WO2008046069A2 - Nucleotide sequences and polypetides encoded thereby useful for increasing tolerance to oxidative stress in plants - Google Patents

Nucleotide sequences and polypetides encoded thereby useful for increasing tolerance to oxidative stress in plants Download PDF

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Publication number
WO2008046069A2
WO2008046069A2 PCT/US2007/081301 US2007081301W WO2008046069A2 WO 2008046069 A2 WO2008046069 A2 WO 2008046069A2 US 2007081301 W US2007081301 W US 2007081301W WO 2008046069 A2 WO2008046069 A2 WO 2008046069A2
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Prior art keywords
plant
oxidative stress
nucleic acid
tolerance
polypeptide
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PCT/US2007/081301
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French (fr)
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WO2008046069A3 (en
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Fasong Zhou
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Ceres, Inc.
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Priority to US12/445,005 priority Critical patent/US20110265199A1/en
Publication of WO2008046069A2 publication Critical patent/WO2008046069A2/en
Publication of WO2008046069A3 publication Critical patent/WO2008046069A3/en
Priority to US13/644,359 priority patent/US9777287B2/en
Priority to US14/627,544 priority patent/US10428344B2/en
Priority to US15/689,941 priority patent/US10815494B2/en
Priority to US16/551,347 priority patent/US11624075B2/en
Priority to US16/554,116 priority patent/US11396659B2/en
Priority to US16/991,897 priority patent/US20210087576A1/en
Priority to US16/991,904 priority patent/US20210079416A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • NUCLEOTIDE SEQUENCES AND POLYPEPTIDES ENCODED THEREBY USEFUL FOR INCREASING TOLERANCE TO OXIDATIVE STRESS IN PLANTS
  • the present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able to enhance plant growth under oxidative stress conditions.
  • the present invention further relates to using the nucleic acid molecules and polypeptides to make transgenic plants, plant cells, plant materials or seeds of a plant having improved growth rate, vegetative growth, seedling vigor and/or biomass under oxidative stress conditions as compared to wild-type plants grown under similar conditions.
  • the present invention also relates to novel screening methods which comprise using sodium salicylate to induce endogenous hydrogen peroxide production and cell death (oxidative stress) or nitric oxide synthase (NOS) to induce excessive amount of nitric oxide (NO) production and stunted growth and to subsequently screen for genes and plant lines that enhance plant growth under oxidative stress conditions or high NO conditions
  • ROS reactive oxygen species
  • ROI reactive oxygen intermediates
  • AOS activated oxygen species
  • ROS/ROI/AOS include the oxygen-centered superoxide (O 2 ) and hydroxyl ( 1 OH) free radicals as well as hydrogen peroxide (H 2 O 2 ), nitric oxide (NO) and O 2 1 .
  • Oxidative stress damages cell structure and affects cell metabolism and catabolism.
  • Membrane lipids are subject to oxidation by ROS/ROI/AOS, resulting in accumulation of high molecular weight, cross-linked fatty acids and phospholipids.
  • Oxidative attack on proteins results in site-specific amino acid modifications, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electrical charge and increased susceptibility to proteolysis, all of which frequently leads to elimination of enzyme activity.
  • ROS/ROI/AOS that generate oxygen free radicals, such as ionizing radiation, also induce numerous lesions in DNA at both the sugar and base moieties which cause deletions, mutation and other lethal genetic effects such as base degradation, single strand breakage and cross-linking to proteins. Morphologically, the adverse effects of high levels of ROS accumulation are manifested as stunted growth and necrotic lesions.
  • ROS/ROI/AOS are also key regulators of metabolic and defense pathways, playing roles as signaling or secondary messenger molecules.
  • pathogen-induced ROS/ROI/AOS production is critical in disease resistance where these molecules are involved at three different levels: penetration resistance, hypersensitive response (HR) and systemic acquired resistance (Levine et al. (1994); Lamb and Dixon (1997); Zhou et al. (2000); Aviv et al. (2002)).
  • HR hypersensitive response
  • ROS/ROI/AOS function by reinforcing cell walls through polyphenols cross-linking.
  • H 2 O 2 is an active signaling molecule whose effect is dose dependent.
  • SA salicylic acid
  • SA as a phytohormone, also promotes early flowering (Martinez et al. (2004)).
  • SA at various levels may play different roles in plant growth and stress responses. However, most of the time, the increased tolerance to high levels of SA appears to be beneficial, since it reduces the side effects of SA accumulation while stimulating SA-mediated stress responses.
  • NO is capable of generating ROS/ROI/AOS and is a plant signaling molecule involved in the regulation of seed germination, stomatal closure (Mata and Lamattina (2001); Desikan et al (2002)), flowering time (He et al. (2004)), antioxidant reactions to suppress cell death (Beligni et al.
  • ROS/ROI/AOS In order to control the two-fold nature of ROS/ROI/AOS molecules, plants have developed a sophisticated regulatory system which involves both production and scavenging of ROS/ROI/AOS in cells. During normal growth and development, this pathway monitors the level of ROS/ROI/AOS produced by metabolism and controls the expression and activity of ROS/ROI/AOS scavenging pathways.
  • the major ROS/ROI/AOS scavenging mechanisms include the action of the superoxide dismutase (SOD), ascorbate perioxidase (APX) and catalase (CAT) enzymes as well as nonenzymatic components such as ascorbic acid, ⁇ - tocopherol and glutathione.
  • SOD superoxide dismutase
  • APX ascorbate perioxidase
  • CAT catalase
  • the antioxidant enzymes are believed to be critical components in preventing oxidative stress, in part because pretreatment of plants with one form of stress, and which SUMMARY
  • This document provides methods and materials related to plants having modulated levels of tolerance to oxidative stress.
  • this document provides transgenic plants and plant cells having increased levels of tolerance to oxidative stress, nucleic acids used to generate transgenic plants and plant cells having increased levels of tolerance to oxidative stress, and methods for making plants and plant cells having increased levels of tolerance to oxidative stress.
  • Such plants and plant cells provide the opportunity to produce crops or plants under oxidative stress conditions without stunted growth and diminished yields.
  • Increased levels of tolerance to oxidative stress may be useful to produce biomass which may be converted to a liquid fuel or other chemicals and/or to produce food and feed on land that is currently marginally productive, resulting in an overall expansion of arable land.
  • a method comprises growing a plant cell comprising an exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide.
  • the Hidden Markov Model (HMM) bit score of the amino acid sequence of the polypeptide is greater than about 30 using an HMM generated from the amino acid sequences depicted in one of Figures 3, 5 and 8.
  • the plant and/or plant tissue has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise the exogenous nucleic acid.
  • the amino acid sequence of the polypeptide has an HMM bit score greater than about 45 using an HMM generated from the amino acid sequences depicted in Figure 3. In some embodiments the amino acid sequence of the polypeptide has an HMM bit score greater than about 120 using an HMM generated from the amino acid sequences depicted in Figure 5. In some embodiments the amino acid sequence of the polypeptide has an HMM bit score greater than about 115 using an HMM generated from the amino acid sequences depicted in Figure 8.
  • a method comprises growing a plant cell comprising an exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85 percent or greater sequence identity to an amino acid sequence set forth in SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 11 1, 112, 1 14, 116, 117, 118, 119, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165,
  • a method comprises growing a plant cell comprising an exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to at least a fragment of a nucleotide sequence set forth in SEQ ID NOs.
  • a method comprises introducing into a plant cell an exogenous nucleic acid, that comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide.
  • the HMM bit score of the amino acid sequence of the polypeptide is greater than 30, using an HMM generated from the amino acid sequences depicted in one of Figures 3, 5 and 8.
  • a plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
  • a method comprises introducing into a plant cell an exogenous nucleic acid that comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85% percent or greater sequence identity to an amino acid sequence set forth in SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 11 1, 112, 1 14, 1 16, 1 17, 1 18, 1 19, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170
  • the methods comprise introducing into the plant cell an exogenous nucleic acid encoding polypeptides selected from the group consisting of SEQ ID NOs: 79, 94, 102 and 107.
  • a plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
  • a method comprises introducing into a plant cell an exogenous nucleic acid, that comprises a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to a nucleotide sequence set forth in SEQ ID NOs: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 1 13, 1 15, 121, 123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 30
  • Plant cells comprising an exogenous nucleic acid are provided herein.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide.
  • the HMM bit score of the amino acid sequence of the polypeptide is greater than 30, using an HMM based on the amino acid sequences depicted in one of Figures 3, 5 and 8.
  • the plant and/or plant tissue has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 11 1, 1 12, 114, 116, 1 17, 1 18, 119, 120,
  • a plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to at least a fragment of a nucleotide sequence selected from the group consisting of SEQ ID Nos. 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 113, 1 15, 121,
  • a plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
  • a transgenic plant comprising such a plant cell is also provided.
  • the transgenic plant is a member of a species selected from the group consisting of Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp.
  • Some embodiments are related to products comprising seed or vegetative tissue from transgenic plants as described above. Some embodiments relate to food or feed products from transgenic plants as described above.
  • an isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide having 80% or greater sequence identity to the amino acid sequence set forth in SEQ ID Nos. 79, 94, 102 or 107.
  • methods of identifying a genetic polymorphism associated with variation in the level of oxidative stress tolerance include providing a population of plants, and determining whether one or more genetic polymorphisms in the population are genetically linked to the locus for a polypeptide selected from the group consisting of the polypeptides depicted in Figures 3, 5, 8, or SEQ ID NO: 107 and functional homologs thereof.
  • the correlation between variation in the level of oxidative stress tolerance in plants and/or plant tissues of the population and the presence of the one or more polymorphisms in plants of the population is measured, thereby permitting identification of whether or not the one or more polymorphisms are associated with such variation.
  • methods of making a plant line include determining whether one or more genetic polymorphisms in a population of plants is associated with the locus for a polypeptide selected from the group consisting of the polypeptides depicted in Figures 3, 5, 8, or SEQ ID NO: 107 and functional homologs thereof, identifying one or more plants in the population in which the presence of at least one allele at the one or more polymorphisms is associated with variation in oxidative stress tolerance, crossing each of the one or more identified plants with itself or a different plant to produce seed, crossing at least one progeny plant grown from said seed with itself or a different plant, and repeating the crossing steps for an additional 0-5 generations to make the plant line.
  • the at least one allele will be present in the plant line.
  • the method of making a plant line may be applied, for example, to a population of switchgrass plants.
  • FIG. 1 Growth of six independent transgenic events of ME02077; T 2 generation transgenic and non-transgenic plants grown under salicylic acid stress conditions.
  • Figure 2. Growth of two selected transgenic events of ME02077; T 2 and T 3 generation transgenic and non-transgenic plants grown under salicylic acid stress conditions.
  • Figure 3. Amino acid sequence alignment of homologues of ME02077 (SEQ ID NO: 79). conserveed regions are enclosed in a box.
  • Figure 4 Growth of two selected transgenic events of ME06123; transgenic and non- transgenic plants in two consecutive generations grown under salicylic acid stress conditions. [034] Figure 5. Amino acid sequence alignment of homologues of ME06123 (SEQ ID NO: 94). conserveed regions are enclosed in a box.
  • FIG. 1 Figure 6. Growth of three selected transgenic events of ME00922; T 2 and T 3 generation transgenic and non-transgenic plants grown under L-arginine stress conditions. [036] Figure 7. Growth of two selected transgenic events of ME00922; T 3 generation transgenic and non-transgenic plants grown under L-arginine and SNP stress conditions. [037] Figure 8. Amino acid sequence alignment of homologues of ME00922 (SEQ ID NO: 102). conserveed regions are enclosed in a box.
  • FIG. 1 Growth of three transgenic events of ME12485; T 2 and T 3 generation transgenic and non-transgenic plants grown under salicylic acid stress conditions.
  • the invention features methods and materials related to modulating oxidative stress tolerance levels in plants and/or plant tissues.
  • the plants may also have increased biomass and/or yield.
  • the methods can include transforming a plant cell with a nucleic acid encoding an oxidative stress tolerance-modulating polypeptide, wherein expression of the polypeptide results in a modulated level of oxidative stress tolerance.
  • Plant cells produced using such methods can be grown to produce plants having an increased oxidative stress tolerance, and/or biomass, in comparison to wild type plants grown under the same conditions.
  • Such plants, and the seeds of such plants may be used to produce, for example, yield and/or biomass utilized for biofuel production, such as, but not limited to, ethanol and butanol.
  • amino acid refers to one of the twenty biologically occurring amino acids and to synthetic amino acids, including D/L optical isomers.
  • Cell type-preferential promoter or “tissue-preferential promoter” refers to a promoter that drives expression preferentially in a target cell type or tissue, respectively, but may also lead to some transcription in other cell types or tissues as well.
  • Control plant refers to a plant that does not contain the exogenous nucleic acid present in a transgenic plant of interest, but otherwise has the same or similar genetic background as such a transgenic plant.
  • a suitable control plant can be a non-transgenic wild type plant, a non-transgenic segregant from a transformation experiment, or a transgenic plant that contains an exogenous nucleic acid other than the exogenous nucleic acid of interest.
  • Domains are groups of substantially contiguous amino acids in a polypeptide that can be used to characterize protein families and/or parts of proteins. Such domains have a "fingerprint” or “signature” that can comprise conserved primary sequence, secondary structure, and/or three-dimensional conformation. Generally, domains are correlated with specific in vitro and/or in vivo activities.
  • a domain can have a length of from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400 amino acids.
  • Down-regulation refers to regulation that decreases production of expression products (mRNA, polypeptide, or both) relative to basal or native states.
  • Exogenous with respect to a nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not in its natural environment.
  • an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct.
  • An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism.
  • An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. It will be appreciated that an exogenous nucleic acid may have been introduced into a progenitor and not into the cell under consideration.
  • a transgenic plant containing an exogenous nucleic acid can be the progeny of a cross between a stably transformed plant and a non-transgenic plant. Such progeny are considered to contain the exogenous nucleic acid.
  • “Expression” refers to the process of converting genetic information of a polynucleotide into RNA through transcription, which is catalyzed by an enzyme, RNA polymerase, and into protein, through translation of mRNA on ribosomes.
  • “Heterologous polypeptide” as used herein refers to a polypeptide that is not a naturally occurring polypeptide in a plant cell, e.g., a transgenic Panicum virgatum plant transformed with and expressing the coding sequence for a nitrogen transporter polypeptide from a Zea mays plant.
  • isolated nucleic acid includes a naturally-occurring nucleic acid, provided one or both of the sequences immediately flanking that nucleic acid in its naturally- occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a nucleic acid that exists as a purified molecule or a nucleic acid molecule that is incorporated into a vector or a virus.
  • Modulation of the level of a compound or constituent refers to the change in the level of the indicated compound or constituent that is observed as a result of expression of, or transcription from, an exogenous nucleic acid in a plant cell. The change in level is measured relative to the corresponding level in control plants.
  • Nucleic acid and polynucleotide are used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA or RNA containing nucleic acid analogs. Polynucleotides can have any three-dimensional structure. A nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand).
  • Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes and nucleic acid primers.
  • a polynucleotide may contain unconventional or modified nucleotides.
  • "Operably linked” refers to the positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the regulatory region.
  • a regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
  • Polypeptide refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics, regardless of post-translational modification, e.g., phosphorylation or glycosylation.
  • the subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds.
  • Full-length polypeptides, truncated polypeptides, point mutants, insertion mutants, splice variants, chimeric proteins, and fragments thereof are encompassed by this definition.
  • Progeny includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F], F 2 , F 3 , F 4 , F 5 , F 6 and subsequent generation plants, or seeds formed on BCi, BC 2 , BC 3 , and subsequent generation plants, or seeds formed on FiBCi, FiBC 2 , F 1 BC 3 , and subsequent generation plants.
  • the designation Fi refers to the progeny of a cross between two parents that are genetically distinct.
  • the designations F 2 , F 3 , F 4 , F 5 and F 6 refer to subsequent generations of self- or sib-pollinated progeny of an Fi plant.
  • regulatory region refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof.
  • a regulatory region typically comprises at least a core (basal) promoter.
  • a regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • a suitable enhancer is a cis-regulatory element (-212 to -154) from the upstream region of the octopine synthase (ocs) gene. Fromm et al., The Plant Cell, 1 :977-984 (1989).
  • Up-regulation refers to regulation that increases the level of an expression product (mRNA, polypeptide, or both) relative to basal or native states.
  • Vector refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • воднк includes cloning and expression vectors, as well as viral vectors and integrating vectors.
  • An "expression vector” is a vector that includes a regulatory region.
  • Oxidative stress Plant species vary in their capacity to tolerate ROS/ROI/AOS. "Oxidative stress” can be defined as the set of environmental conditions under which a plant will begin to suffer the effects of elevated ROS/ROI/AOS concentration, such as decreases in enzymatic activity, DNA breakage, DNA-protein crosslinking, necrosis and stunted growth. For these reasons, plants experiencing oxidative stress typically exhibit a significant reduction in biomass and/or yield.
  • Elevated oxidative stress may be caused by natural, geological processes and by human activities, such as pollution. Since plant species vary in their capacity to tolerate oxidative stress, the precise environmental conditions that cause stress cannot be generalized. However, under oxidative stress conditions, oxidative stress tolerant plants produce higher biomass, yield and survivorship than plants that are not oxidative stress tolerant. Differences in physical appearance, recovery and yield can be quantified
  • Photosynthetic efficiency photosynthetic efficiency, or electron transport via photosystem II, is estimated by the relationship between Fm, the maximum fluorescence signal and the variable fluorescence, Fv. A reduction in the optimum quantum yield (Fv/Fm) indicates stress and can be used to monitor the performance of transgenic plants compared to non-transgenic plants under oxidative stress conditions.
  • SAGI Salicylic Acid Growth Index
  • Oxidative stress tolerance-modulating polypeptides described herein include oxidative stress tolerance-modulating polypeptides.
  • Oxidative stress tolerance-modulating polypeptides can be effective to modulate oxidative stress tolerance levels when expressed in a plant or plant cell.
  • Such polypeptides typically contain at least one domain indicative of oxidative stress tolerance- modulating polypeptides, as described in more detail herein.
  • Oxidative stress tolerance- modulating polypeptides typically have an HMM bit score that is greater than 30, as described in more detail herein.
  • oxidative stress tolerance-modulating polypeptides have greater than 85 % identity to SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 111, 112, 114, 116, 117, 118, 1 19, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142,
  • An oxidative stress tolerance-modulating polypeptide can contain an AP2 domain, which is predicted to be characteristic of an oxidative stress tolerance-modulating polypeptide. These polypeptides typically bind to the GCC-box pathogenesis-related promoter element and activates the plant's defense genes. Ethylene, chemically the simplest plant hormone, participates in a number of stress responses and developmental processes: e.g., fruit ripening, inhibition of stem and root elongation, promotion of seed germination and flowering, senescence of leaves and flowers, and sex determination.
  • Ethylene responsive element binding proteins have now been identified in a variety of plants. The proteins share a similar domain of around 59 amino acids, which interacts directly with the GCC box in the ERE (see e.g. PUBMED:7732375). For example, SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143,
  • An oxidative stress tolerance-modulating polypeptide can contain a transmembrane amino acid transporter protein domain, which is predicted to be characteristic of an oxidative stress tolerance-modulating polypeptide.
  • SEQ ID NOs: 94, 96, 97, 98, 99 100, 249, 251, 252, 253, 255, 255, 256, 258, 260, 261, 263, 263, 264, 266, 266, 267, 269, 270, 271, 272, 273, 274, 275, , 277, 279, , 281, 282, 284, 286, 286, 288, 289, 290, 291, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339
  • An oxidative stress tolerance-modulating polypeptide can contain a Rubisco LSMT substrate-binding domain, which is predicted to be characteristic of an oxidative stress tolerance-modulating polypeptide.
  • Members of this family adopt a multihelical structure, with an irregular array of long and short alpha-helices. They allow binding of the protein to substrate, such as the N-terminal tails of histones H3 and H4 and the large subunit of the Rubisco holoenzyme complex.
  • SEQ ID NOs: 102, 104, 105, 109, 1 10, 1 11, 1 12, 1 14, 116, 1 17, 118, 119, 120, 122, and 124 exemplify polypeptide sequences having Rubisco LSMT substrate-binding domains.
  • An oxidative stress tolerance-modulating polypeptide can contain a SET domain, which is predicted to be characteristic of an oxidative stress tolerance-modulating polypeptide.
