be associated with their constitutive overexpression. In addition to the damaging effect of heat stress itself, high temperature environment may result in higher transpiration of plants which leads to a water deficit growth condition. Promoters that drive protein expression in response to high temperature or water deficit condition can be used to regulate the expression of the disclosed polypeptides to confer heat tolerance to plants. Such promoters include but are not limited to the sequences located in the promoter regions of At5g52310 (RD29A), At5g52300, AT1G16850, At3g46230, AT1G52690, At2g37870, AT5G43840, At5g66780, At3gl7520, and

At4g09600. The exemplar stress-inducible promoter sequences are listed in the sequence listing, see SEQ ID NO: 104-113.

(2) Knock-out knock-down

Where available, T-DNA insertion lines from either the public or the in-house collections were analyzed.

In cases where a T-DNA insertion line was unavailable, an RNA interference (RNAi) strategy was sometimes used. At the outset of the program, the system was tested with two well-characterized genes [LEAFY (Weigel et al., 1992) and CONSTANS (Putterill et al., 1995)] that gave clear morphological phenotypes when mutated. In each case, RNAi lines were obtained that exhibited characters seen in the null mutants.

It is known that overexpression of one or more of the CBF clade members can produce a comparable phenotype to that obtained from reducing expression (for example, by mutation or knockdown approaches such as antisense or RNA interference) of one or more of the family members. For instance, overexpression of the CBF family proteins has been widely demonstrated to confer tolerance to drought and low temperature stress (Jaglo et al., 2001). Nonetheless, Novillo 2004 reported that a null or severely hypomorphic allele mutation in cbf2, i.e., G41, resulted in an accumulation of CBFl (G40) and CBF3 (G42) transcripts and stronger and more sustained expression of CBFl -regulated genes. The cbf2 mutant plants showed higher capacity to freezing tolerance and higher drought and cold tolerance, which correlates with the increased expression of CBFl . Such results can be accounted for by cross regulation between the genes encoding different transcription factor family members. In the study by Novillo et al, (2004) supra, CBF2 was shown to be a negative transcriptional regulator of the CBFl and CBF3 genes. Therefore, overexpression of some members of this clade can be achieved through knocking down their suppressor gene expression.

A number of constructs were used to modulate the activity of sequences described herein. An individual project was defined as the analysis of lines for a particular construct (for example, this might include G912 lines that constitutively overexpressed a sequence described herein). Either a direct promoter fusion construct or a two component expression construct were used in these projects. In some studies, a full-length wild-type version of a gene was directly fused to a promoter that drove its expression in transgenic plants. Such a promoter could be the native promoter of that gene, or a constitutive promoter such as the cauliflower mosaic virus 35S promoter. In other studies, a promoter that drives tissue specific or conditional expression was used.

A list of constructs (these expression vectors are identified by a "PID" designation provided in the second column) used to created plants overexpressing CBF clade and superclade members is provided in Table 5 and in the Sequence Listing.

Table 5. Ex ression constructs used to create lants overex ressin disclosed sequences

Os/G3377 19/20 35 S Direct promoter-fusion

AI/G2107 21/22 35 S Direct promoter-fusion

AI/G2107 21/22 RD29A 2 -co mponents - sup Tfn

AI/G2513 23/24 35 S Direct promoter-fusion

Gm/G3468 25/26 35 S Direct promoter-fusion

Os/G3373 27/28 35 S Direct promoter-fusion

Species abbreviations for Table 5: At - Arabidopsis thaliana; Gm - Glycine max; Ms: Medicago sativa; Os - Oryza sativa; Zm - Zea mays

*: CUT1 promoter drives gene expression predominantly in shoot epidermal and guard-cells

** The plasmid construction was described in Gilmour et al.,(2000; see page 1862, 1^st col, ¾3), the content of which is hereby incorporated by reference.

Example II. Transformation

Transformation of Arabidopsis was performed by an Agrobacterium- ediated protocol based on the method of Bechtold and Pelletier (1998). Unless otherwise specified, all experimental work was done using the Columbia ecotype.

Plant preparation. Arabidopsis seeds were sown on mesh covered pots. The seedlings were thinned so that 6-10 evenly spaced plants remained on each pot 10 days after planting. The primary bolts were cut off a week before transformation to break apical dominance and encourage auxiliary shoots to form. Transformation was typically performed at 4-5 weeks after sowing.

Bacterial culture preparation. Agrobacterium stocks were inoculated from single colony plates or from glycerol stocks and grown with the appropriate antibiotics and grown until saturation. On the morning of transformation, the saturated cultures were centrifuged and bacterial pellets were re- suspended in Infiltration Media (0.5X MS, IX B5 Vitamins, 5% sucrose, 1 mg/ml benzylaminopurine riboside, 200 μΙ/L Silwet L77) until an A600 reading of 0.8 was reached.

Transformation and harvest of transgenic seeds. The Agrobacterium solution was poured into dipping containers. All flower buds and rosette leaves of the plants were immersed in this solution for 30 seconds. The plants were laid on their side and wrapped to keep the humidity high. The plants were kept this way overnight at 4° C and then the pots were turned upright, unwrapped, and moved to the growth racks.

The plants were maintained on the growth rack under 24-hour light until seeds were ready to be harvested. Seeds were harvested when 80% of the siliques of the transformed plants were ripe

(approximately five weeks after the initial transformation). This seed was deemed TO seed, since it was obtained from the TO generation, and was later plated on selection plates (either kanamycin or sulfonamide). Resistant plants that were identified on such selection plates comprised the Tl generation, from which transgenic seed comprising an expression vector of interest could be derived.

Example III. Morphology analysis

Morphological analysis was performed to determine whether changes in polypeptide levels affect plant growth and development. This was primarily carried out on the Tl generation, when at least 10-20 independent lines were examined. However, in cases where a phenotype required confirmation or detailed characterization, plants from subsequent generations were also analyzed.

Primary transformants were selected on MS medium with 0.3% sucrose and 50 mg/1 kanamycin. T2 and later generation plants were selected in the same manner, except that kanamycin was used at 35 mg/1. In cases where lines carry a sulfonamide marker (as in all lines generated by super-transformation), seeds were selected on MS medium with 0.3% sucrose and 1.5 mg/1 sulfonamide. KO lines were usually germinated on plates without a selection. Seeds were cold-treated (stratified) on plates for three days in the dark (in order to increase germination efficiency) prior to transfer to growth cabinets. Initially, plates were incubated at 22 °C under a light intensity of approximately 100 microEinsteins for seven days. At this stage, transformants were green, possessed the first two true leaves, and were easily distinguished from bleached kanamycin or sulfonamide-susceptible seedlings. Resistant seedlings were then transferred onto soil (Sunshine potting mix). Following transfer to soil, trays of seedlings were covered with plastic lids for 2-3 days to maintain humidity while they became established. Plants were grown on soil under fluorescent light at an intensity of 70-95 microEinsteins and a temperature of 18-23°C. Light conditions consisted of a 24-hour photoperiod unless otherwise stated. In instances where alterations in flowering time were apparent, flowering time was re-examined under both 12-hour and 24-hour light to assess whether the phenotype was photoperiod dependent. Under our 24-hour light growth conditions, the typical generation time (seed to seed) was approximately 14 weeks.

Because many aspects of Arabidopsis development are dependent on localized environmental conditions, in all cases plants were evaluated in comparison to controls in the same flat. As noted below, controls for transgenic lines were wild-type plants or transgenic plants harboring an empty transformation vector selected on kanamycin or sulfonamide. Careful examination was made at the following stages: seedling (1 week), rosette (2-3 weeks), flowering (4-7 weeks), and late seed set (8-12 weeks). Seed was also inspected. Seedling morphology was assessed on selection plates. At all other stages, plants were macroscopically evaluated while growing on soil. All significant differences (including alterations in growth rate, size, leaf and flower morphology, coloration, and flowering time) were recorded, but routine measurements were not taken if no differences were apparent. In certain cases, stem sections were stained to reveal lignin distribution. In these instances, hand-sectioned stems were mounted in phloroglucinol saturated 2M HC1 (which stains lignin pink) and viewed immediately under a dissection microscope.

Note that for a given project (gene-promoter combination, GAL4 fusion lines, RNAi lines etc.), ten lines were typically examined in subsequent plate based physiology assays.