  • SET domains are protein lysine methyltransferase enzymes. SET domains appear to be protein-protein interaction domains. SET domains sometimes mediate interactions with a family of proteins that display similarity with dual-specificity phosphatases.
  • the SET domain consists of two regions known as SET-N and SET-C. SET-C forms an unusual and conserved knot-like structure of probably functional importance. Additionally to SET-N and SET-C, an insert region (SET-I) and flanking regions of high structural variability form part of the overall structure.
  • SEQ ID NOs: 102, 104, 105, 109, 1 10, 111, 1 12, 114, 1 16, 1 17, 1 18, 1 19, 120, 122, 124, 126, and 127 exemplify polypeptide sequences having SET domains.
  • an oxidative stress tolerance-modulating polypeptide is truncated at the amino- or carboxy-terminal end of a naturally occurring polypeptide.
  • a truncated polypeptide may retain certain domains of the naturally occurring polypeptide while lacking others.
  • length variants that are up to 5 amino acids shorter or longer typically exhibit the salinity tolerance and/or oxidative stress tolerance-modulating activity of a truncated polypeptide.
  • Expression in a plant of such a truncated polypeptide confers a difference in the level of oxidative stress tolerance in a plant and/or plant tissue as compared to the corresponding level a control plant and/or tissue thereof that does not comprise the truncation.
  • one or more functional homologs of a reference oxidative stress tolerance-modulating polypeptide defined by one or more of the pfam descriptions indicated above are suitable for use as oxidative stress tolerance-modulating polypeptides.
  • a functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide.
  • a functional homolog and the reference polypeptide may be natural occurring polypeptides, and the sequence similarity may be due to convergent or divergent evolutionary events.
  • functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs.
  • Variants of a naturally occurring functional homolog such as polypeptides encoded by mutants of a wild type coding sequence, may themselves be functional homologs.
  • Functional homologs can also be created via site-directed mutagenesis of the coding sequence for an oxidative stress tolerance-modulating polypeptide, or by combining domains from the coding sequences for different naturally-occurring oxidative stress tolerance-modulating polypeptides ("domain swapping").
  • domain swapping domain swapping
  • Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of oxidative stress tolerance-modulating polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundant databases using an oxidative stress tolerance-modulating polypeptide amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as an oxidative stress tolerance-modulating polypeptide.
  • Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in oxidative stress tolerance-modulating polypeptides, e.g., conserved functional domains.
  • conserveed regions can be identified by locating a region within the primary amino acid sequence of an oxidative stress tolerance-modulating polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. A description of the information included at the Pfam database is described in Sonnhammer et al., Nucl.
  • conserveed regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate.
  • polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions.
  • conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity).
  • a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
  • Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 79 are provided in Figure 3 and in the Sequence Listing.
  • Such functional homologs include SEQ ID NO: 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242,
  • a functional homolog of SEQ ID NO: 79 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 79.
  • amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO- 94 are provided in Figure 5.
  • Such functional homologs include SEQ ID NO: 96, 97, 98, 99, 100, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 311, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352 and 353.
  • a functional homolog of SEQ ID NO: 94 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 94.
  • Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 102 are provided in Figure 8.
  • Such functional homologs include SEQ ID NO: 104, 105, 109, 110, 11 1, 112, 1 14, 116, 117, 118, 119, 120, 122, 124, 126 and 127.
  • a functional homolog of SEQ ID NO: 102 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 102.
  • amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 107 are provided in the Sequence Listing. Such functional homologs include SEQ ID NO: 354, 356 and 357).
  • a functional homolog of SEQ ID NO: 107 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 107.
  • oxidative stress tolerance-modulating polypeptide facilitates production of variants of oxidative stress tolerance-modulating polypeptides.
  • Variants of oxidative stress tolerance-modulating polypeptides typically have 10 or fewer conservative amino acid substitutions within the primary amino acid sequence, e.g., 7 or fewer conservative amino acid substitutions, 5 or fewer conservative amino acid substitutions, or between 1 and 5 conservative substitutions.
  • a useful variant polypeptide can be constructed based on one of the alignments set forth in Figures 3, 5 and 8. Such a polypeptide includes the conserved regions, arranged in the order depicted in the Figure from amino-terminal end to carboxy-terminal end.
  • Such a polypeptide may also include zero, one, or more than one amino acid in positions marked by dashes.
  • the length of such a polypeptide is the sum of the amino acid residues in all conserved regions.
  • amino acids are present at all positions marked by dashes, such a polypeptide has a length that is the sum of the amino acid residues in all conserved regions and all dashes.
  • useful oxidative stress tolerance-modulating polypeptides include those that fit a Hidden Markov Model based on the polypeptides set forth in any one of Figures 3, 5 and 8.
  • a Hidden Markov Model is a statistical model of a consensus sequence for a group of functional homologs. See, Durbin et al., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids ⁇ Cambridge University Press, [068] Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 102 are provided in Figure 8.
  • Such functional homologs include SEQ ID NO: 104, 105, 109, 110, 11 1, 1 12, 1 14, 116, 117, 118, 119, 120, 122, 124, 126 and 127.
  • a functional homolog of SEQ ID NO: 102 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 102.
  • amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 107 are provided in the Sequence Listing. Such functional homologs include SEQ ID NO: 354, 356 and 357).
  • a functional homolog of SEQ ID NO: 107 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 107.
  • oxidative stress tolerance-modulating polypeptide facilitates production of variants of oxidative stress tolerance-modulating polypeptides.
  • Variants of oxidative stress tolerance-modulating polypeptides typically have 10 or fewer conservative amino acid substitutions within the primary amino acid sequence, e.g., 7 or fewer conservative amino acid substitutions, 5 or fewer conservative amino acid substitutions, or between 1 and 5 conservative substitutions.
  • a useful variant polypeptide can be constructed based on one of the alignments set forth in Figures 3, 5 and 8. Such a polypeptide includes the conserved regions, arranged in the order depicted in the Figure from amino-terminal end to carboxy-terminal end.
  • Such a polypeptide may also include zero, one, or more than one amino acid in positions marked by dashes.
  • the length of such a polypeptide is the sum of the amino acid residues in all conserved regions.
  • amino acids are present at all positions marked by dashes, such a polypeptide has a length that is the sum of the amino acid residues in all conserved regions and all dashes.
  • useful oxidative stress tolerance-modulating polypeptides include those that fit a Hidden Markov Model based on the polypeptides set forth in any one of Figures 3, 5 and 8.
  • a Hidden Markov Model is a statistical model of a consensus sequence for a group of functional homologs. See, Durbin et al., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids ⁇ Cambridge University Press, Cambridge, UK (1998). An HMM is generated by the program HMMER 2.3.2 with default program parameters, using the sequences of the group of functional homologs as input.
  • ProbCons Do et al., Genome Res., 15(2):330- 40 (2005)) version 1.11 using a set of default parameters: -c, —consistency REPS of 2; -ir, — iterative-refinement REPS of 100; -pre, ⁇ pre-training REPS of 0.
  • ProbCons is a public domain software program provided by Stanford University.
  • HMM The default parameters for building an HMM (hmmbuild) are as follows: the default "architecture prior" (archpri) used by MAP architecture construction is 0.85, and the default cutoff threshold (idlevel) used to determine the effective sequence number is 0.62.
  • HMMER 2.3.2 was released October 3, 2003 under a GNU general public license, and is available from various sources on the World Wide Web.
  • Hmmbuild outputs the model as a text file.
  • the HMM for a group of functional homologs can be used to determine the likelihood that a candidate oxidative stress tolerance-modulating polypeptide sequence is a better fit to that particular HMM than to a null HMM generated using a group of sequences that are not structurally or functionally related.
  • the likelihood that a subject polypeptide sequence is a better fit to an HMM than to a null HMM is indicated by the HMM bit score, a number generated when the candidate sequence is fitted to the HMM profile using the HMMER hmmsearch program.
  • the following default parameters are used when running hmmsearch: the default E-value cutoff (E) is 10.0, the default bit score cutoff (T) is negative infinity, the default number of sequences in a database (Z) is the real number of sequences in the database, the default E-value cutoff for the per-domain ranked hit list (domE) is infinity, and the default bit score cutoff for the per-domain ranked hit list (domT) is negative infinity.
  • a high HMM bit score indicates a greater likelihood that the subject sequence carries out one or more of the biochemical or physiological function(s) of the polypeptides used to generate the HMM.
  • a high HMM bit score is at least 20, and often is higher. Slight variations in the HMM bit score of a particular sequence can occur due to factors such as the order in which sequences are processed for alignment by multiple sequence alignment algorithms such as the ProbCons program. Nevertheless, such HMM bit score variation is minor. [074] As those of skill in the art would appreciate, the HMM scores provided in the sequence listing are merely exemplary. Since multiple sequence alignment algorithms, such as ProbCons, can only generate near-optimal results, slight variations of the model can arise due to factors such as the order in which sequences are processed for alignment.
  • HMM score variability is minor, and so the HMM scores in the sequence listing are representative of models made with the respective sequences.
  • the oxidative stress-modulating polypeptides discussed below fit the indicated HMM with an HMM bit score greater than 20 (e.g., greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500).
  • the HMM bit score of a salinity and/or oxidative stress-modulating polypeptide discussed below is about 50%, 60%, 70%, 80%, 90%, or 95% of the HMM bit score of a functional homolog provided in the Sequence Listing.
  • an oxidative stress-modulating polypeptide discussed below fits the indicated HMM with an HMM bit score greater than 20, and has a domain indicative of an oxidative stress-modulating polypeptide.
  • an oxidative stress- modulating polypeptide discussed below fits the indicated HMM with an HMM bit score greater than 20, and has 85% or greater sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or 100% sequence identity) to an amino acid sequence shown in any one of Figures 3, 5 and 8 or to an amino acid sequence correlated in the Sequence Listing to a any one of Figures 3, 5 and 8.
  • polypeptides are provided that have HMM bit scores greater than 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 550 600, 650, 700 or 725, when fitted to an HMM generated from the amino acid sequences set forth in Figure 3.
  • Such polypeptides include Ceres SEEDLINE ID no.ME02077, Public GI ID no. 89257562, Ceres CLONE ID no.1725082, Public GI ID no. 92878368, Ceres CLONE ID no.1661 141, Public GI ID no. 92878365, Ceres CLONE ID no. 1894778, Public GI ID no. 50927523, Public GI ID no.
  • polypeptides are provided that have HMM bit scores greater than 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1200, 1250, 1300, 1350 or 1400 when fitted to an HMM generated from the amino acid sequences set forth in Figure 5.
  • Such polypeptides include Ceres SEEDLINE ID no.ME06123, Ceres ANNOT ID no. 1450631, Ceres CLONE ID no. 1658212, Public GI ID no 50927941 , Ceres CLONE ID no. 383013, Ceres CLONE ID no. 788118, Public GI ID no.
  • 10006534 (SEQ ID NO: 94, 96, 97, 98, 99, 100, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352 and 353, respectively).
  • polypeptides are provided that have HMM bit scores greater than 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1 150, 1200 or 1250 when fitted to an HMM generated from the amino acid sequences set forth in Figure 8.
  • Such polypeptides include Ceres SEEDLINE ID no ME00922, Ceres ANNOT ID no. 1536088. Public GI ID no. 77554044, Ceres CLONE ID no. 479625, Public GI ID no. 22326803, Public GI ID no. 18377718, Public GI ID no.
  • an oxidative stress tolerance-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one of the amino acid sequences set forth in SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94,
  • Polypeptides having such a percent sequence identity often have a domain indicative of an oxidative stress-modulating polypeptide and/or have an HMM bit score that is greater than 20, as discussed above.
  • amino acid sequences of oxidative stress tolerance-modulating polypeptides having at least 85% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs: 79, 80, 83, 84, 89, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 109 and 122, are provided in Figures 3, 5 and 8.
  • Percent sequence identity refers to the degree of sequence identity between any given reference sequence, e.g., SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96,
  • a candidate sequence typically has a length that is from 80 percent to 200 percent of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200 percent of the length of the reference sequence.
  • a percent identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows.
  • a reference sequence e.g., a nucleic acid sequence or an amino acid sequence
  • ClustalW version 1.83, default parameters
  • ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5.
  • gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
  • the ClustalW output is a sequence alignment that reflects the relationship between sequences.
  • ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).
  • searchlauncher.bcm.tmc.edu/multi-align/multi-align.html searchlauncher.bcm.tmc.edu/multi-align/multi-align.html
  • European Bioinformatics Institute site on the World Wide Web ebi.ac.uk/clustalw.
  • 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • an oxidative stress tolerance-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one or more of the amino acid sequence set forth in SEQ ID NO: 79 Amino acid sequences of polypeptides having high sequence identity to the polypeptide set forth in SEQ ID NO: 79 are provided in the Sequence Listing.
  • Such polypeptides include SEQ ID NO: 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242,
  • an oxidative stress tolerance-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 94.
  • Amino acid sequences of polypeptides having high sequence identity to the polypeptide set forth in SEQ ID NO: 94 are provided in the Sequence Listing.
  • Such polypeptides include SEQ ID NO: SEQ ID NO: 96, 97, 98, 99, 100, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352 and 353.
  • an oxidative stress-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 102.
  • Amino acid sequences of polypeptides having high sequence identity to the polypeptide set forth in SEQ ID NO: 102 are provided in the Sequence Listing.
  • Such polypeptides include SEQ ID NO: 102, 104, 105, 109, 1 10, 1 1 1, 1 12. 114. 116. 117, 118, 119, 120, 122, 124, 126 and 127.
  • an oxidative stress-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 107.
  • Amino acid sequences of polypeptides having high sequence identity to the polypeptide set forth in SEQ ID NO: 107 are provided in the Sequence Listing.
  • Such polypeptides include SEQ ID NO: 354, 356 and 357.
  • an oxidative stress tolerance-modulating polypeptide can include additional amino acids that are not involved in oxidative stress tolerance modulation, and thus such a polypeptide can be longer than would otherwise be the case.
  • an oxidative stress- tolerance modulating polypeptide can include a purification tag, a chloroplast transit peptide, an amyloplast transit peptide, a mitochondrial transit peptide, or a leader sequence added to the amino or carboxy terminus.
  • an oxidative stress- tolerance modulating polypeptide includes an amino acid sequence that functions as a reporter, e.g., a green fluorescent protein or yellow fluorescent protein.
  • Nucleic acids described herein include nucleic acids that are effective to modulate oxidative stress tolerance levels when transcribed in a plant or plant cell. Such nucleic acids include, without limitation, those that encode an oxidative stress tolerance-modulating polypeptide and those that can be used to inhibit expression of an oxidative stress tolerance- modulating polypeptide via a nucleic acid based method.
  • Nucleic acids encoding oxidative stress tolerance-modulating polypeptides are described herein. Such nucleic acids include SEQ ID NOs: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 113, 1 15, 121, 123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317,
  • An oxidative stress tolerance-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 78.
  • an oxidative stress tolerance- modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 78.
  • an oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 78, 81, 86, 127, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236 and 243.
  • An oxidative stress tolerance-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 93.
  • an oxidative stress tolerance- modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 93.
  • an oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 93, 95, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346 and 348.
  • An oxidative stress tolerance-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 101.
  • an oxidative stress tolerance- modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 101.
  • an oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 101, 103, 106, 108, 113, 115, 121, 123 and 125.
  • An oxidative stress tolerance-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 355.
  • an oxidative stress tolerance- modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 355.
  • an oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 355.
  • Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • PCR polymerase chain reaction
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
  • Isolated nucleic acids of the invention also can be obtained by mutagenesis of, e.g., a naturally occurring DNA.
  • a nucleic acid encoding one of the oxidative stress tolerance-modulating polypeptides described herein can be used to express the polypeptide in a plant species of interest, typically by transforming a plant cell with a nucleic acid having the coding sequence for the polypeptide operably linked in sense orientation to one or more regulatory regions.
  • nucleic acids can encode a particular oxidative stress tolerance-modulating polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
  • codons in the coding sequence for a given oxidative stress tolerance-modulating polypeptide can be modified such that optimal expression in a particular plant species is obtained, using appropriate codon bias tables for that species.
  • expression of an oxidative stress tolerance-modulating polypeptide inhibits one or more functions of an endogenous polypeptide.
  • a nucleic acid that encodes a dominant negative polypeptide can be used to inhibit protein function.
  • a dominant negative polypeptide typically is mutated or truncated relative to an endogenous wild type polypeptide, and its presence in a cell inhibits one or more functions of the wild type polypeptide in that cell, i.e., the dominant negative polypeptide is genetically dominant and confers a loss of function.
  • the mechanism by which a dominant negative polypeptide confers such a phenotype can vary but often involves a protein-protein interaction or a protein-DNA interaction.
  • a dominant negative polypeptide can be an enzyme that is truncated relative to a native wild type enzyme, such that the truncated polypeptide retains domains involved in binding a first protein but lacks domains involved in binding a second protein. The truncated polypeptide is thus unable to properly modulate the activity of the second protein. See, e.g., US 2007/0056058.
  • a point mutation that results in a non-conservative amino acid substitution in a catalytic domain can result in a dominant negative polypeptide. See, e.g., US 2005/032221.
  • a dominant negative polypeptide can be a transcription factor that is truncated relative to a native wild type transcription factor, such that the truncated polypeptide retains the DNA binding domain(s) but lacks the activation domain(s).
  • a truncated polypeptide can inhibit the wild type transcription factor from binding DNA, thereby inhibiting transcription activation.
  • RNA interference collection Oct. 2005 at nature.com/reviews/focus/mai.
  • a number of nucleic acid based methods including antisense RNA, ribozyme directed RNA cleavage, post-transcriptional gene silencing (PTGS), e.g., RNA interference (RNAi), and transcriptional gene silencing (TGS) are known to inhibit gene expression in plants.
  • PTGS post-transcriptional gene silencing
  • RNAi RNA interference
  • TLS transcriptional gene silencing
  • Antisense technology is one well-known method.
  • a nucleic acid segment from a gene to be repressed is cloned and operably linked to a regulatory region and a transcription termination sequence so that the antisense strand of RNA is transcribed.
  • the recombinant construct is then transformed into plants, as described herein, and the antisense strand of RNA is produced.
  • the nucleic acid segment need not be the entire sequence of the gene to be repressed, but typically will be substantially complementary to at least a portion of the sense strand of the gene to be repressed. Generally, higher homology can be used to compensate for the use of a shorter sequence.
  • a sequence of at least 30 nucleotides is used, e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more.
  • a nucleic acid in another method, can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA.
  • Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA.
  • Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide.
  • Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site- specific recognition sequences can be used.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contains a 5'-UG-3' nucleotide sequence.
  • the construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Patent No. 5,254,678 and WO 02/46449 and references cited therein.
  • Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.
  • tRNA transfer RNA
  • RNA endoribonucleases which have been described, such as the one that occurs naturally in Tetrahymena thermophila, can be useful. See, for example, U.S. Patent No. 4,987,071 and 6,423,885.
  • RNAi can also be used to inhibit the expression of a gene.
  • a construct can be prepared that includes a sequence that is transcribed into an RNA that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure.
  • one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of an oxidative stress tolerance- modulating polypeptide, and that is from about 10 nucleotides to about 2,500 nucleotides in length.
  • the length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides.
  • the other strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the antisense strand of the coding sequence of the oxidative stress tolerance- modulating polypeptide, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence.
  • one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the 3' or 5' untranslated region of an mRNA encoding an oxidative stress tolerance-modulating polypeptide
  • the other strand of the stem portion of the double stranded RNA comprises a sequence that is similar or identical to the sequence that is complementary to the 3' or 5' untranslated region, respectively, of the mRNA encoding the oxidative stress tolerance- modulating polypeptide.
  • one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sequence of an intron in the pre-mRNA encoding an oxidative stress tolerance-modulating polypeptide
  • the other strand of the stem portion comprises a sequence that is similar or identical to the sequence that is complementary to the sequence of the intron in the pre-mRNA.
  • the loop portion of a double stranded RNA can be from 3 nucleotides to 5,000 nucleotides, e.g., from 3 nucleotides to 25 nucleotides, from 15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides.
  • the loop portion of the RNA can include an intron.
  • a double stranded RNA can have zero, one, two, three, four, five, six, seven, eight, nine, ten, or more stem-loop structures.
  • Methods for using RNAi to inhibit the expression of a gene are known to those of skill in the art. See, e.g., U.S. Patents 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. See also WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S. Patent Publications 20030175965, 20030175783, 20040214330, and 20030180945.