Example IV. Physiology Experimental Methods

In subsequent Examples, unless otherwise indicted, morphological and physiological traits are disclosed in comparison to wild-type control plants. That is, a transformed plant that is described as heat tolerant was large or showed better performance in heat stress conditions with respect to a control plant, the latter including wild-type plants, parental lines and lines transformed with a vector that does not contain a sequence of interest. When a plant is said to have a better performance than controls, it generally is larger, has greater vigor, has greater yield, and/or shows less stress symptoms than control plants. The better performing lines may, for example, produce less anthocyanin, or are larger, greener, or more vigorous in response to a particular stress, as noted below. Better performance generally implies greater size or yield, or tolerance to a particular biotic or abiotic stress, less sensitivity to ABA, or better recovery from a stress (as in the case of a soil-based drought treatment) than controls.

Plate Assays. Plate-based physiological assays (shown below), heat-stress related conditions were used as a pre-screen to identify top performing lines (i.e. lines from transformation with a particular construct). Typically, ten or more lines were subjected to plate assays, although in some cases fewer or more lines were tested.

Germination assays. All germination assays were performed in tissue culture with Arabidopsis overexpressors of CBF clade and superclade sequences. Growing the plants under controlled temperature and humidity on sterile medium produces uniform plant material that has not been exposed to additional stresses (such as water stress) which could cause variability in the results obtained. All assays were designed to detect plants that were more tolerant or less tolerant to the particular stress condition and were developed with reference to the following publications: Jang et al. (1997), Smeekens (1998), Liu and Zhu (1997), Saleki et al. (1993), Wu et al. (1996), Zhu et al. (1998), Alia et al. (1998), Xin and Browse, (1998), Leon-Kloosterziel et al. (1996). Where possible, assay conditions were originally tested in a blind experiment with controls that had phenotypes related to the condition tested.

Prior to plating, seed for all experiments were surface sterilized in the following manner: (l)five minute incubation with mixing in 70 % ethanol, (2) 20 minute incubation with mixing in 30% bleach,

0.01 % triton-X 100, (3) 5X rinses with sterile water, (4) Seeds were re-suspended in 0.1 % sterile agarose and stratified at 4° C for 3-4 days.

All germination assays follow modifications of the same basic protocol. Sterile seeds were sown on the conditional media that has a basal composition of 80% MS + Vitamins. For heat sensitivity assay, plates were typically incubated at 32° C under 24-hour light (120-130 μΕ m^"2 s^"1) n a growth chamber. Evaluation of germination and seedling vigor was performed five days after planting.

Growth assays. The following growth assays were conducted with Arabidopsis overexpressors of CBF related sequences. For heat sensitivity growth assays, seeds were germinated and grown for ten days on a medium comprising 0.8X strength Murashige and Skoog (MS) basal salt , 0.05% MES, 0.6% Phytoblend agar (Caisson Laboratories, North Logan, UT), and 1 % sucrose at 22 °C. The ten-day old seedlings were then transplanted to a medium comprising half strength MS basal salt, 0.05% MES, 0.6% Phytoblend agar in 22 °C for one day to relieve transplanting stress. Heat stress (34 °C) was applied for five days, after which the plants were transferred back to 22 °C for recovery and evaluated after a further four days. Unless otherwise stated, all experiments were performed with the Arabidopsis thaliana ecotype Columbia (col-0), soybean or maize plants. Assays were usually conducted on non-selected segregating T2 populations (in order to avoid the extra stress of selection). Control plants for assays on lines containing direct promoter-fusion constructs were Col-0 plants transformed an empty transformation vector (pMEN65). Controls for 2-component lines (generated by supertransformation) were the background promoter-driver lines (i.e. promoter ::LexA-GAL4TA lines), into which the supertransformations were initially performed.

Data interpretation:

At the time of evaluation, plants were given one of the following scores:

(++) Substantially enhanced performance compared to controls. The phenotype was very consistent and growth was significantly above the normal levels of variability observed for that assay.

(+) Enhanced performance compared to controls. The response was consistent but was only moderately above the normal levels of variability observed for that assay.

(wt) No detectable difference from wild-type controls.

(-) Impaired performance compared to controls. The response was consistent but was only moderately above the normal levels of variability observed for that assay.

(- -) Substantially impaired performance compared to controls. The phenotype was consistent and growth was significantly above the normal levels of variability observed for that assay.

(n/d) Experiment failed, data not obtained, or assay not performed.