  • Constructs containing regulatory regions operably linked to nucleic acid molecules in sense orientation can also be used to inhibit the expression of a gene.
  • the transcription product can be similar or identical to the sense coding sequence of an oxidative stress tolerance-modulating polypeptide.
  • the transcription product can also be unpolyadenylated, lack a 5' cap structure, or contain an unsplicable intron.
  • a construct containing a nucleic acid having at least one strand that is a template for both sense and antisense sequences that are complementary to each other is used to inhibit the expression of a gene.
  • the sense and antisense sequences can be part of a larger nucleic acid molecule or can be part of separate nucleic acid molecules having sequences that are not complementary.
  • the sense or antisense sequence can be a sequence that is identical or complementary to the sequence of an mRNA, the 3' or 5' untranslated region of an mRNA, or an intron in a pre-mRNA encoding an oxidative stress tolerance- modulating polypeptide.
  • the sense or antisense sequence is identical or complementary to a sequence of the regulatory region that drives transcription of the gene encoding an oxidative stress tolerance-modulating polypeptide.
  • the sense sequence is the sequence that is complementary to the antisense sequence.
  • the sense and antisense sequences can be any length greater than about 12 nucleotides (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides).
  • an antisense sequence can be 21 or 22 nucleotides in length.
  • the sense and antisense sequences range in length from about 15 nucleotides to about 30 nucleotides, e.g., from about 18 nucleotides to about 28 nucleotides, or from about 21 nucleotides to about 25 nucleotides.
  • an antisense sequence is a sequence complementary to an mRNA sequence encoding an oxidative stress tolerance-modulating polypeptide described herein.
  • the sense sequence complementary to the antisense sequence can be a sequence present within the mRNA of the oxidative stress tolerance-modulating polypeptide.
  • sense and antisense sequences are designed to correspond to a 15-30 nucleotide sequence of a target mRNA such that the level of that target mRNA is reduced.
  • a construct containing a nucleic acid having at least one strand that is a template for more than one sense sequence can be used to inhibit the expression of a gene.
  • a construct containing a nucleic acid having at least one strand that is a template for more than one antisense sequence can be used to inhibit the expression of a gene.
  • a construct can contain a nucleic acid having at least one strand that is a template for two sense sequences and two antisense sequences.
  • the multiple sense sequences can be identical or different, and the multiple antisense sequences can be identical or different.
  • a construct can have a nucleic acid having one strand that is a template for two identical sense sequences and two identical antisense sequences that are complementary to the two identical sense sequences.
  • an isolated nucleic acid can have one strand that is a template for (1) two identical sense sequences 20 nucleotides in length, (2) one antisense sequence that is complementary to the two identical sense sequences 20 nucleotides in length, (3) a sense sequence 30 nucleotides in length, and (4) three identical antisense sequences that are complementary to the sense sequence 30 nucleotides in length.
  • the constructs provided herein can be designed to have any arrangement of sense and antisense sequences. For example, two identical sense sequences can be followed by two identical antisense sequences or can be positioned between two identical antisense sequences.
  • a nucleic acid having at least one strand that is a template for one or more sense and/or antisense sequences can be operably linked to a regulatory region to drive transcription of an RNA molecule containing the sense and/or antisense sequence(s).
  • a nucleic acid can be operably linked to a transcription terminator sequence, such as the terminator of the nopaline synthase (nos) gene.
  • two regulatory regions can direct transcription of two transcripts: one from the top strand, and one from the bottom strand. See, for example, Yan et al, Plant Physiol, 141 : 1508-1518 (2006). The two regulatory regions can be the same or different.
  • the two transcripts can form double- stranded RNA molecules that induce degradation of the target RNA.
  • a nucleic acid can be positioned within a T-DNA or plant-derived transfer DNA (P-DNA) such that the left and right T-DNA border sequences, or the left and right border-like sequences of the P- DNA, flank or are on either side of the nucleic acid. See, US 2006/0265788.
  • the nucleic acid sequence between the two regulatory regions can be from about 15 to about 300 nucleotides in length.
  • the nucleic acid sequence between the two regulatory regions is from about 15 to about 200 nucleotides in length, from about 15 to about 100 nucleotides in length, from about 15 to about 50 nucleotides in length, from about 18 to about 50 nucleotides in length, from about 18 to about 40 nucleotides in length, from about 18 to about 30 nucleotides in length, or from about 18 to about 25 nucleotides in length.
  • nucleic-acid based methods for inhibition of gene expression in plants can be a nucleic acid analog.
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'- deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-OaIIyI sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et al, Bioorgan. Med. Chem., 4:5-23 (1996).
  • the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
  • Recombinant constructs provided herein can be used to transform plants or plant cells in order to modulate oxidative stress tolerance levels.
  • a recombinant nucleic acid construct can comprise a nucleic acid encoding an oxidative stress tolerance-modulating polypeptide as described herein, operably linked to a regulatory region suitable for expressing the oxidative stress tolerance-modulating polypeptide in the plant or cell.
  • a nucleic acid can comprise a coding sequence that encodes any of the oxidative stress tolerance-modulating polypeptides as set forth in SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 1 10, 111, 112, 114, 1 16, 1 17, 1 18, 1 19, 120, 122,
  • nucleic acids encoding oxidative stress tolerance-modulating polypeptides are set forth in SEQ ID NOs: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 1 13, 115, 121, 123,
  • the oxidative stress tolerance-modulating polypeptide encoded by a recombinant nucleic acid can be a native oxidative stress tolerance-modulating polypeptide, or can be heterologous to the cell.
  • the recombinant construct contains a nucleic acid that inhibits expression of an oxidative stress tolerance-modulating polypeptide, operably linked to a regulatory region. Examples of suitable regulatory regions are described in the section entitled "Regulatory Regions.”
  • Vectors containing recombinant nucleic acid constructs such as those described herein also are provided.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasm ids. viruses, artificial chromosomes, BACs, YACs, or PACs.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).
  • the vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers.
  • a marker gene can confer a selectable phenotype on a plant cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or an herbicide (e.g., glyphosate, chlorsulfuron or phosphinothricin).
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as luciferase, ⁇ -glucuronidase (GUS), green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FlagTM tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GUS green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine c-myc
  • hemagglutinin hemagglutinin
  • FlagTM tag Kodak, New Haven, CT
  • regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. Transcription of a nucleic acid can be modulated in a similar manner.
  • promoters initiate transcription only, or predominantly, in certain cell types.
  • the choice of regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. Transcription of a nucleic acid can be modulated in a similar manner.
  • Some suitable regulatory regions initiate transcription only, or predominantly, in certain cell types.
  • Methods for identifying and characterizing regulatory regions in plant genomic DNA are known, including, for example, those described in the following references: Jordano et al, Plant Cell, 1 :855-866 (1989); Bustos et al, Plant Cell, 1 :839-854 (1989); Green et al, EMBO J., 7:4035-4044 (1988); Meier et al., Plant Cell, 3:309-316 (1991); and Zhang et al., Plant Physiology, 110:1069-1079 (1996).
  • a promoter can be said to be "broadly expressing” when it promotes transcription in many, but not necessarily all, plant tissues.
  • a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems.
  • a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds.
  • Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326, YPO 144, YPO 190, pl3879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, and PT0633 promoters.
  • CaMV 35S promoter the cauliflower mosaic virus (CaMV) 35S promoter
  • MAS mannopine synthase
  • 1' or 2' promoters derived from T-DNA of Agrobacterium tumefaciens the figwort mosaic virus 34S promoter
  • actin promoters such as the rice actin promoter
  • ubiquitin promoters such as the maize ubiquitin-1 promoter.
  • the CaMV 35S promoter is excluded from the category of broadly expressing promoters. ii. Root Promoters
  • Root-active promoters confer transcription in root tissue, e.g., root endodermis, root epidermis, or root vascular tissues.
  • root-active promoters are root- preferential promoters, i.e., confer transcription only or predominantly in root tissue.
  • Root- preferential promoters include the YP0128, YP0275, PT0625, PT0660, PT0683, and PT0758 promoters.
  • Other root-preferential promoters include the PT0613, PT0672, PT0688, and PT0837 promoters, which drive transcription primarily in root tissue and to a lesser extent in ovules and/or seeds.
  • root-preferential promoters include the root-specific subdomains of the CaMV 35S promoter (Lam et al, Proc. Natl. Acad. Sci. USA, 86:7890- 7894 (1989)), root cell specific promoters reported by Conkling et al., Plant Physiol., 93: 1203-121 1 (1990), and the tobacco RD2 promoter. iii. Maturing Endosperm Promoters
  • promoters that drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Most suitable are promoters that are active predominantly in maturing endosperm, although promoters that are also active in other tissues can sometimes be used.
  • Non-limiting examples of maturing endosperm promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin promoter (Bustos et al., Plant Cell, 1(9):839- 853 (1989)), the soybean trypsin inhibitor promoter (Riggs et al, Plant Cell, l(6):609-621 (1989)), the ACP promoter (Baerson et al, Plant MoI Biol, 22(2):255-267 (1993)), the stearoyl-ACP desaturase promoter (Slocombe et al, Plant Physiol, 104(4):167-176 (1994)), the soybean ⁇ ' subunit of ⁇ -conglycinin promoter (Chen et al, Proc.
  • zein promoters such as the 15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zein promoter.
  • Osgt- 1 promoter from the rice glutelin-1 gene (Zheng et al, MoI Cell Biol, 13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordein promoter.
  • Other maturing endosperm promoters include the YP0092, PT0676, and PT0708 promoters.
  • Promoters that are active in ovary tissues such as the ovule wall and mesocarp can also be useful, e.g., a polygalacturonidase promoter, the banana TRX promoter, the melon actin promoter, YP0396, and PT0623.
  • promoters that are active primarily in ovules include YP0007, YPOl I l, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YPO 115, YPOl 19, YPO 120, and YP0374.
  • regulatory regions can be used that are active in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. Most suitable are promoters that drive expression only or predominantly in polar nuclei or precursors thereto and/or the central cell.
  • a pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac/early endosperm-preferential promoters, although transcription typically decreases significantly in later endosperm development during and after the cellularization phase. Expression in the zygote or developing embryo typically is not present with embryo sac/early endosperm promoters.
  • Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsis atmycl (see, Urao (1996) Plant MoI. Biol., 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Patent 6,906,244).
  • promoters that may be suitable include those derived from the following genes: maize MACl (see, Sheridan (1996) Genetics, 142: 1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) Plant MoI. Biol, 22: 10131-1038).
  • Other promoters include the following Arabidopsis promoters: YP0039, YPOlOl, YPO 102, YPOI lO, YPOl 17, YPOl 19, YP0137, DME, YP0285, and YP0212.
  • promoters that may be useful include the following rice promoters: p530cl0, pOsFIE2-2, pOsMEA, pOsYpl02, and pOsYp285. vi. Embryo Promoters
  • Embryo-preferential promoters include the barley lipid transfer protein (Ltpl) promoter ⁇ Plant Cell Rep (2001) 20:647-654), YP0097, YP0107, YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, and PT0740.
  • Ltpl barley lipid transfer protein
  • Promoters active in photosynthetic tissue confer transcription in green tissues such as leaves and stems. Most suitable are promoters that drive expression only or predominantly in such tissues. Examples of such promoters include the ribulose-l,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Lctrix laricina), the pine cab6 promoter (Yamamoto et al, Plant Cell Physiol., 35:773-778 (1994)), the Cab-1 promoter from wheat (Fejes et al, Plant MoL Biol, 15:921-932 (1990)), the CAB-I promoter from spinach (Lubberstedt et al, Plant Physiol, 104:997-1006 (1994)), the cablR promoter from rice (Luan et al, Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphate dikinase (PPDK) promoter from corn (
  • promoters that have high or preferential activity in vascular bundles include YP0087, YP0093, YPO 108, YP0022, and YP0080.
  • Other vascular tissue-preferential promoters include the glycine-rich cell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell, 3(10): 1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV) promoter (Medberry et al, Plant Cell, 4(2): 185-192 (1992)), and the rice tungro bacilliform virus (RTBV) promoter (Dai et al, Proc. Natl Acad. Sci. USA, 101(2):687-692 (2004)).
  • GRP 1.8 promoter Keller and Baumgartner, Plant Cell, 3(10): 1051-1061 (1991)
  • CoYMV Commelina yellow mottle virus
  • RTBV rice tungro bacilliform virus
  • Inducible promoters confer transcription in response to external stimuli such as chemical agents or environmental stimuli.
  • inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought.
  • drought-inducible promoters include YP0380, PT0848, YP0381, YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384, PT0688, YP0286, YP0377, PD1367, and PD0901.
  • nitrogen-inducible promoters examples include PT0863, PT0829, PT0665, and PT0886.
  • shade-inducible promoters examples include PR0924 and PT0678.
  • An example of a promoter induced by salt is rd29A (Kasuga et al. (1999) Nature Biotech 17: 287-291). x. Basal Promoters
  • a basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation.
  • Basal promoters frequently include a "TATA box” element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation.
  • Basal promoters also may include a "CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
  • CCAAT box typically the sequence CCAAT
  • promoters include, but are not limited to, shoot-preferential, callus- preferential, trichome cell-preferential, guard cell-preferential such as PT0678, tuber- preferential, parenchyma cell-preferential, and senescence-preferential promoters.
  • xii Other Regulatory Regions
  • a 5' untranslated region can be included in nucleic acid constructs described herein.
  • a 5' UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide.
  • a 3' UTR can be positioned between the translation termination codon and the end of the transcript.
  • UTRs can have particular functions such as increasing mRNA stability or attenuating translation. Examples of 3' UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence.
  • more than one regulatory region may be present in a recombinant polynucleotide, e g , introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
  • more than one regulatory region can be operably linked to the sequence of a polynucleotide encoding an oxidative stress tolerance modulating polypeptide.
  • Regulatory regions such as promoters for endogenous genes, can be obtained by chemical synthesis or by subcloning from a genomic DNA that includes such a regulatory region.
  • a nucleic acid comprising such a regulatory region can also include flanking sequences that contain restriction enzyme sites that facilitate subsequent manipulation.
  • misexpression can be accomplished using a two component system, whereby the first component consists of a transgenic plant comprising a transcriptional activator operatively linked to a promoter and the second component consists of a transgenic plant that comprise a nucleic acid molecule of the invention operatively linked to the target- binding sequence/region of the transcriptional activator. The two transgenic plants are crossed and the nucleic acid molecule of the invention is expressed in the progeny of the plant.
  • the misexpression can be accomplished by having the sequences of the two component system transformed in one transgenic plant line.
  • the invention also features transgenic plant cells and plants comprising at least one recombinant nucleic acid construct described herein.
  • a plant or plant cell can be transformed by having a construct integrated into its genome, i.e., can be stably transformed. Stably transformed cells typically retain the introduced nucleic acid with each cell division.
  • a plant or plant cell can also be transiently transformed such that the construct is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced nucleic acid construct with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.
  • Transgenic plant cells used in methods described herein can constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e g , to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. As used herein, a transgenic plant also refers to progeny of an initial transgenic plant having the transgene.
  • Transgenic plants can be grown in suspension culture, or tissue or organ culture.
  • tissue or organ culture For the purposes of this invention, solid and/or liquid tissue culture techniques can be used.
  • transgenic plant cells When using solid medium, transgenic plant cells can be placed directly onto the medium or can be placed onto a filter that is then placed in contact with the medium.
  • transgenic plant cells When using liquid medium, transgenic plant cells can be placed onto a flotation device, e.g., a porous membrane that contacts the liquid medium.
  • a solid medium can be, for example, Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, e.g., 2,4- dichlorophenoxyacetic acid (2,4-D), and a suitable concentration of a cytokinin, e.g., kinetin.
  • a reporter sequence encoding a reporter polypeptide having a reporter activity can be included in the transformation procedure and an assay for reporter activity or expression can be performed at a suitable time after transformation.
  • a suitable time for conducting the assay typically is about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days.
  • the use of transient assays is particularly convenient for rapid analysis in different species, or to confirm expression of a heterologous oxidative stress tolerance-modulating polypeptide whose expression has not previously been confirmed in particular recipient cells.
  • nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrobacterium-mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Patents 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • a population of transgenic plants can be screened and/or selected for those members of the population that have a trait or phenotype conferred by expression of the transgene. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of an oxidative stress tolerance-modulating polypeptide or nucleic acid. Physical and biochemical methods can be used to identify expression levels.
  • RNA transcripts include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, Sl RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides.
  • Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known.
  • a population of plants comprising independent transformation events can be screened for those plants having a desired trait, such as a modulated level of oxidative stress tolerance. Selection and/or screening can be carried out over one or more generations, and/or in more than one geographic location.
  • transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant.
  • selection and/or screening can be applied during a particular developmental stage in which the phenotype is expected to be exhibited by the plant. Selection and/or screening can be carried out to choose those transgenic plants having a statistically significant difference in an oxidative stress tolerance level relative to a control plant that lacks the transgene.
  • transgenic plants have an altered phenotype as compared to a corresponding control plant, as described in the "Transgenic Plant Phenotypes" section herein.
  • a population of transgenic plants can be screened and/or selected for those members of the population that have a trait or phenotype conferred by expression of the transgene. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of an tolerance-modulating polypeptide and/or nucleic acid. Physical and biochemical methods can be used to identify expression levels.
  • RNA transcripts include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, Sl RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides.
  • Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known.
  • a population of plants comprising independent transformation events can be screened for those plants having a desired trait, such as a modulated level of oxidative stress tolerance. Selection and/or screening can be carried out over one or more generations, and/or in more than one geographic location.
  • transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant.
  • selection and/or screening can be applied during a particular developmental stage in which the phenotype is expected to be exhibited by the plant. Selection and/or screening can be carried out to choose those transgenic plants having a statistically significant difference in an oxidative stress tolerance level relative to a control plant that lacks the transgene. Selected or screened transgenic plants have an altered phenotype as compared to a corresponding control plant, as described in the "Transgenic Plant Phenotypes" section herein.
  • the polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae,
  • Suitable species may include members of the genus Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum,
  • Suitable species include Panicum snn.. Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp.
  • Suitable species also include Helianthus annuus (sunflower), Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax), and Brassica juncea.
  • Suitable species also include Beta vulgaris (sugarbeet), and Manihot esculenta (cassava).
  • Suitable species also include Lycopersicon esculentum (tomato), Lactuca sativa (lettuce), Musa paradisiaca (banana), Solarium tuberosum (potato), Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), and
  • Suitable species also include Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, and Alstroemeria spp. [0148] Suitable species also include Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia) and Poinsettia pulcherrima (poinsettia).
  • Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple, Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (ryegrass) and Phleum pratense (timothy).
  • the methods and compositions can be used over a broad range of plant species, including species from the dicot genera Brassica, Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium, Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum, Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale, Sorghum, Triticosecale, Triticum, and Zea.
  • a plant is a member of the species Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearl millet).
  • the polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, wherein such plants are hybrids of different species or varieties of a species (e.g., Saccharum sp. X Miscanthus sp.)
  • a plant in which expression of an oxidative stress modulating polypeptide is modulated can have increased levels of tolerance to oxidative stress.
  • an oxidative stress-modulating polypeptide described herein can be expressed in a transgenic plant, resulting in increased levels of tolerance to oxidative stress.
  • the oxidative stress tolerance levels can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, as compared to those levels in a corresponding control plant that does not express the transgene.
  • nucleic acid molecules and polypeptides of the present invention are of interest because when the nucleic acid molecules are mis-expressed (i.e., when expressed at a non- natural location or in an increased or decreased amount relative to wild-type) they produce plants that exhibit improved oxidation tolerance as compared to wild-type plants, as evidenced in part by the results of various experiments disclosed below.
  • plants transformed with the nucleic acid molecules and polypeptides of the present invention can have any of a number of modified characteristics as compared to wild-type plants. Examples of modified characteristics include photosynthetic efficiency, seedling area, and biomass as it may be measured by plant height, leaf or rosette area, or dry mass. The modified characteristics may be observed and measured at different plant developmental stages, e.g.
  • oxidative stress tolerance can be expressed as ratios or combinations of measurements, such as salicylic acid growth index values.
  • plants transformed with the sequences of the present invention can exhibit increases in SGI, seedling area and/or SAGI values of at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, or even at least 500%.
  • SGI seedling area
  • SAGI values of at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, or even at least 500%.
  • the nucleic acid molecules and polypeptides of the present invention are used to increase the expression of genes that cause the plant to have improved biomass, growth rate and/or seedling vigor in oxidative conditions, in comparison to wild type plants under the same conditions.