Example V. Plate-based experimental results

This report provides experimental observations for transgenic seedlings overexpressing CBF- related polypeptides in plate -based assays, testing for tolerance to heat stress.

Table 6 lists the heat tolerance observed in Arabidopsis, plants overexpressing G912 or orthologs from diverse species of plants, including Arabidopsis, soy, rice, and alfalfa, in experiments conducted to date using methods according to Example IV unless noted otherwise. All observations are made with respect to control plants that did not overexpress a CBF clade or phylogenetically-related transcription factor.

Table 6. Heat tolerance observed in plants that had altered expression of CBF clade or CBF superclade

Col. 1 Col. 2 Col. 3 Col. 4 Col. 5

Species/ Polynucleotide # lines tested on #lines observed to #lines observed to GID No. SEQ ID NO: plates be tolerant in heat be tolerant in heat

/Polypeptide germination assay growth assay SEQ ID NO:

At/G912 7/8 17 1 0

At/G912* 7/8 10 2 0

At G41 * 1/2 10 3 0

At/G41 1/2 2 N/A 2

At/G40** 3/4 1 N/A 1

At/G42 5/6 2 N/A 2

Ms/G3498 9/10 10 1 0

Os/G3375 11/12 1 1 0

Os/G3376 13/14 10 0 1

Ms/G3499 15/16 8 2 0 Gm/G3467 17/18 3 1 1

Os/G3377 19/20 10 2 0

At/G2107 21/22 5 2 0

At/G2107 21/22 6 1 0

At/G2513 23/24 5 0 2

Gm/G3468 25/26 10 0 1

Os/G3373 27/28 7 0 2

Species abbreviations for Table 6: At - Arabidopsis thaliana; Gm - Glycine max; Ms: Medicago sativa; Os - Oryza sativa; Zm - Zea mays

* RNAi nucleic acid constructs

**Seeds were germinated at 22 °C for 11 days as described in Example IV before they were subjected to heat stress (34 °C for five days)

N/A- assay not yet done or completed

The present description thus describes how the transformation of plants, including monocots, with a CBF clade or CBF superclade polypeptide can confer to the transformed plants greater tolerance to heat stress conditions than the level of heat tolerance exhibited by control plants.

Example VI. Utilities of CBF superclade sequences for improving yield or biomass.

Increased heat tolerance to plants may improve yield or biomass. These differences relative to control or untransformed plants may have had a significant positive impact on yield or biomass.

Because of the observed morphological, physiological and stress tolerance similarities among CBF-related sequences, the CBF clade and CBF superclade sequences are expected to increase yield, plant growth, vigor, size, biomass, and/or increase heat tolerance to a variety of crop plants, ornamental plants, and woody plants used in the food, ornamental, paper, pulp, lumber or other industries.

Example VII. Transformation of dicots to produce increased yield and/or heat tolerance

Crop species that overexpress polypeptides of the instant description may produce plants with increased heat stress tolerance and/or yield. Thus, polynucleotide sequences listed in the Sequence Listing recombined into, for example, one of the expression vectors of the instant description, or another suitable expression vector, may be transformed into a plant for the purpose of modifying plant traits for the purpose of improving yield and/or quality. The expression vector may contain a constitutive, tissue- specific or inducible promoter operably linked to the polynucleotide. The cloning vector may be introduced into a variety of plants by means well known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens -mediated transformation. It is now routine to produce transgenic plants using most eudicot plants (see Weissbach and Weissbach (1989); Gelvin et al. (1990); Herrera- Estrella et al. (1983); Bevan (1984); and Klee (1985)). Methods for analysis of traits are routine in the art and examples are disclosed above.

Numerous protocols for the transformation of tomato and soy plants have been previously described, and are well known in the art. Gruber et al. (1993), in Glick and Thompson (1993) describe several expression vectors and culture methods that may be used for cell or tissue transformation and subsequent regeneration. For soybean transformation, methods are described by Miki et al. (1993); and U.S. Pat. No. 5,563,055, (Townsend and Thomas), issued Oct. 8, 1996.

There are a substantial number of alternatives to Agrobacterium- ediated transformation protocols, other methods for the purpose of transferring exogenous genes into soybeans or tomatoes. One such method is microprojectile-mediated transformation, in which DNA on the surface of microprojectile particles is driven into plant tissues with a biolistic device (see, for example, Sanford et al. (1987);

Christou et al. (1992); Sanford (1993); Klein et al. (1987); U.S. Pat. No. 5,015,580 (Christou et al), issued May 14, 1991 ; and U.S. Pat. No. 5,322,783 (Tomes et al.), issued Jun. 21, 1994).

Alternatively, sonication methods (see, for example, Zhang et al. (1991)); direct uptake of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine (see, for example, Hain et al. (1985); Draper et al. (1982)); liposome or spheroplast fusion (see, for example, Deshayes et al. (1985); Christou et al. (1987)); and electroporation of protoplasts and whole cells and tissues (see, for example, Donn et al.(1990); D'Halluin et al. (1992); and Spencer et al. (1994)) have been used to introduce foreign DNA and expression vectors into plants.

After a plant or plant cell is transformed (and the transformed host plant cell then regenerated into a plant), the transformed plant may propagated vegetatively or it may be crossed with itself or a plant from the same line, a non-transformed or wild-type plant, or another transformed plant from a different transgenic line of plants. Crossing provides the advantages of producing new and often stable transgenic varieties. Genes and the traits they confer that have been introduced into a tomato or soybean line may be moved into distinct line of plants using traditional backcrossing techniques well known in the art.

Transformation of tomato plants may be conducted using the protocols of Koornneef et al (1986), and in U.S. Patent 6,613,962, the latter method described in brief here. Eight day old cotyledon explants are precultured for 24 hours in Petri dishes containing a feeder layer of Petunia hybrida suspension cells plated on MS medium with 2% (w/v) sucrose and 0.8% agar supplemented with 10 μΜ α-naphthalene acetic acid and 4.4 μΜ 6-benzylaminopurine. The explants are then infected with a diluted overnight culture of Agrobacterium tumefaciens containing an expression vector comprising a polynucleotide of the instant description for 5-10 minutes, blotted dry on sterile filter paper and cocultured for 48 hours on the original feeder layer plates. Culture conditions are as described above. Overnight cultures of

Agrobacterium tumefaciens are diluted in liquid MS medium with 2% (w/v/) sucrose, pH 5.7) to an OD₆₀₀ of 0.8.

Following cocultivation, the cotyledon explants are transferred to Petri dishes with selective medium comprising MS medium with 4.56 μΜ zeatin, 67.3 μΜ vancomycin, 418.9 μΜ cefotaxime and 171.6 μΜ kanamycin sulfate, and cultured under the culture conditions described above. The explants are subcultured every three weeks onto fresh medium. Emerging shoots are dissected from the underlying callus and transferred to glass jars with selective medium without zeatin to form roots. The formation of roots in a kanamycin sulfate-containing medium is a positive indication of a successful transformation. Transformation of soybean plants may be conducted using the methods found in, for example, U.S. Patent 5,563,055 (Townsend et al., issued October 8, 1996), described in brief here. In this method soybean seed is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Seeds are germinated by plating on 1/10 strength agar solidified medium without plant growth regulators and culturing at 28° C. with a 16 hour day length. After three or four days, seed may be prepared for cocultivation. The seedcoat is removed and the elongating radicle removed 3-4 mm below the cotyledons.

Eucalyptus is now considered an important crop that is grown for example to provide feedstocks for the pulp and paper and biofuel markets. This species is also amenable to transformation as described in PCT patent publication WO/2005/032241.

Crambe has been recognized as a high potential oilseed crop that may be grown for the production of high value oils. An efficient method for transformation of this species has been described in PCT patent publication WO 2009/067398 Al .

Overnight cultures of Agrobacterium tumefaciens harboring the expression vector comprising a polynucleotide of the instant description are grown to log phase, pooled, and concentrated by centrifugation. Inoculations are conducted in batches such that each plate of seed was treated with a newly resuspended pellet of Agrobacterium. The pellets are resuspended in 20 ml inoculation medium. The inoculum is poured into a Petri dish containing prepared seed and the cotyledonary nodes are macerated with a surgical blade. After 30 minutes the explants are transferred to plates of the same medium that has been solidified. Explants are embedded with the adaxial side up and level with the surface of the medium and cultured at 22° C. for three days under white fluorescent light. These plants may then be regenerated according to methods well established in the art, such as by moving the explants after three days to a liquid counter-selection medium (see U.S. Patent 5,563,055).