  • the disclosed sequences and methods increase vegetative growth and growth rate in oxidative conditions
  • the disclosed methods can be used to enhance plant growth in plants grown in oxidative conditions.
  • plants of the present invention show, under oxidative conditions, increased photosynthetic efficiency and increased seedling area as compared to a plant of the same species that is not genetically modified for substantial vegetative growth.
  • increases in biomass production include increases of at least 5%, at least 20%, or even at least 50%, when compared to an amount of biomass production by a wild-type plant of the same species under identical conditions.
  • a difference in the amount of tolerance to oxidative stress in a transgenic plant or cell relative to a control plant or cell is considered statistically significant at p ⁇ 0.05 with an appropriate parametric or non-parametric statistic, e.g., Chi-square test, Student's t- test, Mann-Whitney test, or F-test.
  • a difference in the amount of tolerance to oxidative stress is statistically significant at p ⁇ 0.01, p ⁇ 0.005, or p ⁇ 0.001.
  • the phenotype of a transgenic plant is evaluated relative to a control plant.
  • a plant is said "not to express" a polypeptide when the plant exhibits less than 10%, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount of polypeptide or mRNA encoding the polypeptide exhibited by the plant of interest.
  • Expression can be evaluated using methods including, for example, RT-PCR, Northern blots, Sl RNase protection, primer extensions, Western blots, protein gel electrophoresis, immunoprecipitation, enzyme-linked immunoassays, chip assays, and mass spectrometry.
  • a polypeptide is expressed under the control of a tissue-preferential or broadly expressing promoter, expression can be evaluated in the entire plant or in a selected tissue. Similarly, if a polypeptide is expressed at a particular time, e.g., at a particular time in development or upon induction, expression can be evaluated selectively at a desired time period.
  • Genetic polymorphisms are discrete allelic sequence differences in a population. Typically, an allele that is present at 1% or greater is considered to be a genetic polymorphism.
  • the discovery that polypeptides disclosed herein can modulate oxidative stress tolerance content is useful in plant breeding, because genetic polymorphisms exhibiting a degree of linkage with loci for such polypeptides are more likely to be correlated with variation in an oxidative stress tolerance trait. For example, genetic polymorphisms linked to the loci for such polypeptides are more likely to be useful in marker-assisted breeding programs to create lines having a desired modulation in the oxidative stress tolerance traits.
  • one aspect of the invention includes methods of identifying whether one or more genetic polymorphisms are associated with variation in an oxidative stress tolerance trait. Such methods involve determining whether genetic polymorphisms in a given population exhibit linkage with the locus for one of the polypeptides depicted in Figures 1 thru 6 and/or a functional homolog thereof, such as, but not limited to, those in the Sequence Listing. The correlation is measured between variation in the oxidative stress tolerance traits in plants of the population and the presence of the genetic polymorphism(s) in plants of the population, thereby identifying whether or not the genetic polymorphism(s) are associated with variation for the traits.
  • the allele is associated with variation for one or both of the traits and is useful as a marker for one or more of the traits. If, on the other hand, the presence of a particular allele is not significantly correlated with the desired modulation, the allele is not associated with variation for one or more of the traits and is not useful as a marker.
  • populations suitable for use in the methods may contain a transgene for another, different trait, e.g., herbicide resistance.
  • SSR polymorphisms that are useful in such methods include simple sequence repeats (SSRs, or microsatellites), rapid amplification of polymorphic DNA (RAPDs), single nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and restriction fragment length polymorphisms (RFLPs).
  • SSR polymorphisms can be identified, for example, by making sequence specific probes and amplifying template DNA from individuals in the population of interest by PCR. If the probes flank an SSR in the population, PCR products of different sizes will be produced. See, e.g., U.S. Patent 5,766,847.
  • SSR polymorphisms can be identified by using PCR product(s) as a probe against Southern blots from different individuals in the population. See, U.H. Refseth et al., (1997) Electrophoresis 18: 1519. The identification of RFLPs is discussed, for example, in Alonso-Blanco et al. (Methods in Molecular Biology, vol.82, "Arabidopsis Protocols", pp. 137-146, J.M. Martinez-Zapater and J. Salinas, eds., c. 1998 by Humana Press, Totowa, NJ); Burr ("Mapping Genes with Recombinant Inbreds", pp.
  • the methods are directed to breeding a plant line.
  • Such methods use genetic polymorphisms identified as described above in a marker assisted breeding program to facilitate the development of lines that have a desired alteration in the oxidative stress tolerance trait(s).
  • a suitable genetic polymorphism is identified as being associated with variation for the trait, one or more individual plants are identified that possess the polymorphic allele correlated with the desired variation. Those plants are then used in a breeding program to combine the polymorphic allele with a plurality of other alleles at other loci that are correlated with the desired variation.
  • Techniques suitable for use in a plant breeding program include, without limitation, backcrossing, mass selection, pedigree breeding, bulk selection, crossing to another population and recurrent selection. These techniques can be used alone or in combination with one or more other techniques in a breeding program.
  • each identified plants is selfed or crossed a different plant to produce seed which is then germinated to form progeny plants.
  • At least one such progeny plant is then selfed or crossed with a different plant to form a subsequent progeny generation.
  • the breeding program can repeat the steps of selfing or outcrossing for an additional 0 to 5 generations as appropriate in order to achieve the desired uniformity and stability in the resulting plant line, which retains the polymorphic allele.
  • Transgenic plants provided herein have various uses in the agricultural and energy production industries. For example, transgenic plants described herein can be used to make animal feed and food products. Such plants, however, are often particularly useful as a feedstock for energy production.
  • Transgenic plants described herein often produce higher yields of grain and/or biomass per hectare, relative to control plants that lack the exogenous nucleic acid. In some embodiments, such transgenic plants provide equivalent or even increased yields of grain and/or biomass per hectare relative to control plants when grown under conditions of reduced inputs such as fertilizer and/or water. Thus, such transgenic plants can be used to provide yield stability at a lower input cost and/or under environmentally stressful conditions such as drought. In some embodiments, plants described herein have a composition that permits more efficient processing into free sugars, and subsequently ethanol, for energy production.
  • such plants provide higher yields of ethanol, butanol, other biofuel molecules, and/or sugar-derived co-products per kilogram of plant material, relative to control plants.
  • the transgenic plants described herein improve profitability for farmers and processors as well as decrease costs to consumers.
  • Seeds from transgenic plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture.
  • Packaging material such as paper and cloth are well known in the art.
  • a package of seed can have a label, e.g., a tag or label secured to the packaging material, a label printed on the packaging material, or a label inserted within the package, that describes the nature of the seeds therein.
  • Enhanced oxidative stress tolerance gives the opportunity to grow crops in oxidative stress conditions without stunted growth and diminished yields due to ion imbalance, disruption of water homeostasis, inhibition of metabolism, damage to membranes, and/or cell death.
  • the ability to grow plants in oxidative stress conditions would result in an overall expansion of arable land and increased output of land currently marginally productive due to elevated oxidative stress conditions.
  • Seed or seedling vigor is an important characteristic that can greatly influence successful growth of a plant, such as crop plants.
  • Adverse environmental conditions such as oxidative conditions, can affect a plant growth cycle, germination of seeds and seedling vigor (i.e. vitality and strength under such conditions can differentiate between successful and failed plant growth).
  • Seedling vigor has often been defined to comprise the seed properties that determine "the potential for rapid, uniform emergence and development of normal seedlings under a wide range of field conditions". Hence, it would be advantageous to develop plant seeds with increased vigor, particularly in oxidative stress conditions.
  • increased seedling vigor would be advantageous for cereal plants such as rice, maize, wheat, etc. production. For these crops, germination and growth can often be slowed or stopped by oxidation. Genes associated with increased seed vigor under oxidative stress conditions have therefore been sought for producing improved plant varieties. (Walia et al. (2005) Plant Physiology 139:822-835).
  • Wild-type Arabidopsis thaliana Wassilewskija (WS) plants are transformed with Ti plasmids containing nucleic acid sequences to be expressed, as noted in the respective examples, in the sense orientation relative to the 35S promoter in a Ti plasmid.
  • a Ti plasmid vector useful for these constructs, CRS 338 contains the Ceres- constructed, plant selectable marker gene phosphinothricin acetyltransferase (PAT), which confers herbicide resistance to transformed plants.
  • PAT phosphinothricin acetyltransferase
  • Ten independently transformed events are typically selected and evaluated for their qualitative phenotype in the T 1 generation.
  • Planting Using a 60 mL syringe, 35 mL of the seed mixture is aspirated. 25 drops are added to each pot. Clear propagation domes are placed on top of the pots that are then placed under 55% shade cloth and subirrigated by adding 1 inch of water.
  • Plant Maintenance 3 to 4 days after planting, lids and shade cloth are removed. Plants are watered as needed. After 7-10 days, pots are thinned to 20 plants per pot using forceps. After 2 weeks, all plants are subirrigated with Peters fertilizer at a rate of 1 Tsp per gallon of water. When bolts are about 5-10 cm long, they are clipped between the first node and the base of stem to induce secondary bolts. Dipping infiltration is performed 6 to 7 days after clipping.
  • Agrobacterium starter blocks are obtained (96-well block with Agrobacterium cultures grown to an OD ⁇ oo of approximately 1.0) and inoculated one culture vessel per construct by transferring 1 mL from appropriate well in the starter block. Cultures are then incubated with shaking at 27 0 C. Cultures are spun down after attaining an OD ⁇ oo of approximately 1.0 (about 24 hours). 200 mL infiltration media is added to resuspend Agrobacterium pellets. Infiltration media is prepared by adding 2.2 g MS salts, 50 g sucrose, and 5 ⁇ L 2 mg/mL benzylaminopurine to 900 mL water.
  • Salicylic Acid Screening Screening is routinely performed by agar plate assay using 100 ⁇ M or 150 ⁇ M exogenous sodium salicylate. Media contains 1/2X MS (Sigma), 150 ⁇ L 1 M sodium salicylate (Sigma), 0.5 g MES hydrate (Sigma) and 0.7% phytagar (EM Science), adjusted to pH 5.7 using ION KOH.
  • seedlings are screened daily starting at 6 days. Seedlings that grow larger and stay greener compared to WS control plants are selected as positive candidates and transferred to soil for recovery and seed set.
  • Candidate plants are re-screened by placing 36 seeds from each candidate together with a WS control on the same sodium salicylate plate. Plates are treated as described above and seedling screening begun after 4 days as described. Leaf tissue is harvested from confirmed tolerant candidates for DNA extraction and amplification of the transgene by PCR. [0182] Alternatively, superpool seeds are sown directly on soil and sprayed with 10 mM SA. Leaf tissue is harvested from tolerant candidate plants to isolate DNA for PCR amplification of the transgene and subsequent sequencing of the PCR product. [0183] Traits assessed under sodium salicylate conditions include: seedling area, photosynthesis efficiency, salicylic acid growth index (SAG) and regeneration ability.
  • SAG salicylic acid growth index
  • o Seedling area the total leaf area of a young plant about 2 weeks old.
  • o Photosynthesis efficiency (Fv/Fm) Seedling photosynthetic efficiency, or electron transport via photosystem II, is estimated by the relationship between Fm, the maximum fluorescence signal and the variable fluorescence, Fv.
  • Fv/Fm the maximum fluorescence signal
  • Fv/Fm the maximum fluorescence signal
  • Fv/Fm the maximum fluorescence signal
  • Fv the maximum fluorescence signal
  • Fv variable fluorescence
  • a reduction in the optimum quantum yield (Fv/Fm) indicates stress, and so can be used to monitor the performance of transgenic plants compared to non-transgenic plants under oxidative stress conditions
  • o Salicylic Acid Growth (SAG) Index seedling area (cm 2 ) x photosynthesis efficiency (Fv/Fm).
  • PCR was used to amplify the cDNA insert in one randomly chosen T 2 plant. This PCR product was then sequenced to confirm the sequence in the plants.
  • SAG Salicylic Acid Growth Index
  • T 2 generation transformed plants are tested on BASTA ® plates in order to determine the transgene copy number of each transformed line.
  • a BASTA ® resistant:BASTA ® sensitive segregation ratio of 15: 1 generally indicates two copies of the transgene, and such a segregation ratio of 3: 1 generally indicates one copy of the transgene.
  • Seedlings are screened daily starting at 5 days. Seedlings that grow larger and stay greener compared to WS control plants are selected as positive candidates and transferred to soil for recovery and seed set.
  • Candidate plants are re-screened by placing 36 seeds from each candidate together with a WS control on the same L-arginine plate. Plates are treated as described above and seedling screening begun after 5 days as described. Leaf tissue is harvested from confirmed tolerant candidates for DNA extraction, amplification of the transgene by PCR and sequencing of the PCR product.
  • Traits assessed under L-arginine conditions include: seedling area, photosynthesis efficiency and regeneration ability.
  • o Seedling area the total leaf area of a young plant about 2 weeks old.
  • o Photosynthesis efficiency (Fv/Fm) Seedling photosynthetic efficiency, or electron transport via photosystem II, is estimated by the relationship between Fm, the maximum fluorescence signal and the variable fluorescence, Fv.
  • Fv/Fm the maximum fluorescence signal
  • Fv the maximum fluorescence signal
  • Fv the variable fluorescence
  • PCR is used to amplify the cDNA insert in one randomly chosen T 2 plant. This PCR product is then sequenced to confirm the sequence in the plants.
  • Validation is performed as described above using 60 seeds of each event except that the media is supplemented with 0.5 g/1 MES-hydrate (M8250-Sigma) and the pH adjusted to 5.7. [0194] In some cases, validation is performed using media that is further supplemented with 100 uM SNP.
  • Example 1 ME02077( Ceres cDNA 36505846; Clone 268310; SEQ ID No.78)
  • Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Ti plasmid carrying the 35S promoter operatively linked to Ceres Clone 268310 (SEQ ID No. 79). Wildtype Ws seedlings showed necrotic lesions and stunted growth on plates containing 100 or 150 ⁇ M SA, whereas the transgenic plants showed significantly better growth.
  • the transgene encodes a 301-amino-acid protein that shows similarity to an ethylene responsive element binding factor. Segregation ratios (BASTA ® resistant: BASTA ® sensitive) indicated that ME02077-02 and ME02077-03 each contain one copy of the transgene.
  • Example 2 Ceres cDNA 23537050; Ceres ⁇ nnot. ID 508432; Locus At4s35180; SEQ ID No. 93)
  • Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Ti plasmid carrying the 35S promoter operatively linked to Ceres Annot. ID 508432 (SEQ ID No. 94). Wildtype Ws seedlings showed necrotic lesions and stunted growth on plates containing 100 or 150 ⁇ M SA, whereas the transgenic plants showed significantly better growth.
  • the transgene encodes a 456-amino-acid protein that shows similarity to an amino acid transporter. Consequently, the mechanism of the SA tolerance in ME06123 likely involves the compartmentalization of the toxic molecule.
  • Segregation ratios (BASTA ® resistant: BASTA ® sensitive) indicated that ME06123-01 and ME06123-03 each contain one copy of the transgene.
  • ME18881 an idependent transgenic line with the same sequence as ME06123, was also tested and the results below obtained.
  • Example 3 ME00922 (Ceres cDN ⁇ 23372643; clone 41610; SEQ ID No. 101)
  • Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Ti plasmid carrying the 32449 promoter operatively linked to Ceres cDNA 23372643 (SEQ ID No. 102). Wildtype Ws seedlings showed necrotic lesions and stunted growth on plates containing 10 mM L- arginine, whereas the transgenic plants showed significantly better growth.
  • the transgene encodes a 516 amino-acid protein that contains a SET domain, which has been implicated in transcriptional regulation via histone methylation (Springer et al. 2003).
  • Three transformed lines, ME00922-02, ME00922-03 and ME00922-05 showed the strongest qualitative tolerance to oxidative stress in a prevalidation assay.
  • T 3 lines of ME00922 were also tested for oxidative stress tolerance on growth media supplemented with 100 ⁇ M SNP.
  • five individual lines derived from ME00922-03 and ME00922-04 showed significantly increased seedling area relative to non-transgenic plants.
  • the T 3 generation value for ME00922-03-01, -02 and -03 seedlings increased by 20.95%, 26.98% and 43.51%, respectively.
  • the increase for ME00922-04-01 and -03 seedlings was 26.96% and 102.32%, respectively.
  • Example 4 Example 4: ME12485 (Ceres cDNA 23527804; Ceres Annot. 544535; Locus Atls26710; SEO ID No. 107)
  • Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Ti plasmid carrying the CaMV 35S promoter operatively linked to Ceres Annot. ID 544535 (SEQ ID No. 107). Wildtype Ws seedlings showed necrotic lesions and stunted growth on plates containing 100 ⁇ M SA, whereas the transgenic plants showed significantly better growth. The transgene encodes a 168 amino acid protein of unknown function. Three transformed lines, ME12485- 05, ME12485-08 and ME12485-09, showed the strongest qualitative tolerance to oxidative stress in a prevalidation assay. Their tolerance to 100 ⁇ M SA was further evaluated in a validation assay for two generations. Segregation ratios (BASTA ® resistant: BASTA ® sensitive) indicated that ME12485-05, ME12485-08 and ME12485-09 each contain at least one copy of the transgene.
  • Example 5- Determination of Functional Homologs by Reciprocal BLAST A candidate sequence was considered a functional homolog of a reference sequence if the candidate and reference sequences encoded proteins having a similar function and/or activity.
  • a process known as Reciprocal BLAST (Rivera et al., Proc. Natl. Acad. Sci. USA, 95:6239-6244 (1998)) was used to identify potential functional homolog sequences from databases consisting of all available public and proprietary peptide sequences, including NR from NCBI and peptide translations from Ceres clones.
  • a specific reference polypeptide was searched against all peptides from its source species using BLAST in order to identify polypeptides having BLAST sequence identity of 80% or greater to the reference polypeptide and an alignment length of 85% or greater along the shorter sequence in the alignment.
  • the reference polypeptide and any of the aforementioned identified polypeptides were designated as a cluster.
  • the BLASTP version 2.0 program from Washington University at Saint Louis, Missouri, USA was used to determine BLAST sequence identity and E-value.
  • the BLASTP version 2.0 program includes the following parameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3) the -postsw option.
  • the BLAST sequence identity was calculated based on the alignment of the first BLAST HSP (High-scoring Segment Pairs) of the identified potential functional homolog sequence with a specific reference polypeptide. The number of identically matched residues in the BLAST HSP alignment was divided by the HSP length, and then multiplied by 100 to get the BLAST sequence identity.
  • the HSP length typically included gaps in the alignment, but in some cases gaps were excluded.
  • the main Reciprocal BLAST process consists of two rounds of BLAST searches; forward search and reverse search.
  • a reference polypeptide sequence "polypeptide A”
  • top hits were determined using an E-value cutoff of 10 " 5 and a sequence identity cutoff of 35%.
  • the sequence having the lowest E-value was designated as the best hit, and considered a potential functional homolog or ortholog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original reference polypeptide was considered a potential functional homolog or ortholog as well. This process was repeated for all species of interest.
  • top hits identified in the forward search from all species were BLASTed against all protein sequences from the source species SA.
  • a top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit was also considered as a potential functional homolog.
  • HMMs Hidden Markov Models
  • HMMs were also generated using the sequences shown in Figures 5 and 8 as input. These sequences were input into the respective models and the corresponding HMM bit score for each sequence is shown in the Sequence Listing. Additional sequences were input into the models, and the HMM bit scores for the additional sequences are shown in the Sequence Listing. The results indicate that these additional sequences are functional homologs of SEQ ID NO: 79.
  • HMMs were also generated using the sequences shown in Figures 5 and 8 as input. These sequences were input into the respective models and the corresponding HMM bit score for each sequence is shown in the Sequence Listing. Additional sequences were input into the models, and the HMM bit scores for the additional sequences are shown in the Sequence Listing. The results indicate that these additional sequences are functional homologs of the groups in Figures 5 and 8.
  • CBFl, 2, and 3 is gated by the circadian clock. Plant Physiol 137(3):961-8.
  • Arabidopsis thaliana SOS3 molecular mechanism of sensing calcium for salt stress response J MoI
  • the novel ethylene-regulated gene OsUspl from rice encodes a member of a plant protein family related to prokaryotic universal stress proteins.

Abstract

The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able confer the trait of improved plant size, vegetative growth, growth rate, seedling vigor and/or biomass in plants challenged with oxidative stress conditions. The present invention further relates to the use of these nucleic acid molecules and polypeptides in making transgenic plants, plant cells, plant materials or seeds of a plant having plant size, vegetative growth, growth rate, seedling vigor and/or biomass that are improved in oxidative stress conditions with respect to wild-type plants grown under similar conditions.