The explants may then be picked, embedded and cultured in solidified selection medium. After one month on selective media transformed tissue becomes visible as green sectors of regenerating tissue against a background of bleached, less healthy tissue. Explants with green sectors are transferred to an elongation medium. Culture is continued on this medium with transfers to fresh plates every two weeks. When shoots are 0.5 cm in length they may be excised at the base and placed in a rooting medium.

Example VIII: Transformation of monocots to produce increased yield or heat tolerance

Cereal plants such as, but not limited to, corn, wheat, rice, sorghum, or barley, may be transformed with the present polynucleotide sequences, including monocot or eudicot-derived sequences such as those presented in the present Tables, cloned into a vector such as pGA643 and containing a kanamycin-resistance marker, and expressed constitutively under, for example, the CaMV 35S or COR15 promoters, or with tissue-specific or inducible promoters. The expression vectors may be one found in the Sequence Listing, or any other suitable expression vector may be similarly used. For example, pMEN020 may be modified to replace the Nptll coding region with the BAR gene of Streptomyces hygroscopicus that confers resistance to phosphinothricin. The Kpnl and Bglll sites of the Bar gene are removed by site-directed mutagenesis with silent codon changes.

The cloning vector may be introduced into a variety of cereal plants by means well known in the art including direct DNA transfer or Agrobacterium tumefaciens-mediated transformation. The latter approach may be accomplished by a variety of means, including, for example, that of U.S. Patent No.

5,591,616, in which monocotyledon callus is transformed by contacting dedifferentiating tissue with the

Agrobacterium containing the cloning vector.

The sample tissues are immersed in a suspension of 3x10^~9 cells of Agrobacterium containing the cloning vector for 3-10 minutes. The callus material is cultured on solid medium at 25° C in the dark for several days. The calli grown on this medium are transferred to Regeneration medium. Transfers are continued every two to three weeks (two or three times) until shoots develop. Shoots are then transferred to Shoot-Elongation medium every 2-3 weeks. Healthy looking shoots are transferred to rooting medium and after roots have developed, the plants are placed into moist potting soil.

The transformed plants are then analyzed for the presence of the NPTII gene/ kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Prime Inc. (Boulder, CO).

It is also routine to use other methods to produce transgenic plants of most cereal crops (Vasil

(1994)) such as corn, wheat, rice, sorghum (Cassas et al. (1993)), and barley (Wan and Lemeaux (1994)).

DNA transfer methods such as the microprojectile method can be used for corn (Fromm et al. (1990);

Gordon-Kamm et al. (1990); Ishida (1990)), wheat (Vasil et al. (1992); Vasil et al. (1993); Weeks et al. (1993)), and rice (Christou (1991); Hiei et al. (1994); Aldemita and Hodges (1996); and Hiei et al.

(1997)). For most cereal plants, embryogenic cells derived from immature scute Hum tissues are the preferred cellular targets for transformation (Hiei et al. (1997); Vasil (1994)). For transforming corn embryogenic cells derived from immature scutellar tissue using microprojectile bombardment, the

A188XB73 genotype is the preferred genotype (Fromm et al. (1990); Gordon-Kamm et al. (1990)). After microprojectile bombardment the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm et al. (1990)). Transgenic plants from transformed host plant cells may be regenerated by standard corn regeneration techniques (Fromm et al. (1990); Gordon-Kamm et al.

(1990)).

Example IX: Expression and analysis of increased yield or heat stress tolerance in non-Arabidopsis species

Since CBF closely-related homologs, derived from various diverse plant species, that have been overexpressed in plants have the same functions of conferring increased heat tolerance, it is expected that structurally similar orthologs of the CBF clade or CBF superclade of polypeptide sequences, including SEQ ID NOs: 2n, where n=l-32, can confer increased yield, and/or increased vigor, biomass, size ,or tolerance to heat stresses relative to control plants. As sequences of the instant description have been shown to increase yield or improve heat stress tolerance in Arabidopsis, it is also expected that the sequences listed in the Sequence Listing or in Table 4, including polypeptide sequences comprising one of or any of the conserved AP2 domains provided in Table 4, or CBF clade or CBF superclade of polypeptide sequences, will increase the heat tolerance or yield of transgenic plants including transgenic non-Arabidopsis crop or other commercially important plant species, including, but not limited to, non- Arabidopsis plants and plant species such as monocots and dicots; wheat, corn (maize), teosinte (Zea species which is related to maize), rice, barley; rye; millet; sorghum; sugarcane, miscane, turfgrass, miscanthus, switchgrass; soybean, cotton, rape, oilseed rape including canola, tobacco, tomato, tomatillo, potato, sunflower, alfalfa, clover, banana, blackberry, blueberry, strawberry, raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, watermelon, rosaceous fruits including apple, peach, pear, cherry and plum; and brassicas including broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi; currant; avocado; citrus fruits including oranges, lemons, grapefruit and tangerines, artichoke, cherries; endive; leek; roots such as arrowroot, beet, cassava, turnip, radish, yam, and sweet potato; beans; woody species including pine, poplar, Eucalyptus, mint or other labiates; nuts such as walnut and peanut.

Northern blot analysis, RT-PCR or microarray analysis of the regenerated, transformed plants may be used to show expression of a polypeptide or the instant description and related genes that are capable of inducing abiotic stress tolerance, and/or larger size.

After a eudicot plant, monocot plant or plant cell has been transformed (and the latter plant host cell regenerated into a plant) and shown to have greater heat tolerance, and/or greater size, vigor, biomass, or produce greater yield relative to a control plant under the heat stress conditions, the transformed monocot plant may be crossed with itself or a plant from the same line, a non-transformed or wild-type monocot plant, or another transformed monocot plant from a different transgenic line of plants.

The function of specific polypeptides of the instant description, including closely-related orthologs, have been analyzed and may be further characterized and incorporated into crop plants. The ectopic overexpression of these sequences may be regulated using constitutive, inducible, or tissue specific regulatory elements. Genes that have been examined and have been shown to modify plant traits (including increasing yield and/or abiotic stress tolerance) encode polypeptides found in the Sequence Listing. In addition to these sequences, it is expected that newly discovered polynucleotide and polypeptide sequences closely related to polynucleotide and polypeptide sequences found in the Sequence Listing can also confer alteration of traits in a similar manner to the sequences found in the

Sequence Listing, when transformed into any of a considerable variety of plants of different species, and including dicots and monocots. The polynucleotide and polypeptide sequences derived from monocots (e.g., the rice sequences) may be used to transform both monocot and dicot plants, and those derived from dicots (e.g., the Arabidopsis and soy genes) may be used to transform either group, although it is expected that some of these sequences will function best if the gene is transformed into a plant from the same group as that from which the sequence is derived. As an example of a first step to determine heat tolerance, seeds of these transgenic plants may be subjected to germination assays under heat stress conditions as described above or methods known in the art. Plants overexpressing sequences of the instant description may be found to be more tolerant to heat by having better germination.

The heat tolerances conferred by altered expression (increasing, decreasing, or knocking out the expression level) CBF clade or CBF superclade of polypeptide sequences may contribute to increased yield of commercially available plants. For plants of which biomass is the product of interest, increasing, decreasing, or knocking out the expression level of CBF clade or CBF superclade of polypeptide sequences may increase biomass of the plants. Thus, it is thus expected that these sequences will improve yield and/or biomass in non-Arabidopsis plants relative to control plants.

It is expected that the same methods may be applied to identify other useful and valuable sequences that are functionally-related and/or closely-related to the listed sequences or domains provided in Table 4, and the sequences may be derived from a diverse range of species.

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All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The present description is not limited by the specific embodiments described herein. The instant description now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. Modifications that become apparent from the foregoing description and accompanying figures fall within the scope of the claims.

Claims

CLAIMS What is claimed is:

1. A method of producing and selecting a transgenic plant having enhanced tolerance to heat, the method comprising:

(a) exposing to a heat stress, a plant or plants containing one or more plant cells that overexpress a polypeptide having a percent identity to the AP2 domain of any of SEQ ID NOs: 2n, where n=l-32; wherein the percent identity is selected from the group consisting of at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41 %, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51 %, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%, at least 81 %, at least 82%, at least 83%, at least

84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%, at least 98%, at least 99%, and about 100%; and

(b) selecting from the plant or plants a transgenic plant line that expresses the polypeptide which, when expressed in the transgenic plant, confers greater heat stress tolerance to the transgenic plant than the heat stress tolerance of a control plant that does not overexpress the recombinant polypeptide.