Description

NUCLEOTIDE SEQUENCES AND POLYPEPTIDES ENCODED THEREBY USEFUL FOR INCREASING TOLERANCE TO OXIDATIVE STRESS IN PLANTS
TECHNICAL FIELD
[001] The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able to enhance plant growth under oxidative stress conditions. The present invention further relates to using the nucleic acid molecules and polypeptides to make transgenic plants, plant cells, plant materials or seeds of a plant having improved growth rate, vegetative growth, seedling vigor and/or biomass under oxidative stress conditions as compared to wild-type plants grown under similar conditions. The present invention also relates to novel screening methods which comprise using sodium salicylate to induce endogenous hydrogen peroxide production and cell death (oxidative stress) or nitric oxide synthase (NOS) to induce excessive amount of nitric oxide (NO) production and stunted growth and to subsequently screen for genes and plant lines that enhance plant growth under oxidative stress conditions or high NO conditions
BACKGROUND
[002] Plants lead a sessile lifestyle and so are generally destined to reside where their seed germinates. Consequently, they can be exposed to unfavorable environmental conditions arising from weather, pollution and location. Stress conditions, such as extremes in temperature, drought and desiccation, salinity, soil nutrient content, heavy metals, UV radiation, pollutants such as ozone and SO2, mechanical stress, high light and pathogen attack, have a large impact on plant growth and development. These types of stress exposure induce formation of toxic oxygen species, which are generated in all aerobic cells and are associated with oxidative damage at the cellular level. Several recently published reports have characterized toxic oxygen species generation and the subsequent oxidative damage caused by abiotic stresses (see Larkindale and Knight (2002); Borsani et al. (2001); Lee et al (2004); Aroca et al (2005); Luna et al (2005); and Noctor et al (2002)). [003] The toxic oxygen species are referred to as reactive oxygen species (ROS), reactive oxygen intermediates (ROI) or activated oxygen species (AOS) and are partially reduced or activated derivatives of oxygen. ROS/ROI/AOS include the oxygen-centered superoxide (O2) and hydroxyl (1OH) free radicals as well as hydrogen peroxide (H2O2), nitric oxide (NO) and O2 1. These oxygen species are generated as byproducts from reactions that occur during photosynthesis, respiration and photorespiration, and are predominantly formed in the chloroplasts, mitochondria, endoplasmic reticulum, microbodies (e.g. peroxisomes and glyoxysomes), plasma membranes and cell walls. While the toxicity of O2 " and H2O2 themselves is relatively low, their metal-dependent conversion to highly toxic -OH is thought to be responsible for the majority of the biological damage associated with these molecules. [004] Oxidative stress damages cell structure and affects cell metabolism and catabolism. Membrane lipids are subject to oxidation by ROS/ROI/AOS, resulting in accumulation of high molecular weight, cross-linked fatty acids and phospholipids. Oxidative attack on proteins results in site-specific amino acid modifications, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electrical charge and increased susceptibility to proteolysis, all of which frequently leads to elimination of enzyme activity. ROS/ROI/AOS that generate oxygen free radicals, such as ionizing radiation, also induce numerous lesions in DNA at both the sugar and base moieties which cause deletions, mutation and other lethal genetic effects such as base degradation, single strand breakage and cross-linking to proteins. Morphologically, the adverse effects of high levels of ROS accumulation are manifested as stunted growth and necrotic lesions.
[005] Although capable of producing damage, ROS/ROI/AOS are also key regulators of metabolic and defense pathways, playing roles as signaling or secondary messenger molecules. For example, pathogen-induced ROS/ROI/AOS production is critical in disease resistance where these molecules are involved at three different levels: penetration resistance, hypersensitive response (HR) and systemic acquired resistance (Levine et al. (1994); Lamb and Dixon (1997); Zhou et al. (2000); Aviv et al. (2002)). In penetration resistance, ROS/ROI/AOS function by reinforcing cell walls through polyphenols cross-linking. With respect to hypersensitive response, H2O2 is an active signaling molecule whose effect is dose dependent. At high dosages, H2O2 triggers hypersensitive cell death and thus restricts the pathogen to local infection sites (Lamb and Dixon (1997)) while low dosages block cell cycle progression (Reichheld et al. (1999)) and signal secondary wall differentiation (Potikha et al. (1999)). Lastly, ROS/ROI/AOS molecules play a role in broad-spectrum systemic acquired disease resistance by triggering micro-HR systematically after the first pathogen inoculation. [006] In the signal cascades leading to oxidative stress, salicylic acid (SA) has been identified as an important signaling molecule to mediate ROS/ROI/AOS accumulation in various stress conditions, such as salt and osmotic stress (Borsani et al. (2001)), drought (Senaratna et al. (2000)), heat (Dat et al. (1998)), cold (Scott et al. (2004)), UV-light (Surplus et al. (1998)), paraquat (Kim et al. (2003)) and disease resistance against different pathogens (Zhou et al. (2004)). High levels of SA induce H2O2 production as well as cell death. [007] Several signaling components required for SA-mediated ROS/ROI/AOS accumulation and gene expression have been characterized. For example, NPi?/ is required for SA-induced PR gene expression and disease resistance (Cao et al. (1994)). The mutations in edsl and eds5 block SA-mediated signaling and enhance disease susceptibility (Rusterucci et al. (2001)). Over-expression of NahG in various plant species also suppresses SA-induced responses to both abiotic and biotic stresses (Delaney et al. (1994)). Recently, Scott and colleagues (2004) reported that chilling treatment induced accumulation of SA in Arabidopsis and the degradation of SA by overexpression of NahG enhanced cold tolerance in a transgenic plant.
[008] SA, as a phytohormone, also promotes early flowering (Martinez et al. (2004)). SA at various levels may play different roles in plant growth and stress responses. However, most of the time, the increased tolerance to high levels of SA appears to be beneficial, since it reduces the side effects of SA accumulation while stimulating SA-mediated stress responses. [009] Similarly, NO is capable of generating ROS/ROI/AOS and is a plant signaling molecule involved in the regulation of seed germination, stomatal closure (Mata and Lamattina (2001); Desikan et al (2002)), flowering time (He et al. (2004)), antioxidant reactions to suppress cell death (Beligni et al. (2002)) and tolerance to biotic and abiotic stress conditions (Mata and Lamattina (2001)). While the effects of NO can be mimicked through the application of sodium nitroprusside (SNP), endogenous NO production in plants results from the activity of a nitric oxide synthase that uses L-arginine (Guo et al. (2003)) as well as nitrate reductase-mediated reactions (Desikan et al (2002)). NO can react with redox centers in proteins and membranes, thereby causing cell damage and inducing cell death. [010] In order to control the two-fold nature of ROS/ROI/AOS molecules, plants have developed a sophisticated regulatory system which involves both production and scavenging of ROS/ROI/AOS in cells. During normal growth and development, this pathway monitors the level of ROS/ROI/AOS produced by metabolism and controls the expression and activity of ROS/ROI/AOS scavenging pathways. The major ROS/ROI/AOS scavenging mechanisms include the action of the superoxide dismutase (SOD), ascorbate perioxidase (APX) and catalase (CAT) enzymes as well as nonenzymatic components such as ascorbic acid, α- tocopherol and glutathione.
[011] The antioxidant enzymes are believed to be critical components in preventing oxidative stress, in part because pretreatment of plants with one form of stress, and which SUMMARY
[016] This document provides methods and materials related to plants having modulated levels of tolerance to oxidative stress. For example, this document provides transgenic plants and plant cells having increased levels of tolerance to oxidative stress, nucleic acids used to generate transgenic plants and plant cells having increased levels of tolerance to oxidative stress, and methods for making plants and plant cells having increased levels of tolerance to oxidative stress. Such plants and plant cells provide the opportunity to produce crops or plants under oxidative stress conditions without stunted growth and diminished yields. Increased levels of tolerance to oxidative stress may be useful to produce biomass which may be converted to a liquid fuel or other chemicals and/or to produce food and feed on land that is currently marginally productive, resulting in an overall expansion of arable land. [017] Methods of producing a plant and/or plant tissue are provided herein. In one aspect, a method comprises growing a plant cell comprising an exogenous nucleic acid. The exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide. The Hidden Markov Model (HMM) bit score of the amino acid sequence of the polypeptide is greater than about 30 using an HMM generated from the amino acid sequences depicted in one of Figures 3, 5 and 8. The plant and/or plant tissue has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise the exogenous nucleic acid. In some embodiments the amino acid sequence of the polypeptide has an HMM bit score greater than about 45 using an HMM generated from the amino acid sequences depicted in Figure 3. In some embodiments the amino acid sequence of the polypeptide has an HMM bit score greater than about 120 using an HMM generated from the amino acid sequences depicted in Figure 5. In some embodiments the amino acid sequence of the polypeptide has an HMM bit score greater than about 115 using an HMM generated from the amino acid sequences depicted in Figure 8.
[018] In another aspect, a method comprises growing a plant cell comprising an exogenous nucleic acid. The exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85 percent or greater sequence identity to an amino acid sequence set forth in SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 11 1, 112, 1 14, 116, 117, 118, 119, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241 , 242, 244, 245, 246, 247, 248, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291 , 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357. A plant produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
[019] In another aspect, a method comprises growing a plant cell comprising an exogenous nucleic acid. The exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to at least a fragment of a nucleotide sequence set forth in SEQ ID NOs. 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 113, 1 15, 121, 123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346, 348, 350 and 355 and to a nucleotide sequence encoding any of the amino acid sequences set forth in the sequence listing. A plant and/or plant tissue produced from the plant cell has a difference in the level of oxidative stress tolerance as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
[020] Methods of modulating the level of oxidative stress tolerance in a plant are provided herein. In one aspect, a method comprises introducing into a plant cell an exogenous nucleic acid, that comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide. The HMM bit score of the amino acid sequence of the polypeptide is greater than 30, using an HMM generated from the amino acid sequences depicted in one of Figures 3, 5 and 8. A plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
[021] In another aspect, a method comprises introducing into a plant cell an exogenous nucleic acid that comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85% percent or greater sequence identity to an amino acid sequence set forth in SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 11 1, 112, 1 14, 1 16, 1 17, 1 18, 1 19, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 311, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357. A plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
[022] In some embodiments, the methods comprise introducing into the plant cell an exogenous nucleic acid encoding polypeptides selected from the group consisting of SEQ ID NOs: 79, 94, 102 and 107. A plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
[023] In another aspect, a method comprises introducing into a plant cell an exogenous nucleic acid, that comprises a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to a nucleotide sequence set forth in SEQ ID NOs: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 1 13, 1 15, 121, 123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346, 348, 350 and 355 and to a nucleotide sequence encoding any of the amino acid sequences set forth in the sequence listing. A plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid.
[024] Plant cells comprising an exogenous nucleic acid are provided herein. In one aspect, the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide. The HMM bit score of the amino acid sequence of the polypeptide is greater than 30, using an HMM based on the amino acid sequences depicted in one of Figures 3, 5 and 8. The plant and/or plant tissue has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid. In another aspect, the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 11 1, 1 12, 114, 116, 1 17, 1 18, 119, 120,
122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301,
302, 304, 306, 308, 309, 311, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357. A plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid. In another aspect, the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to at least a fragment of a nucleotide sequence selected from the group consisting of SEQ ID Nos. 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 113, 1 15, 121,
123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298,
303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346, 348, 350 and 355, and to a nucleotide sequence encoding any of the amino acid sequences set forth in the sequence listing. A plant and/or plant tissue produced from the plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise the exogenous nucleic acid. A transgenic plant comprising such a plant cell is also provided. In some embodiments, the transgenic plant is a member of a species selected from the group consisting of Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearl millet). Some embodiments are related to products comprising seed or vegetative tissue from transgenic plants as described above. Some embodiments relate to food or feed products from transgenic plants as described above. [025] In another aspect, an isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide having 80% or greater sequence identity to the amino acid sequence set forth in SEQ ID Nos. 79, 94, 102 or 107.
[026] In another aspect, methods of identifying a genetic polymorphism associated with variation in the level of oxidative stress tolerance are provided. The methods include providing a population of plants, and determining whether one or more genetic polymorphisms in the population are genetically linked to the locus for a polypeptide selected from the group consisting of the polypeptides depicted in Figures 3, 5, 8, or SEQ ID NO: 107 and functional homologs thereof. The correlation between variation in the level of oxidative stress tolerance in plants and/or plant tissues of the population and the presence of the one or more polymorphisms in plants of the population is measured, thereby permitting identification of whether or not the one or more polymorphisms are associated with such variation.
[027] In another aspect, methods of making a plant line are provided. The methods include determining whether one or more genetic polymorphisms in a population of plants is associated with the locus for a polypeptide selected from the group consisting of the polypeptides depicted in Figures 3, 5, 8, or SEQ ID NO: 107 and functional homologs thereof, identifying one or more plants in the population in which the presence of at least one allele at the one or more polymorphisms is associated with variation in oxidative stress tolerance, crossing each of the one or more identified plants with itself or a different plant to produce seed, crossing at least one progeny plant grown from said seed with itself or a different plant, and repeating the crossing steps for an additional 0-5 generations to make the plant line. The at least one allele will be present in the plant line. The method of making a plant line may be applied, for example, to a population of switchgrass plants. [028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[029] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[030] Figure 1. Growth of six independent transgenic events of ME02077; T2 generation transgenic and non-transgenic plants grown under salicylic acid stress conditions. [031] Figure 2. Growth of two selected transgenic events of ME02077; T2 and T3 generation transgenic and non-transgenic plants grown under salicylic acid stress conditions. [032] Figure 3. Amino acid sequence alignment of homologues of ME02077 (SEQ ID NO: 79). Conserved regions are enclosed in a box.
[033] Figure 4. Growth of two selected transgenic events of ME06123; transgenic and non- transgenic plants in two consecutive generations grown under salicylic acid stress conditions. [034] Figure 5. Amino acid sequence alignment of homologues of ME06123 (SEQ ID NO: 94). Conserved regions are enclosed in a box.
[035] Figure 6. Growth of three selected transgenic events of ME00922; T2 and T3 generation transgenic and non-transgenic plants grown under L-arginine stress conditions. [036] Figure 7. Growth of two selected transgenic events of ME00922; T3 generation transgenic and non-transgenic plants grown under L-arginine and SNP stress conditions. [037] Figure 8. Amino acid sequence alignment of homologues of ME00922 (SEQ ID NO: 102). Conserved regions are enclosed in a box.
[038] Figure 9. Growth of three transgenic events of ME12485; T2 and T3 generation transgenic and non-transgenic plants grown under salicylic acid stress conditions.
DETAILED DESCRIPTION
[039] The invention features methods and materials related to modulating oxidative stress tolerance levels in plants and/or plant tissues. In some embodiments, the plants may also have increased biomass and/or yield. The methods can include transforming a plant cell with a nucleic acid encoding an oxidative stress tolerance-modulating polypeptide, wherein expression of the polypeptide results in a modulated level of oxidative stress tolerance. Plant cells produced using such methods can be grown to produce plants having an increased oxidative stress tolerance, and/or biomass, in comparison to wild type plants grown under the same conditions. Such plants, and the seeds of such plants, may be used to produce, for example, yield and/or biomass utilized for biofuel production, such as, but not limited to, ethanol and butanol.
I. Definitions
[040] "Amino acid" refers to one of the twenty biologically occurring amino acids and to synthetic amino acids, including D/L optical isomers.
[041] "Cell type-preferential promoter" or "tissue-preferential promoter" refers to a promoter that drives expression preferentially in a target cell type or tissue, respectively, but may also lead to some transcription in other cell types or tissues as well. [042] "Control plant" refers to a plant that does not contain the exogenous nucleic acid present in a transgenic plant of interest, but otherwise has the same or similar genetic background as such a transgenic plant. A suitable control plant can be a non-transgenic wild type plant, a non-transgenic segregant from a transformation experiment, or a transgenic plant that contains an exogenous nucleic acid other than the exogenous nucleic acid of interest. [043] "Domains" are groups of substantially contiguous amino acids in a polypeptide that can be used to characterize protein families and/or parts of proteins. Such domains have a "fingerprint" or "signature" that can comprise conserved primary sequence, secondary structure, and/or three-dimensional conformation. Generally, domains are correlated with specific in vitro and/or in vivo activities. A domain can have a length of from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400 amino acids.
[044] "Down-regulation" refers to regulation that decreases production of expression products (mRNA, polypeptide, or both) relative to basal or native states. [045] "Exogenous" with respect to a nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism. An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. It will be appreciated that an exogenous nucleic acid may have been introduced into a progenitor and not into the cell under consideration. For example, a transgenic plant containing an exogenous nucleic acid can be the progeny of a cross between a stably transformed plant and a non-transgenic plant. Such progeny are considered to contain the exogenous nucleic acid.
[046] "Expression" refers to the process of converting genetic information of a polynucleotide into RNA through transcription, which is catalyzed by an enzyme, RNA polymerase, and into protein, through translation of mRNA on ribosomes. [047] "Heterologous polypeptide" as used herein refers to a polypeptide that is not a naturally occurring polypeptide in a plant cell, e.g., a transgenic Panicum virgatum plant transformed with and expressing the coding sequence for a nitrogen transporter polypeptide from a Zea mays plant.
[048] "Isolated nucleic acid" as used herein includes a naturally-occurring nucleic acid, provided one or both of the sequences immediately flanking that nucleic acid in its naturally- occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a nucleic acid that exists as a purified molecule or a nucleic acid molecule that is incorporated into a vector or a virus. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries, genomic libraries, or gel slices containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid. [049] "Modulation" of the level of a compound or constituent refers to the change in the level of the indicated compound or constituent that is observed as a result of expression of, or transcription from, an exogenous nucleic acid in a plant cell. The change in level is measured relative to the corresponding level in control plants.
[050] "Nucleic acid" and "polynucleotide" are used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA or RNA containing nucleic acid analogs. Polynucleotides can have any three-dimensional structure. A nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes and nucleic acid primers. A polynucleotide may contain unconventional or modified nucleotides. [051] "Operably linked" refers to the positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a regulatory region, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the regulatory region. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
[052] "Polypeptide" as used herein refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics, regardless of post-translational modification, e.g., phosphorylation or glycosylation. The subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds. Full-length polypeptides, truncated polypeptides, point mutants, insertion mutants, splice variants, chimeric proteins, and fragments thereof are encompassed by this definition.
[053] "Progeny" includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F], F2, F3, F4, F5, F6 and subsequent generation plants, or seeds formed on BCi, BC2, BC3, and subsequent generation plants, or seeds formed on FiBCi, FiBC2, F1BC3, and subsequent generation plants. The designation Fi refers to the progeny of a cross between two parents that are genetically distinct. The designations F2, F3, F4, F5 and F6 refer to subsequent generations of self- or sib-pollinated progeny of an Fi plant. [054] "Regulatory region" refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). For example, a suitable enhancer is a cis-regulatory element (-212 to -154) from the upstream region of the octopine synthase (ocs) gene. Fromm et al., The Plant Cell, 1 :977-984 (1989). [055] "Up-regulation" refers to regulation that increases the level of an expression product (mRNA, polypeptide, or both) relative to basal or native states. [056] "Vector" refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. The term "vector" includes cloning and expression vectors, as well as viral vectors and integrating vectors. An "expression vector" is a vector that includes a regulatory region. [057] Oxidative stress: Plant species vary in their capacity to tolerate ROS/ROI/AOS. "Oxidative stress" can be defined as the set of environmental conditions under which a plant will begin to suffer the effects of elevated ROS/ROI/AOS concentration, such as decreases in enzymatic activity, DNA breakage, DNA-protein crosslinking, necrosis and stunted growth. For these reasons, plants experiencing oxidative stress typically exhibit a significant reduction in biomass and/or yield.
[058] Elevated oxidative stress may be caused by natural, geological processes and by human activities, such as pollution. Since plant species vary in their capacity to tolerate oxidative stress, the precise environmental conditions that cause stress cannot be generalized. However, under oxidative stress conditions, oxidative stress tolerant plants produce higher biomass, yield and survivorship than plants that are not oxidative stress tolerant. Differences in physical appearance, recovery and yield can be quantified
[059] Photosynthetic efficiency: photosynthetic efficiency, or electron transport via photosystem II, is estimated by the relationship between Fm, the maximum fluorescence signal and the variable fluorescence, Fv. A reduction in the optimum quantum yield (Fv/Fm) indicates stress and can be used to monitor the performance of transgenic plants compared to non-transgenic plants under oxidative stress conditions. [060] Salicylic Acid Growth Index (SAGI): Photosynthetic efficiency x seedling area.
II. Polypeptides
[061] Polypeptides described herein include oxidative stress tolerance-modulating polypeptides. Oxidative stress tolerance-modulating polypeptides can be effective to modulate oxidative stress tolerance levels when expressed in a plant or plant cell. Such polypeptides typically contain at least one domain indicative of oxidative stress tolerance- modulating polypeptides, as described in more detail herein. Oxidative stress tolerance- modulating polypeptides typically have an HMM bit score that is greater than 30, as described in more detail herein. In some embodiments, oxidative stress tolerance-modulating polypeptides have greater than 85 % identity to SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 111, 112, 114, 116, 117, 118, 1 19, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142,
143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165,
167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192,
194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357as described in more detail herein.
A. Domains Indicative of Oxidative Stress Tolerance-Modulating Polypeptides [062] An oxidative stress tolerance-modulating polypeptide can contain an AP2 domain, which is predicted to be characteristic of an oxidative stress tolerance-modulating polypeptide. These polypeptides typically bind to the GCC-box pathogenesis-related promoter element and activates the plant's defense genes. Ethylene, chemically the simplest plant hormone, participates in a number of stress responses and developmental processes: e.g., fruit ripening, inhibition of stem and root elongation, promotion of seed germination and flowering, senescence of leaves and flowers, and sex determination. DNA sequence elements that confer ethylene responsiveness have been shown to contain two 1 lbp GCC boxes, which are necessary and sufficient for transcriptional control by ethylene. Ethylene responsive element binding proteins (EREBPs) have now been identified in a variety of plants. The proteins share a similar domain of around 59 amino acids, which interacts directly with the GCC box in the ERE (see e.g. PUBMED:7732375). For example, SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143,
144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167,
168, 169, 170, 171, 173, 175, 176, 178, 1 8O5 181 , 182, 1 83, 184, 186, 188, 190, 192, 194,
195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, and 248 exemplify polypeptide sequences having AP2 domains.
[063] An oxidative stress tolerance-modulating polypeptide can contain a transmembrane amino acid transporter protein domain, which is predicted to be characteristic of an oxidative stress tolerance-modulating polypeptide. For example, SEQ ID NOs: 94, 96, 97, 98, 99, 100, 249, 251, 252, 253, 255, 255, 256, 258, 260, 261, 263, 263, 264, 266, 266, 267, 269, 270, 271, 272, 273, 274, 275, , 277, 279, , 281, 282, 284, 286, 286, 288, 289, 290, 291, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, and 353 exemplify polypeptide sequences having transmembrane amino acid transporter protein domains.
[064] An oxidative stress tolerance-modulating polypeptide can contain a Rubisco LSMT substrate-binding domain, which is predicted to be characteristic of an oxidative stress tolerance-modulating polypeptide. Members of this family adopt a multihelical structure, with an irregular array of long and short alpha-helices. They allow binding of the protein to substrate, such as the N-terminal tails of histones H3 and H4 and the large subunit of the Rubisco holoenzyme complex. For example, SEQ ID NOs: 102, 104, 105, 109, 1 10, 1 11, 1 12, 1 14, 116, 1 17, 118, 119, 120, 122, and 124 exemplify polypeptide sequences having Rubisco LSMT substrate-binding domains.
[065] An oxidative stress tolerance-modulating polypeptide can contain a SET domain, which is predicted to be characteristic of an oxidative stress tolerance-modulating polypeptide. SET domains are protein lysine methyltransferase enzymes. SET domains appear to be protein-protein interaction domains. SET domains sometimes mediate interactions with a family of proteins that display similarity with dual-specificity phosphatases. The SET domain consists of two regions known as SET-N and SET-C. SET-C forms an unusual and conserved knot-like structure of probably functional importance. Additionally to SET-N and SET-C, an insert region (SET-I) and flanking regions of high structural variability form part of the overall structure. For example, SEQ ID NOs: 102, 104, 105, 109, 1 10, 111, 1 12, 114, 1 16, 1 17, 1 18, 1 19, 120, 122, 124, 126, and 127 exemplify polypeptide sequences having SET domains.
[066] In some embodiments, an oxidative stress tolerance-modulating polypeptide is truncated at the amino- or carboxy-terminal end of a naturally occurring polypeptide. A truncated polypeptide may retain certain domains of the naturally occurring polypeptide while lacking others. Thus, length variants that are up to 5 amino acids shorter or longer typically exhibit the salinity tolerance and/or oxidative stress tolerance-modulating activity of a truncated polypeptide. Expression in a plant of such a truncated polypeptide confers a difference in the level of oxidative stress tolerance in a plant and/or plant tissue as compared to the corresponding level a control plant and/or tissue thereof that does not comprise the truncation.
B. Functional Homologs Identified by Reciprocal BLAST [062] In some embodiments, one or more functional homologs of a reference oxidative stress tolerance-modulating polypeptide defined by one or more of the pfam descriptions indicated above are suitable for use as oxidative stress tolerance-modulating polypeptides. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide may be natural occurring polypeptides, and the sequence similarity may be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, may themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for an oxidative stress tolerance-modulating polypeptide, or by combining domains from the coding sequences for different naturally-occurring oxidative stress tolerance-modulating polypeptides ("domain swapping"). The term "functional homolog" is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
[063] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of oxidative stress tolerance-modulating polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundant databases using an oxidative stress tolerance-modulating polypeptide amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as an oxidative stress tolerance-modulating polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in oxidative stress tolerance-modulating polypeptides, e.g., conserved functional domains.
[064] Conserved regions can be identified by locating a region within the primary amino acid sequence of an oxidative stress tolerance-modulating polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. A description of the information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al., Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate.
[065] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity. [066] Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 79 are provided in Figure 3 and in the Sequence Listing. Such functional homologs include SEQ ID NO: 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247 and 248. In some cases, a functional homolog of SEQ ID NO: 79 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 79.
[067] Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO- 94 are provided in Figure 5. Such functional homologs include SEQ ID NO: 96, 97, 98, 99, 100, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 311, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352 and 353. In some cases, a functional homolog of SEQ ID NO: 94 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 94. [068] Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 102 are provided in Figure 8. Such functional homologs include SEQ ID NO: 104, 105, 109, 110, 11 1, 112, 1 14, 116, 117, 118, 119, 120, 122, 124, 126 and 127. In some cases, a functional homolog of SEQ ID NO: 102 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 102.
[069] Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 107 are provided in the Sequence Listing. Such functional homologs include SEQ ID NO: 354, 356 and 357). In some cases, a functional homolog of SEQ ID NO: 107 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 107.
[070] The identification of conserved regions in an oxidative stress tolerance-modulating polypeptide facilitates production of variants of oxidative stress tolerance-modulating polypeptides. Variants of oxidative stress tolerance-modulating polypeptides typically have 10 or fewer conservative amino acid substitutions within the primary amino acid sequence, e.g., 7 or fewer conservative amino acid substitutions, 5 or fewer conservative amino acid substitutions, or between 1 and 5 conservative substitutions. A useful variant polypeptide can be constructed based on one of the alignments set forth in Figures 3, 5 and 8. Such a polypeptide includes the conserved regions, arranged in the order depicted in the Figure from amino-terminal end to carboxy-terminal end. Such a polypeptide may also include zero, one, or more than one amino acid in positions marked by dashes. When no amino acids are present at positions marked by dashes, the length of such a polypeptide is the sum of the amino acid residues in all conserved regions. When amino acids are present at all positions marked by dashes, such a polypeptide has a length that is the sum of the amino acid residues in all conserved regions and all dashes.
C. Functional Homologies Identified by HMM
[071] In some embodiments, useful oxidative stress tolerance-modulating polypeptides include those that fit a Hidden Markov Model based on the polypeptides set forth in any one of Figures 3, 5 and 8. A Hidden Markov Model (HMM) is a statistical model of a consensus sequence for a group of functional homologs. See, Durbin et al., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids± Cambridge University Press, [068] Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 102 are provided in Figure 8. Such functional homologs include SEQ ID NO: 104, 105, 109, 110, 11 1, 1 12, 1 14, 116, 117, 118, 119, 120, 122, 124, 126 and 127. In some cases, a functional homolog of SEQ ID NO: 102 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 102.
[069] Amino acid sequences of functional homologs of the polypeptide set forth in SEQ ID NO: 107 are provided in the Sequence Listing. Such functional homologs include SEQ ID NO: 354, 356 and 357). In some cases, a functional homolog of SEQ ID NO: 107 has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 107.
[070] The identification of conserved regions in an oxidative stress tolerance-modulating polypeptide facilitates production of variants of oxidative stress tolerance-modulating polypeptides. Variants of oxidative stress tolerance-modulating polypeptides typically have 10 or fewer conservative amino acid substitutions within the primary amino acid sequence, e.g., 7 or fewer conservative amino acid substitutions, 5 or fewer conservative amino acid substitutions, or between 1 and 5 conservative substitutions. A useful variant polypeptide can be constructed based on one of the alignments set forth in Figures 3, 5 and 8. Such a polypeptide includes the conserved regions, arranged in the order depicted in the Figure from amino-terminal end to carboxy-terminal end. Such a polypeptide may also include zero, one, or more than one amino acid in positions marked by dashes. When no amino acids are present at positions marked by dashes, the length of such a polypeptide is the sum of the amino acid residues in all conserved regions. When amino acids are present at all positions marked by dashes, such a polypeptide has a length that is the sum of the amino acid residues in all conserved regions and all dashes.
C. Functional Homologues Identified by HMM
[071] In some embodiments, useful oxidative stress tolerance-modulating polypeptides include those that fit a Hidden Markov Model based on the polypeptides set forth in any one of Figures 3, 5 and 8. A Hidden Markov Model (HMM) is a statistical model of a consensus sequence for a group of functional homologs. See, Durbin et al., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids^ Cambridge University Press, Cambridge, UK (1998). An HMM is generated by the program HMMER 2.3.2 with default program parameters, using the sequences of the group of functional homologs as input. The multiple sequence alignment is generated by ProbCons (Do et al., Genome Res., 15(2):330- 40 (2005)) version 1.11 using a set of default parameters: -c, —consistency REPS of 2; -ir, — iterative-refinement REPS of 100; -pre, ~pre-training REPS of 0. ProbCons is a public domain software program provided by Stanford University.
[072] The default parameters for building an HMM (hmmbuild) are as follows: the default "architecture prior" (archpri) used by MAP architecture construction is 0.85, and the default cutoff threshold (idlevel) used to determine the effective sequence number is 0.62. HMMER 2.3.2 was released October 3, 2003 under a GNU general public license, and is available from various sources on the World Wide Web. Hmmbuild outputs the model as a text file. [073] The HMM for a group of functional homologs can be used to determine the likelihood that a candidate oxidative stress tolerance-modulating polypeptide sequence is a better fit to that particular HMM than to a null HMM generated using a group of sequences that are not structurally or functionally related. The likelihood that a subject polypeptide sequence is a better fit to an HMM than to a null HMM is indicated by the HMM bit score, a number generated when the candidate sequence is fitted to the HMM profile using the HMMER hmmsearch program. The following default parameters are used when running hmmsearch: the default E-value cutoff (E) is 10.0, the default bit score cutoff (T) is negative infinity, the default number of sequences in a database (Z) is the real number of sequences in the database, the default E-value cutoff for the per-domain ranked hit list (domE) is infinity, and the default bit score cutoff for the per-domain ranked hit list (domT) is negative infinity. A high HMM bit score indicates a greater likelihood that the subject sequence carries out one or more of the biochemical or physiological function(s) of the polypeptides used to generate the HMM. A high HMM bit score is at least 20, and often is higher. Slight variations in the HMM bit score of a particular sequence can occur due to factors such as the order in which sequences are processed for alignment by multiple sequence alignment algorithms such as the ProbCons program. Nevertheless, such HMM bit score variation is minor. [074] As those of skill in the art would appreciate, the HMM scores provided in the sequence listing are merely exemplary. Since multiple sequence alignment algorithms, such as ProbCons, can only generate near-optimal results, slight variations of the model can arise due to factors such as the order in which sequences are processed for alignment. Nevertheless, HMM score variability is minor, and so the HMM scores in the sequence listing are representative of models made with the respective sequences. [075] The oxidative stress-modulating polypeptides discussed below fit the indicated HMM with an HMM bit score greater than 20 (e.g., greater than 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500). In some embodiments, the HMM bit score of a salinity and/or oxidative stress-modulating polypeptide discussed below is about 50%, 60%, 70%, 80%, 90%, or 95% of the HMM bit score of a functional homolog provided in the Sequence Listing. In some embodiments, an oxidative stress-modulating polypeptide discussed below fits the indicated HMM with an HMM bit score greater than 20, and has a domain indicative of an oxidative stress-modulating polypeptide. In some embodiments, an oxidative stress- modulating polypeptide discussed below fits the indicated HMM with an HMM bit score greater than 20, and has 85% or greater sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or 100% sequence identity) to an amino acid sequence shown in any one of Figures 3, 5 and 8 or to an amino acid sequence correlated in the Sequence Listing to a any one of Figures 3, 5 and 8.
[076] In the Sequence Listing polypeptides are provided that have HMM bit scores greater than 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 550 600, 650, 700 or 725, when fitted to an HMM generated from the amino acid sequences set forth in Figure 3. Such polypeptides include Ceres SEEDLINE ID no.ME02077, Public GI ID no. 89257562, Ceres CLONE ID no.1725082, Public GI ID no. 92878368, Ceres CLONE ID no.1661 141, Public GI ID no. 92878365, Ceres CLONE ID no. 1894778, Public GI ID no. 50927523, Public GI ID no. 8809575, Public GI ID no. 1208497, Public GI ID no. 60459383, Ceres SEEDLINE ID no. ME02077, Ceres CLONE ID no. 1935323, Public GI ID no. 12003376, Public GI ID no. 8843855, Ceres CLONE ID no. 1850283, Public GI ID no. 34013890, Ceres CLONE ID no. 1428135, Ceres CLONE ID no. 1605871, Public GI ID no. 38260669, Public GI ID no. 38260631, Public GI ID no. 38260685, Public GI ID no. 14140143, Public GI ID no. 115447673, Public GI ID no. 125540543, Public GI ID no. 38260649, Public GI ID no. 38260618, Public GI ID no. 38196019, Ceres CLONE ID no. 540272, Public GI ID no.7576196, Public GI ID no.79508406, Public GI ID no.32490143, Ceres CLONE ID no. 1335966, Ceres CLONE ID no. 998737, Public CLONE ID no. 10093608, Ceres ANNOT ID no. 1537977, Public GI ID no. 115459750, Public GI ID no. 125549239, Public GI ID no. 125549238, Ceres CLONE ID no. 927860, Ceres CLONE ID no. 68144, Public GI ID no. 21264420, Public GI ID no. 115493452, Public GI ID no. 7528276, Public GI ID no. 37730469, Ceres CLONE ID no. 907605, Ceres CLONE ID no. 582684, Public GI ID no. 28274834, Ceres CLONE ID no. 1926437, Ceres ANNOT ID no. 14477726, Public GI ID no. 115459748, Public GI ID no. 1 16310255, Public GI ID no. 125549236, Public GI ID no. 125591182, Ceres CLONE ID no. 1848864, Ceres CLONE ID no. 1807978, Ceres CLONE ID no. 1933466, Ceres CLONE ID no. 1834552, Ceres CLONE ID no. 1614474, Public GI ID no. 40060531, Ceres ANNOT ID no. 1438401, Ceres CLONE ID no. 1847074, Ceres CLONE ID no. 1855621, Ceres ANNOT ID no. 1484557, Ceres CLONE ID no. 1846882, Ceres CLONE ID no. 1854513, Public GI ID no. 18423250, Public GI ID no. 21592411, Ceres CLONE ID no. 2347, Ceres CLONE ID no. 1080500, Ceres CLONE ID no. 1849534, Ceres CLONE ID no. 965028, Ceres CLONE ID no. 464504, Public GI ID no. 12231294, Public GI ID no. 15240297, Public GI ID no. 13430474, Public GI ID no. 21617964, Ceres ANNOT ID no. 1438399, Ceres ANNOT ID no. 1484559, Ceres CLONE ID no. 548557, Public GI ID no. 18422770, Public GI ID no. 8809606, Public GI ID no. 89257455, Public GI ID no. 89257468, Ceres ANNOT ID no. 1486129, Ceres ANNOT ID no. 1446832, Public GI ID no. 40060533, Public GI ID no. 76097531, Public GI ID no. 60459379, Public GI ID no.45642990, Public GI ID no. 3298498, Ceres CLONE ID no. 268310, Public GI ID no. 18414895, Public GI ID no. 17473792, Public GI ID no. 57012758 and Public GI ID no. 57012874 (SEQ ID NO: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247 and 248, respectively).
[077] In the Sequence Listing polypeptides are provided that have HMM bit scores greater than 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1200, 1250, 1300, 1350 or 1400 when fitted to an HMM generated from the amino acid sequences set forth in Figure 5. Such polypeptides include Ceres SEEDLINE ID no.ME06123, Ceres ANNOT ID no. 1450631, Ceres CLONE ID no. 1658212, Public GI ID no 50927941 , Ceres CLONE ID no. 383013, Ceres CLONE ID no. 788118, Public GI ID no.
92871428, Ceres ANNOT ID no. 1473784, Public GI ID no. 7239491, Public GI ID no.
92871429, Ceres ANNOT ID no.1516369, Public GI ID no. 125548592, Ceres ANNOT ID no. 1517322, Ceres ANNOT ID no. 1459650, Public GI ID no. 125593423, Ceres CLONE ID no. 1796001, Public GI ID no. 115474609, Ceres CLONE ID no. 684177, Public GI ID no. 30409136, Ceres CLONE ID no.195207, Public GI ID no. 125536241, Public GI ID no. 22330117, Public GI ID no. 12597815, Public GI ID no. 115488002, Public GI ID no. 125579355, Public GI ID no. 976402, Ceres ANNOT ID no. 1493584, Ceres CLONE ID no. 15711 17, Ceres CLONE ID no. 786667, Public GI ID no. 15219896, Ceres CLONE ID no.914964, Ceres ANNOT ID no. 1440705, Ceres CLONE ID no.264227, Public GI ID no. 30693666, Public GI ID no. 30693663, Public GI ID no. 2443875, Ceres CLONE ID no.1257761, Ceres CLONE ID no.l 109755, Public GI ID no. 15232176, Public GI ID no. 6091720, Ceres ANNOT ID no. 1512922, Public GI ID no.15220504, Public GI ID no. 116830999, Public GI ID no. 18395471, Ceres ANNOT ID no. 1463076, Ceres CLONE ID no. 1904659, Ceres ANNOT ID no. 1458889, Public GI ID no. 15222615, Ceres ANNOT ID no. 1527205, Public GI ID no. 53749301, Public GI ID no. 115458804, Public GI ID no. 125590641, Public GI ID no. 2576361, Public GI ID no. 21593132, Ceres CLONE ID no. 36461, Public GI ID no.4455344, Ceres ANNOT ID no. 1450630, Public GI ID no. 452593, Ceres ANNOT ID no. 1495347, Ceres ANNOT ID no. 1442446, Ceres CLONE ID no. 573806, Public GI ID no. 115459914, Public GI ID no. 116310155, Public GI ID no. 1 15488592, Public GI ID no. 15220283, Public GI ID no. 5688864, Ceres ANNOT ID no. 1539815, Public GI ID no. 125591283, Public GI ID no. 125549341, Ceres CLONE ID no. 833489, Ceres ANNOT ID no. 1450632, Ceres ANNOT ID no. 1460559, Ceres CLONE ID no. 1990904, Ceres ANNOT ID no. 1486399, Ceres CLONE ID no. 30044, Ceres CLONE ID no. 910080, Public GI ID no. 125536645 and Public PUBLICCLONE ID no. 10006534 (SEQ ID NO: 94, 96, 97, 98, 99, 100, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352 and 353, respectively).