2. The method of claim 1, wherein the polypeptide is a member of the CBF superclade and comprises SEQ ID NO: 114.

3. The method of claim 1, wherein the AP2 domain is at least 90% identical to the AP2 domain of SEQ ID NOs: 2n, where n=l-32.

4. The method of claim 1, wherein the polypeptide comprises SEQ ID NOs: 2n, where n=l-32.

5. The method of claim 1, wherein the transgenic plant is more tolerant to a temperature of greater than 25 °C than the control plant.

6. The method of claim 1, wherein the transgenic plant is more tolerant to a temperature of greater than 32°C than the control plant.

7. The method of claim 1, wherein the transgenic plant is selected from the group consisting of a corn, wheat, rice, Miscanthus, switchgrass, ryegrass, sugarcane, miscane, barley, sorghum, soy, cotton, canola, rapeseed, Crambe, Camelina, sugar beet, alfalfa, tomato, Eucalyptus, poplar, willow, pine, birch and a woody plant.

8. The method of claim 1, wherein expression of the polypeptide in the transgenic plant is regulated by a constitutive, tissue-specific or inducible promoter.

9. The method of claim 7, wherein the inducible promoter is selected from the group consisting of an AT5G52310 (RD29A) promoter, an At5g52300 promoter, an AT1G16850 promoter, an At3g46230 promoter, an AT1G52690 promoter, an At2g37870 promoter, an AT5G43840 promoter, an At5g66780 promoter, an At3gl7520 promoter, and an At4g09600 promoter.

10. The method of claim 1, wherein the transgenic plant is a transgenic seed or a host plant cell comprising the nucleic acid construct.

11. The method of claim 1, wherein the transgenic plant produces a greater biomass than the control plant.

12. A method of producing and selecting a transgenic plant having enhanced tolerance to heat, the method comprising:

(a) providing one or more plant cells comprising a recombinant polynucleotide encoding a polypeptide that is at least 30% identical to SEQ ID NO: 2, and has an AP2 domain that is at least 70% identical to the AP2 domain of SEQ ID NOs: 2n, where n=l-32,; and

(b) exposing a plant or plants containing the one or more plant cells to a heat stress; and

(c) selecting from the plant or plants a transformed plant that overexpresses the polypeptide which, when overexpressed in the transformed plant, confers greater heat tolerance, greater biomass, vigor or size relative to a control plant that does not contain the recombinant polynucleotide.

13. The method of claim 12, wherein the AP2 domain is at least 90% identical to the AP2 domain of SEQ ID NO: 2n, where n=l-32.

14. The method of claim 12, wherein the polypeptide comprises SEQ ID NO: 2n, where n=l-32.

15. The method of claim 12, wherein the transgenic plant is more tolerant to a temperature of greater than 25 °C than the control plant.

16. The method of claim 12, wherein the transgenic plant is more tolerant to a temperature of greater than 32°C than the control plant.

17. The method of claim 12, wherein the transgenic plant is a transgenic seed or a host plant cell comprising the nucleic acid construct.

18. The method of claim 12, wherein expression of the polypeptide in the transgenic plant is regulated by a constitutive, tissue-specific or inducible promoter

19. A method of increasing yield of plant comprising the step of;

(a) providing one or more plant cells comprising a recombinant polynucleotide encoding a polypeptide that is at least 30% identical to SEQ ID NO: 2, and has an AP2 domain that is at least 70% identical to the AP2 domain of SEQ ID NOs: 2n, where n=l-32; (b) exposing a plant or plants containing the one or more plant cells to a heat stress; and

(c) selecting a plant or plants that have increased yield as compared to a control plant.

20. The method of claim 19, wherein the increased yield is increased seed yield, increased vegetative yield, increased biomass, or increased yield of a plant product.

21. A method of producing and selecting a transgenic plant having enhanced tolerance to heat, the method comprising:

(a) providing one or more plant cells comprising a recombinant polynucleotide encoding a polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 114;

(b) exposing a plant or plants containing the one or more plant cells to a heat stress; and

(c) selecting from the plant or plants a transformed plant that overexpresses the polypeptide which, when overexpressed in the transformed plant, confers greater heat tolerance, greater biomass, vigor or size relative to a control plant that does not contain the recombinant polynucleotide.