[078] In the Sequence Listing polypeptides are provided that have HMM bit scores greater than 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1 150, 1200 or 1250 when fitted to an HMM generated from the amino acid sequences set forth in Figure 8. Such polypeptides include Ceres SEEDLINE ID no ME00922, Ceres ANNOT ID no. 1536088. Public GI ID no. 77554044, Ceres CLONE ID no. 479625, Public GI ID no. 22326803, Public GI ID no. 18377718, Public GI ID no. 7573451, Ceres CLONE ID no. 24159, Ceres ANNOT ID no. 1482610, Public GI ID no. 92872502, Public GI ID no.l 15487958, Public GI ID no.125536207, Public GI ID no.125578929, Ceres CLONE ID no. 26560_ME22365, Ceres CLONE ID no. 463156, Ceres CLONE ID no. 1998672 and Public PUBLICCLONE ID no. 10092646 (SEQ ID NO: 102, 104, 105, 109, 110, 111, 112, 114, 116, 117, 118, 119, 120, 122, 124, 126 and 127, respectively). D. Percent Identity
[079] In some embodiments, an oxidative stress tolerance-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one of the amino acid sequences set forth in SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94,
96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 1 1 1, 1 12, 114, 116, 1 17, 118, 1 19, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145,
146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169,
170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227,
229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249,
251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271 , 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 311, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328,
329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357. Polypeptides having such a percent sequence identity often have a domain indicative of an oxidative stress-modulating polypeptide and/or have an HMM bit score that is greater than 20, as discussed above. Examples of amino acid sequences of oxidative stress tolerance-modulating polypeptides having at least 85% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs: 79, 80, 83, 84, 89, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 109 and 122, are provided in Figures 3, 5 and 8.
[080] "Percent sequence identity" refers to the degree of sequence identity between any given reference sequence, e.g., SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96,
97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 111, 112, 114, 116, 117, 1 18, 119, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146,
147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170,
171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229,
230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249, 251,
252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 311, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329,
330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357, and a candidate oxidative stress-modulating sequence. A candidate sequence typically has a length that is from 80 percent to 200 percent of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200 percent of the length of the reference sequence. A percent identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A reference sequence (e.g., a nucleic acid sequence or an amino acid sequence) is aligned to one or more candidate sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., Nucleic Acids Res., 31(13):3497-500 (2003).
[081] ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: GIy, Pro, Ser, Asn, Asp, GIn, GIu, Arg, and Lys; residue-specific gap penalties: on. The ClustalW output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw). [082] To determine percent identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using ClustalW, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[083] In some cases, an oxidative stress tolerance-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one or more of the amino acid sequence set forth in SEQ ID NO: 79 Amino acid sequences of polypeptides having high sequence identity to the polypeptide set forth in SEQ ID NO: 79 are provided in the Sequence Listing. Such polypeptides include SEQ ID NO: 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247 and 248. [084] In some cases, an oxidative stress tolerance-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 94. Amino acid sequences of polypeptides having high sequence identity to the polypeptide set forth in SEQ ID NO: 94 are provided in the Sequence Listing. Such polypeptides include SEQ ID NO: SEQ ID NO: 96, 97, 98, 99, 100, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352 and 353. [085] In some cases, an oxidative stress-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 102. Amino acid sequences of polypeptides having high sequence identity to the polypeptide set forth in SEQ ID NO: 102 are provided in the Sequence Listing. Such polypeptides include SEQ ID NO: 102, 104, 105, 109, 1 10, 1 1 1, 1 12. 114. 116. 117, 118, 119, 120, 122, 124, 126 and 127.
[086] In some cases, an oxidative stress-modulating polypeptide has an amino acid sequence with at least 50% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 107. Amino acid sequences of polypeptides having high sequence identity to the polypeptide set forth in SEQ ID NO: 107 are provided in the Sequence Listing. Such polypeptides include SEQ ID NO: 354, 356 and 357.
E. Other Sequences [087] It should be appreciated that an oxidative stress tolerance-modulating polypeptide can include additional amino acids that are not involved in oxidative stress tolerance modulation, and thus such a polypeptide can be longer than would otherwise be the case. For example, an oxidative stress- tolerance modulating polypeptide can include a purification tag, a chloroplast transit peptide, an amyloplast transit peptide, a mitochondrial transit peptide, or a leader sequence added to the amino or carboxy terminus. In some embodiments, an oxidative stress- tolerance modulating polypeptide includes an amino acid sequence that functions as a reporter, e.g., a green fluorescent protein or yellow fluorescent protein.
III. Nucleic Acids
[088] Nucleic acids described herein include nucleic acids that are effective to modulate oxidative stress tolerance levels when transcribed in a plant or plant cell. Such nucleic acids include, without limitation, those that encode an oxidative stress tolerance-modulating polypeptide and those that can be used to inhibit expression of an oxidative stress tolerance- modulating polypeptide via a nucleic acid based method.
A. Nucleic acids encoding oxidative stress tolerance-modulating polypeptides [089] Nucleic acids encoding oxidative stress tolerance-modulating polypeptides are described herein. Such nucleic acids include SEQ ID NOs: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 113, 1 15, 121, 123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346, 348, 350 and 355, as described in more detail below.
[090] An oxidative stress tolerance-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 78. Alternatively, an oxidative stress tolerance- modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 78. For example, an oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 78, 81, 86, 127, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236 and 243. [091] An oxidative stress tolerance-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 93. Alternatively, an oxidative stress tolerance- modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 93. For example, an oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 93, 95, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346 and 348. [092] An oxidative stress tolerance-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 101. Alternatively, an oxidative stress tolerance- modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 101. For example, an oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 101, 103, 106, 108, 113, 115, 121, 123 and 125.
[093] An oxidative stress tolerance-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 355. Alternatively, an oxidative stress tolerance- modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 355. For example, an oxidative stress tolerance-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 355.
[094] Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids of the invention also can be obtained by mutagenesis of, e.g., a naturally occurring DNA.
B. Use of Nucleic Acids to Modulate Expression of Polypeptides i. Expression of an oxidative stress tolerance-Modulating Polypeptide [095] A nucleic acid encoding one of the oxidative stress tolerance-modulating polypeptides described herein can be used to express the polypeptide in a plant species of interest, typically by transforming a plant cell with a nucleic acid having the coding sequence for the polypeptide operably linked in sense orientation to one or more regulatory regions. It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular oxidative stress tolerance-modulating polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given oxidative stress tolerance-modulating polypeptide can be modified such that optimal expression in a particular plant species is obtained, using appropriate codon bias tables for that species.
[096] In some cases, expression of an oxidative stress tolerance-modulating polypeptide inhibits one or more functions of an endogenous polypeptide. For example, a nucleic acid that encodes a dominant negative polypeptide can be used to inhibit protein function. A dominant negative polypeptide typically is mutated or truncated relative to an endogenous wild type polypeptide, and its presence in a cell inhibits one or more functions of the wild type polypeptide in that cell, i.e., the dominant negative polypeptide is genetically dominant and confers a loss of function. The mechanism by which a dominant negative polypeptide confers such a phenotype can vary but often involves a protein-protein interaction or a protein-DNA interaction. For example, a dominant negative polypeptide can be an enzyme that is truncated relative to a native wild type enzyme, such that the truncated polypeptide retains domains involved in binding a first protein but lacks domains involved in binding a second protein. The truncated polypeptide is thus unable to properly modulate the activity of the second protein. See, e.g., US 2007/0056058. As another example, a point mutation that results in a non-conservative amino acid substitution in a catalytic domain can result in a dominant negative polypeptide. See, e.g., US 2005/032221. As another example, a dominant negative polypeptide can be a transcription factor that is truncated relative to a native wild type transcription factor, such that the truncated polypeptide retains the DNA binding domain(s) but lacks the activation domain(s). Such a truncated polypeptide can inhibit the wild type transcription factor from binding DNA, thereby inhibiting transcription activation. ii. Inhibition of Expression of an oxidative stress tolerance-Modulating Polypeptide [097] Polynucleotides and recombinant constructs described herein can be used to inhibit expression of an oxidative stress tolerance-modulating polypeptide in a plant species of interest. See, e.g., Matzke and Birchler, Nature Reviews Genetics 6:24-35 (2005); Akashi et al., Nature Reviews MoI. Cell Biology 6:413-422 (2005); Mittal, Nature Reviews Genetics 5:355-365 (2004); Dorsett and Tuschl, Nature Reviews Drug Discovery 3: 318-329 (2004); and Nature Reviews RNA interference collection. Oct. 2005 at nature.com/reviews/focus/mai. A number of nucleic acid based methods, including antisense RNA, ribozyme directed RNA cleavage, post-transcriptional gene silencing (PTGS), e.g., RNA interference (RNAi), and transcriptional gene silencing (TGS) are known to inhibit gene expression in plants. Antisense technology is one well-known method. In this method, a nucleic acid segment from a gene to be repressed is cloned and operably linked to a regulatory region and a transcription termination sequence so that the antisense strand of RNA is transcribed. The recombinant construct is then transformed into plants, as described herein, and the antisense strand of RNA is produced. The nucleic acid segment need not be the entire sequence of the gene to be repressed, but typically will be substantially complementary to at least a portion of the sense strand of the gene to be repressed. Generally, higher homology can be used to compensate for the use of a shorter sequence. Typically, a sequence of at least 30 nucleotides is used, e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more.
[098] In another method, a nucleic acid can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA. See, U.S. Patent No. 6,423,885. Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide. Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site- specific recognition sequences can be used. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contains a 5'-UG-3' nucleotide sequence. The construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Patent No. 5,254,678 and WO 02/46449 and references cited therein. Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo. Perriman et al, Proc. Natl. Acad. Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, "Expressing Ribozymes in Plants", Edited by Turner, P.C., Humana Press Inc., Totowa, NJ. RNA endoribonucleases which have been described, such as the one that occurs naturally in Tetrahymena thermophila, can be useful. See, for example, U.S. Patent No. 4,987,071 and 6,423,885.
[099] PTGS, e.g., RNAi, can also be used to inhibit the expression of a gene. For example, a construct can be prepared that includes a sequence that is transcribed into an RNA that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure. In some embodiments, one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of an oxidative stress tolerance- modulating polypeptide, and that is from about 10 nucleotides to about 2,500 nucleotides in length. The length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides. The other strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the antisense strand of the coding sequence of the oxidative stress tolerance- modulating polypeptide, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence. In some cases, one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the 3' or 5' untranslated region of an mRNA encoding an oxidative stress tolerance-modulating polypeptide, and the other strand of the stem portion of the double stranded RNA comprises a sequence that is similar or identical to the sequence that is complementary to the 3' or 5' untranslated region, respectively, of the mRNA encoding the oxidative stress tolerance- modulating polypeptide. In other embodiments, one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sequence of an intron in the pre-mRNA encoding an oxidative stress tolerance-modulating polypeptide, and the other strand of the stem portion comprises a sequence that is similar or identical to the sequence that is complementary to the sequence of the intron in the pre-mRNA. The loop portion of a double stranded RNA can be from 3 nucleotides to 5,000 nucleotides, e.g., from 3 nucleotides to 25 nucleotides, from 15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides. The loop portion of the RNA can include an intron. A double stranded RNA can have zero, one, two, three, four, five, six, seven, eight, nine, ten, or more stem-loop structures. A construct including a sequence that is operably linked to a regulatory region and a transcription termination sequence, and that is transcribed into an RNA that can form a double stranded RNA, is transformed into plants as described herein. Methods for using RNAi to inhibit the expression of a gene are known to those of skill in the art. See, e.g., U.S. Patents 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. See also WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S. Patent Publications 20030175965, 20030175783, 20040214330, and 20030180945.
[0100] Constructs containing regulatory regions operably linked to nucleic acid molecules in sense orientation can also be used to inhibit the expression of a gene. The transcription product can be similar or identical to the sense coding sequence of an oxidative stress tolerance-modulating polypeptide. The transcription product can also be unpolyadenylated, lack a 5' cap structure, or contain an unsplicable intron. Methods of inhibiting gene expression using a full-length cDNA as well as a partial cDNA sequence are known in the art. See, e.g., U.S. Patent No. 5,231,020.
[0101] In some embodiments, a construct containing a nucleic acid having at least one strand that is a template for both sense and antisense sequences that are complementary to each other is used to inhibit the expression of a gene. The sense and antisense sequences can be part of a larger nucleic acid molecule or can be part of separate nucleic acid molecules having sequences that are not complementary. The sense or antisense sequence can be a sequence that is identical or complementary to the sequence of an mRNA, the 3' or 5' untranslated region of an mRNA, or an intron in a pre-mRNA encoding an oxidative stress tolerance- modulating polypeptide. In some embodiments, the sense or antisense sequence is identical or complementary to a sequence of the regulatory region that drives transcription of the gene encoding an oxidative stress tolerance-modulating polypeptide. In each case, the sense sequence is the sequence that is complementary to the antisense sequence. [0102] The sense and antisense sequences can be any length greater than about 12 nucleotides (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides). For example, an antisense sequence can be 21 or 22 nucleotides in length. Typically, the sense and antisense sequences range in length from about 15 nucleotides to about 30 nucleotides, e.g., from about 18 nucleotides to about 28 nucleotides, or from about 21 nucleotides to about 25 nucleotides.
[0103] In some embodiments, an antisense sequence is a sequence complementary to an mRNA sequence encoding an oxidative stress tolerance-modulating polypeptide described herein. The sense sequence complementary to the antisense sequence can be a sequence present within the mRNA of the oxidative stress tolerance-modulating polypeptide. Typically, sense and antisense sequences are designed to correspond to a 15-30 nucleotide sequence of a target mRNA such that the level of that target mRNA is reduced. [0104] In some embodiments, a construct containing a nucleic acid having at least one strand that is a template for more than one sense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sense sequences) can be used to inhibit the expression of a gene. Likewise, a construct containing a nucleic acid having at least one strand that is a template for more than one antisense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antisense sequences) can be used to inhibit the expression of a gene. For example, a construct can contain a nucleic acid having at least one strand that is a template for two sense sequences and two antisense sequences. The multiple sense sequences can be identical or different, and the multiple antisense sequences can be identical or different. For example, a construct can have a nucleic acid having one strand that is a template for two identical sense sequences and two identical antisense sequences that are complementary to the two identical sense sequences. Alternatively, an isolated nucleic acid can have one strand that is a template for (1) two identical sense sequences 20 nucleotides in length, (2) one antisense sequence that is complementary to the two identical sense sequences 20 nucleotides in length, (3) a sense sequence 30 nucleotides in length, and (4) three identical antisense sequences that are complementary to the sense sequence 30 nucleotides in length. The constructs provided herein can be designed to have any arrangement of sense and antisense sequences. For example, two identical sense sequences can be followed by two identical antisense sequences or can be positioned between two identical antisense sequences.
[0105] A nucleic acid having at least one strand that is a template for one or more sense and/or antisense sequences can be operably linked to a regulatory region to drive transcription of an RNA molecule containing the sense and/or antisense sequence(s). In addition, such a nucleic acid can be operably linked to a transcription terminator sequence, such as the terminator of the nopaline synthase (nos) gene. In some cases, two regulatory regions can direct transcription of two transcripts: one from the top strand, and one from the bottom strand. See, for example, Yan et al, Plant Physiol, 141 : 1508-1518 (2006). The two regulatory regions can be the same or different. The two transcripts can form double- stranded RNA molecules that induce degradation of the target RNA. In some cases, a nucleic acid can be positioned within a T-DNA or plant-derived transfer DNA (P-DNA) such that the left and right T-DNA border sequences, or the left and right border-like sequences of the P- DNA, flank or are on either side of the nucleic acid. See, US 2006/0265788. The nucleic acid sequence between the two regulatory regions can be from about 15 to about 300 nucleotides in length. In some embodiments, the nucleic acid sequence between the two regulatory regions is from about 15 to about 200 nucleotides in length, from about 15 to about 100 nucleotides in length, from about 15 to about 50 nucleotides in length, from about 18 to about 50 nucleotides in length, from about 18 to about 40 nucleotides in length, from about 18 to about 30 nucleotides in length, or from about 18 to about 25 nucleotides in length.
[0106] In some nucleic-acid based methods for inhibition of gene expression in plants, a suitable nucleic acid can be a nucleic acid analog. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'- deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-OaIIyI sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et al, Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
C. Constructs/Vectors
[0107] Recombinant constructs provided herein can be used to transform plants or plant cells in order to modulate oxidative stress tolerance levels. A recombinant nucleic acid construct can comprise a nucleic acid encoding an oxidative stress tolerance-modulating polypeptide as described herein, operably linked to a regulatory region suitable for expressing the oxidative stress tolerance-modulating polypeptide in the plant or cell. Thus, a nucleic acid can comprise a coding sequence that encodes any of the oxidative stress tolerance-modulating polypeptides as set forth in SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 1 10, 111, 112, 114, 1 16, 1 17, 1 18, 1 19, 120, 122,
124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302,
304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357. Examples of nucleic acids encoding oxidative stress tolerance-modulating polypeptides are set forth in SEQ ID NOs: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 1 13, 115, 121, 123,
125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303,
305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346, 348, 350 and 355. The oxidative stress tolerance-modulating polypeptide encoded by a recombinant nucleic acid can be a native oxidative stress tolerance-modulating polypeptide, or can be heterologous to the cell. In some cases, the recombinant construct contains a nucleic acid that inhibits expression of an oxidative stress tolerance-modulating polypeptide, operably linked to a regulatory region. Examples of suitable regulatory regions are described in the section entitled "Regulatory Regions."
[0108] Vectors containing recombinant nucleic acid constructs such as those described herein also are provided. Suitable vector backbones include, for example, those routinely used in the art such as plasm ids. viruses, artificial chromosomes, BACs, YACs, or PACs. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).
[0109] The vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers. A marker gene can confer a selectable phenotype on a plant cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or an herbicide (e.g., glyphosate, chlorsulfuron or phosphinothricin). In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as luciferase, β-glucuronidase (GUS), green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. [0110]
D. Regulatory regions
[0111] The choice of regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. Transcription of a nucleic acid can be modulated in a similar manner.
[0112] Some suitable promoters initiate transcription only, or predominantly, in certain cell types. The choice of regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. Transcription of a nucleic acid can be modulated in a similar manner.
[0113] Some suitable regulatory regions initiate transcription only, or predominantly, in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known, including, for example, those described in the following references: Jordano et al, Plant Cell, 1 :855-866 (1989); Bustos et al, Plant Cell, 1 :839-854 (1989); Green et al, EMBO J., 7:4035-4044 (1988); Meier et al., Plant Cell, 3:309-316 (1991); and Zhang et al., Plant Physiology, 110:1069-1079 (1996).
[0114] Examples of various classes of regulatory regions are described below. Some of the regulatory regions indicated below as well as additional regulatory regions are described in more detail in U.S. Patent Application Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140; 60/757,544; 60/776,307; 10/957,569; 11/058,689; 11/172,703; 11/208,308; 11/274,890; 60/583,609; 60/612,891 ; 11/097,589; 11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017; PCT/US05/011105; PCT/US05/23639; PCT/US05/034308; PCT/US05/034343; and PCT/US06/038236; PCT/US06/040572; and PCT/US07/62762.
[0115] For example, the sequences of regulatory regions p326, YP0144, YP0190, pl3879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633, YP0128, YP0275, PT0660, PT0683, PT0758, PT0613, PT0672, PT0688, PT0837, YP0092, PT0676, PT0708, YP0396, YP0007, YPOl I l, YPO 103, YP0028, YPOl 21, YP0008, YP0039, YPOl 15, YPO 119, YP0120, YP0374, YPOlOl, YP0102, YPOI lO, YPOl 17, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143, YPOl 56, PT0650, PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886, PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286, YP0377, PD 1367, PT0863, PT0829, PT0665, PT0678, YP0086, YPO 188, YP0263, PT0743 and YP0096 are set forth in the sequence listing of PCT/US06/040572; the sequence of regulatory region PT0625 is set forth in the sequence listing of PCT/US05/034343; the sequences of regulatory regions PT0623, YP0388, YP0087, YP0093, YP0108, YP0022 and YP0080 are set forth in the sequence listing of U.S. Patent Application Ser. No. 11/172,703; the sequence of regulatory region PR0924 is set forth in the sequence listing of PCT/US07/62762; and the sequences of regulatory regions p530cl0, pOsFIE2-2, pOsMEA, pOsYpl02, and pOsYp285 are set forth in the sequence listing of PCT/US06/038236. [0116] It will be appreciated that a regulatory region may meet criteria for one classification based on its activity in one plant species, and yet meet criteria for a different classification based on its activity in another plant species. i. Broadly Expressing Promoters
[0117] A promoter can be said to be "broadly expressing" when it promotes transcription in many, but not necessarily all, plant tissues. For example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems. As another example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds. Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326, YPO 144, YPO 190, pl3879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, and PT0633 promoters. Additional examples include the cauliflower mosaic virus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the 1' or 2' promoters derived from T-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34S promoter, actin promoters such as the rice actin promoter, and ubiquitin promoters such as the maize ubiquitin-1 promoter. In some cases, the CaMV 35S promoter is excluded from the category of broadly expressing promoters. ii. Root Promoters
[0118] Root-active promoters confer transcription in root tissue, e.g., root endodermis, root epidermis, or root vascular tissues. In some embodiments, root-active promoters are root- preferential promoters, i.e., confer transcription only or predominantly in root tissue. Root- preferential promoters include the YP0128, YP0275, PT0625, PT0660, PT0683, and PT0758 promoters. Other root-preferential promoters include the PT0613, PT0672, PT0688, and PT0837 promoters, which drive transcription primarily in root tissue and to a lesser extent in ovules and/or seeds. Other examples of root-preferential promoters include the root-specific subdomains of the CaMV 35S promoter (Lam et al, Proc. Natl. Acad. Sci. USA, 86:7890- 7894 (1989)), root cell specific promoters reported by Conkling et al., Plant Physiol., 93: 1203-121 1 (1990), and the tobacco RD2 promoter. iii. Maturing Endosperm Promoters
[0119] In some embodiments, promoters that drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Most suitable are promoters that are active predominantly in maturing endosperm, although promoters that are also active in other tissues can sometimes be used. Non-limiting examples of maturing endosperm promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin promoter (Bustos et al., Plant Cell, 1(9):839- 853 (1989)), the soybean trypsin inhibitor promoter (Riggs et al, Plant Cell, l(6):609-621 (1989)), the ACP promoter (Baerson et al, Plant MoI Biol, 22(2):255-267 (1993)), the stearoyl-ACP desaturase promoter (Slocombe et al, Plant Physiol, 104(4):167-176 (1994)), the soybean α' subunit of β-conglycinin promoter (Chen et al, Proc. Natl. Acad. Sci. USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al, Plant MoI. Biol, 34(3):549-555 (1997)), and zein promoters, such as the 15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zein promoter. Also suitable are the Osgt- 1 promoter from the rice glutelin-1 gene (Zheng et al, MoI Cell Biol, 13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordein promoter. Other maturing endosperm promoters include the YP0092, PT0676, and PT0708 promoters. iv. Ovary Tissue Promoters
[0120] Promoters that are active in ovary tissues such as the ovule wall and mesocarp can also be useful, e.g., a polygalacturonidase promoter, the banana TRX promoter, the melon actin promoter, YP0396, and PT0623. Examples of promoters that are active primarily in ovules include YP0007, YPOl I l, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YPO 115, YPOl 19, YPO 120, and YP0374. v. Embryo Sac/Early Endosperm Promoters
[0121] To achieve expression in embryo sac/early endosperm, regulatory regions can be used that are active in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. Most suitable are promoters that drive expression only or predominantly in polar nuclei or precursors thereto and/or the central cell. A pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac/early endosperm-preferential promoters, although transcription typically decreases significantly in later endosperm development during and after the cellularization phase. Expression in the zygote or developing embryo typically is not present with embryo sac/early endosperm promoters.
[0122] Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsis atmycl (see, Urao (1996) Plant MoI. Biol., 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Patent 6,906,244). Other promoters that may be suitable include those derived from the following genes: maize MACl (see, Sheridan (1996) Genetics, 142: 1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) Plant MoI. Biol, 22: 10131-1038). Other promoters include the following Arabidopsis promoters: YP0039, YPOlOl, YPO 102, YPOI lO, YPOl 17, YPOl 19, YP0137, DME, YP0285, and YP0212. Other promoters that may be useful include the following rice promoters: p530cl0, pOsFIE2-2, pOsMEA, pOsYpl02, and pOsYp285. vi. Embryo Promoters
[0123] Regulatory regions that preferentially drive transcription in zygotic cells following fertilization can provide embryo-preferential expression. Most suitable are promoters that preferentially drive transcription in early stage embryos prior to the heart stage, but expression in late stage and maturing embryos is also suitable. Embryo-preferential promoters include the barley lipid transfer protein (Ltpl) promoter {Plant Cell Rep (2001) 20:647-654), YP0097, YP0107, YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, and PT0740. vii. Photosynthetic Tissue Promoters
[0124] Promoters active in photosynthetic tissue confer transcription in green tissues such as leaves and stems. Most suitable are promoters that drive expression only or predominantly in such tissues. Examples of such promoters include the ribulose-l,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Lctrix laricina), the pine cab6 promoter (Yamamoto et al, Plant Cell Physiol., 35:773-778 (1994)), the Cab-1 promoter from wheat (Fejes et al, Plant MoL Biol, 15:921-932 (1990)), the CAB-I promoter from spinach (Lubberstedt et al, Plant Physiol, 104:997-1006 (1994)), the cablR promoter from rice (Luan et al, Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al, Proc. Natl Acad. Sci. USA, 90:9586-9590 (1993)), the tobacco Lhcbl *2 promoter (Cerdan et al, Plant MoI Biol, 33:245-255 (1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al, Planta, 196:564-570 (1995)), and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissue promoters include PT0535, PT0668, PT0886, YP0144, YP0380 and PT0585. viii. Vascular Tissue Promoters
[0125] Examples of promoters that have high or preferential activity in vascular bundles include YP0087, YP0093, YPO 108, YP0022, and YP0080. Other vascular tissue-preferential promoters include the glycine-rich cell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell, 3(10): 1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV) promoter (Medberry et al, Plant Cell, 4(2): 185-192 (1992)), and the rice tungro bacilliform virus (RTBV) promoter (Dai et al, Proc. Natl Acad. Sci. USA, 101(2):687-692 (2004)). ix. Inducible Promoters
[0126] Inducible promoters confer transcription in response to external stimuli such as chemical agents or environmental stimuli. For example, inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought. Examples of drought-inducible promoters include YP0380, PT0848, YP0381, YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384, PT0688, YP0286, YP0377, PD1367, and PD0901. Examples of nitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886. Examples of shade-inducible promoters include PR0924 and PT0678. An example of a promoter induced by salt is rd29A (Kasuga et al. (1999) Nature Biotech 17: 287-291). x. Basal Promoters
[0127] A basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation. Basal promoters frequently include a "TATA box" element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation. Basal promoters also may include a "CCAAT box" element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site. xi. Other Promoters
[0128] Other classes of promoters include, but are not limited to, shoot-preferential, callus- preferential, trichome cell-preferential, guard cell-preferential such as PT0678, tuber- preferential, parenchyma cell-preferential, and senescence-preferential promoters. Promoters designated YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YPOl 19, and YP0096, as described in the above-referenced patent applications, may also be useful. xii. Other Regulatory Regions
[0129] A 5' untranslated region (UTR) can be included in nucleic acid constructs described herein. A 5' UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide. A 3' UTR can be positioned between the translation termination codon and the end of the transcript. UTRs can have particular functions such as increasing mRNA stability or attenuating translation. Examples of 3' UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence. [0130] It will be understood that more than one regulatory region may be present in a recombinant polynucleotide, e g , introns, enhancers, upstream activation regions, transcription terminators, and inducible elements. Thus, for example, more than one regulatory region can be operably linked to the sequence of a polynucleotide encoding an oxidative stress tolerance modulating polypeptide.
[0131] Regulatory regions, such as promoters for endogenous genes, can be obtained by chemical synthesis or by subcloning from a genomic DNA that includes such a regulatory region. A nucleic acid comprising such a regulatory region can also include flanking sequences that contain restriction enzyme sites that facilitate subsequent manipulation. [0132] Alternatively, misexpression can be accomplished using a two component system, whereby the first component consists of a transgenic plant comprising a transcriptional activator operatively linked to a promoter and the second component consists of a transgenic plant that comprise a nucleic acid molecule of the invention operatively linked to the target- binding sequence/region of the transcriptional activator. The two transgenic plants are crossed and the nucleic acid molecule of the invention is expressed in the progeny of the plant. In another alternative embodiment of the present invention, the misexpression can be accomplished by having the sequences of the two component system transformed in one transgenic plant line.
IV. Transgenic Plants and Plant Cells
A. Transformation
[0133] The invention also features transgenic plant cells and plants comprising at least one recombinant nucleic acid construct described herein. A plant or plant cell can be transformed by having a construct integrated into its genome, i.e., can be stably transformed. Stably transformed cells typically retain the introduced nucleic acid with each cell division. A plant or plant cell can also be transiently transformed such that the construct is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced nucleic acid construct with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.
[0134] Transgenic plant cells used in methods described herein can constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e g , to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. As used herein, a transgenic plant also refers to progeny of an initial transgenic plant having the transgene. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct. [0135] Transgenic plants can be grown in suspension culture, or tissue or organ culture. For the purposes of this invention, solid and/or liquid tissue culture techniques can be used. When using solid medium, transgenic plant cells can be placed directly onto the medium or can be placed onto a filter that is then placed in contact with the medium. When using liquid medium, transgenic plant cells can be placed onto a flotation device, e.g., a porous membrane that contacts the liquid medium. A solid medium can be, for example, Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, e.g., 2,4- dichlorophenoxyacetic acid (2,4-D), and a suitable concentration of a cytokinin, e.g., kinetin. [0136] When transiently transformed plant cells are used, a reporter sequence encoding a reporter polypeptide having a reporter activity can be included in the transformation procedure and an assay for reporter activity or expression can be performed at a suitable time after transformation. A suitable time for conducting the assay typically is about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days. The use of transient assays is particularly convenient for rapid analysis in different species, or to confirm expression of a heterologous oxidative stress tolerance-modulating polypeptide whose expression has not previously been confirmed in particular recipient cells.
[0137] Techniques for introducing nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrobacterium-mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Patents 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
B. Screening/selection
[0138] A population of transgenic plants can be screened and/or selected for those members of the population that have a trait or phenotype conferred by expression of the transgene. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of an oxidative stress tolerance-modulating polypeptide or nucleic acid. Physical and biochemical methods can be used to identify expression levels. These include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, Sl RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known. As an alternative, a population of plants comprising independent transformation events can be screened for those plants having a desired trait, such as a modulated level of oxidative stress tolerance. Selection and/or screening can be carried out over one or more generations, and/or in more than one geographic location. In some cases, transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant. In addition, selection and/or screening can be applied during a particular developmental stage in which the phenotype is expected to be exhibited by the plant. Selection and/or screening can be carried out to choose those transgenic plants having a statistically significant difference in an oxidative stress tolerance level relative to a control plant that lacks the transgene. Selected or screened transgenic plants have an altered phenotype as compared to a corresponding control plant, as described in the "Transgenic Plant Phenotypes" section herein. [0139] A population of transgenic plants can be screened and/or selected for those members of the population that have a trait or phenotype conferred by expression of the transgene. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of an tolerance-modulating polypeptide and/or nucleic acid. Physical and biochemical methods can be used to identify expression levels. These include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, Sl RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known. As an alternative, a population of plants comprising independent transformation events can be screened for those plants having a desired trait, such as a modulated level of oxidative stress tolerance. Selection and/or screening can be carried out over one or more generations, and/or in more than one geographic location. In some cases, transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant. In addition, selection and/or screening can be applied during a particular developmental stage in which the phenotype is expected to be exhibited by the plant. Selection and/or screening can be carried out to choose those transgenic plants having a statistically significant difference in an oxidative stress tolerance level relative to a control plant that lacks the transgene. Selected or screened transgenic plants have an altered phenotype as compared to a corresponding control plant, as described in the "Transgenic Plant Phenotypes" section herein.
C, Plant Species
[0140] The polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae, Theaceae, or Vitaceae.
[0141] Suitable species may include members of the genus Abelmoschus, Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolβa, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea. [0142] Suitable species include Panicum snn.. Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale (triticum - wheat X rye) and bamboo. [0143] Suitable species also include Helianthus annuus (sunflower), Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax), and Brassica juncea. [0144] Suitable species also include Beta vulgaris (sugarbeet), and Manihot esculenta (cassava).
[0145] Suitable species also include Lycopersicon esculentum (tomato), Lactuca sativa (lettuce), Musa paradisiaca (banana), Solarium tuberosum (potato), Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), and Solanum melongena (eggplant).
[0146] Suitable species also include Papaver somniferum (opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis sativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Colchicum autumnale, Veratrum californica., Digitalis lanata, Digitalis purpurea, Dioscorea spp,, Andrographis paniculata, Atropa belladonna, Datura stomonium, Berber is spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (= Huperzia serrata), Lycopodium spp., Rauwolβa serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, and Tanacetum parthenium.
[0147] Suitable species also include Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, and Alstroemeria spp. [0148] Suitable species also include Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia) and Poinsettia pulcherrima (poinsettia).
[0149] Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple, Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (ryegrass) and Phleum pratense (timothy).
[0150] Thus, the methods and compositions can be used over a broad range of plant species, including species from the dicot genera Brassica, Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium, Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum, Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale, Sorghum, Triticosecale, Triticum, and Zea. In some embodiments, a plant is a member of the species Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearl millet).
[0151] In certain embodiments, the polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, wherein such plants are hybrids of different species or varieties of a species (e.g., Saccharum sp. X Miscanthus sp.)
D. Transgenic Plant Phenotypes
[0152] In some embodiments, a plant in which expression of an oxidative stress modulating polypeptide is modulated can have increased levels of tolerance to oxidative stress. For example, an oxidative stress-modulating polypeptide described herein can be expressed in a transgenic plant, resulting in increased levels of tolerance to oxidative stress. The oxidative stress tolerance levels can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, as compared to those levels in a corresponding control plant that does not express the transgene. [0153] The nucleic acid molecules and polypeptides of the present invention are of interest because when the nucleic acid molecules are mis-expressed (i.e., when expressed at a non- natural location or in an increased or decreased amount relative to wild-type) they produce plants that exhibit improved oxidation tolerance as compared to wild-type plants, as evidenced in part by the results of various experiments disclosed below. In particular, plants transformed with the nucleic acid molecules and polypeptides of the present invention can have any of a number of modified characteristics as compared to wild-type plants. Examples of modified characteristics include photosynthetic efficiency, seedling area, and biomass as it may be measured by plant height, leaf or rosette area, or dry mass. The modified characteristics may be observed and measured at different plant developmental stages, e.g. seed, seedling, bolting, senescense, etc. Often, oxidative stress tolerance can be expressed as ratios or combinations of measurements, such as salicylic acid growth index values. For example, plants transformed with the sequences of the present invention can exhibit increases in SGI, seedling area and/or SAGI values of at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, or even at least 500%. These traits can be used to exploit or maximize plant products. For example, the nucleic acid molecules and polypeptides of the present invention are used to increase the expression of genes that cause the plant to have improved biomass, growth rate and/or seedling vigor in oxidative conditions, in comparison to wild type plants under the same conditions.
[0154] Because the disclosed sequences and methods increase vegetative growth and growth rate in oxidative conditions, the disclosed methods can be used to enhance plant growth in plants grown in oxidative conditions. For example, plants of the present invention show, under oxidative conditions, increased photosynthetic efficiency and increased seedling area as compared to a plant of the same species that is not genetically modified for substantial vegetative growth. Examples of increases in biomass production include increases of at least 5%, at least 20%, or even at least 50%, when compared to an amount of biomass production by a wild-type plant of the same species under identical conditions.
[0155] Typically, a difference in the amount of tolerance to oxidative stress in a transgenic plant or cell relative to a control plant or cell is considered statistically significant at p < 0.05 with an appropriate parametric or non-parametric statistic, e.g., Chi-square test, Student's t- test, Mann-Whitney test, or F-test. In some embodiments, a difference in the amount of tolerance to oxidative stress is statistically significant at p < 0.01, p < 0.005, or p < 0.001. [0156] The phenotype of a transgenic plant is evaluated relative to a control plant. A plant is said "not to express" a polypeptide when the plant exhibits less than 10%, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount of polypeptide or mRNA encoding the polypeptide exhibited by the plant of interest. Expression can be evaluated using methods including, for example, RT-PCR, Northern blots, Sl RNase protection, primer extensions, Western blots, protein gel electrophoresis, immunoprecipitation, enzyme-linked immunoassays, chip assays, and mass spectrometry. It should be noted that if a polypeptide is expressed under the control of a tissue-preferential or broadly expressing promoter, expression can be evaluated in the entire plant or in a selected tissue. Similarly, if a polypeptide is expressed at a particular time, e.g., at a particular time in development or upon induction, expression can be evaluated selectively at a desired time period.
V. Plant Breeding
[0157] Genetic polymorphisms are discrete allelic sequence differences in a population. Typically, an allele that is present at 1% or greater is considered to be a genetic polymorphism. The discovery that polypeptides disclosed herein can modulate oxidative stress tolerance content is useful in plant breeding, because genetic polymorphisms exhibiting a degree of linkage with loci for such polypeptides are more likely to be correlated with variation in an oxidative stress tolerance trait. For example, genetic polymorphisms linked to the loci for such polypeptides are more likely to be useful in marker-assisted breeding programs to create lines having a desired modulation in the oxidative stress tolerance traits. [0158] Thus, one aspect of the invention includes methods of identifying whether one or more genetic polymorphisms are associated with variation in an oxidative stress tolerance trait. Such methods involve determining whether genetic polymorphisms in a given population exhibit linkage with the locus for one of the polypeptides depicted in Figures 1 thru 6 and/or a functional homolog thereof, such as, but not limited to, those in the Sequence Listing. The correlation is measured between variation in the oxidative stress tolerance traits in plants of the population and the presence of the genetic polymorphism(s) in plants of the population, thereby identifying whether or not the genetic polymorphism(s) are associated with variation for the traits. If the presence of a particular allele is statistically significantly correlated with a desired modulation in the oxidative stress tolerance traits, the allele is associated with variation for one or both of the traits and is useful as a marker for one or more of the traits. If, on the other hand, the presence of a particular allele is not significantly correlated with the desired modulation, the allele is not associated with variation for one or more of the traits and is not useful as a marker.
[0159] Such methods are applicable to populations containing the naturally occurring endogenous polypeptide rather than an exogenous nucleic acid encoding the polypeptide, i.e., populations that are not transgenic for the exogenous nucleic acid. It will be appreciated, however, that populations suitable for use in the methods may contain a transgene for another, different trait, e.g., herbicide resistance.
[0160] Genetic polymorphisms that are useful in such methods include simple sequence repeats (SSRs, or microsatellites), rapid amplification of polymorphic DNA (RAPDs), single nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and restriction fragment length polymorphisms (RFLPs). SSR polymorphisms can be identified, for example, by making sequence specific probes and amplifying template DNA from individuals in the population of interest by PCR. If the probes flank an SSR in the population, PCR products of different sizes will be produced. See, e.g., U.S. Patent 5,766,847. Alternatively, SSR polymorphisms can be identified by using PCR product(s) as a probe against Southern blots from different individuals in the population. See, U.H. Refseth et al., (1997) Electrophoresis 18: 1519. The identification of RFLPs is discussed, for example, in Alonso-Blanco et al. (Methods in Molecular Biology, vol.82, "Arabidopsis Protocols", pp. 137-146, J.M. Martinez-Zapater and J. Salinas, eds., c. 1998 by Humana Press, Totowa, NJ); Burr ("Mapping Genes with Recombinant Inbreds", pp. 249-254, in Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c. 1994 by Springer- Verlag New York, Inc.: New York, NY, USA; Berlin Germany; Burr et al. Genetics (1998) 118: 519; and Gardiner, J. et al., (1993) Genetics 134: 917). The identification of AFLPs is discussed, for example, in EP 0 534 858 and US Pat. 5,878,215.
[0161] In some embodiments, the methods are directed to breeding a plant line. Such methods use genetic polymorphisms identified as described above in a marker assisted breeding program to facilitate the development of lines that have a desired alteration in the oxidative stress tolerance trait(s). Once a suitable genetic polymorphism is identified as being associated with variation for the trait, one or more individual plants are identified that possess the polymorphic allele correlated with the desired variation. Those plants are then used in a breeding program to combine the polymorphic allele with a plurality of other alleles at other loci that are correlated with the desired variation. Techniques suitable for use in a plant breeding program are known in the art and include, without limitation, backcrossing, mass selection, pedigree breeding, bulk selection, crossing to another population and recurrent selection. These techniques can be used alone or in combination with one or more other techniques in a breeding program. Thus, each identified plants is selfed or crossed a different plant to produce seed which is then germinated to form progeny plants. At least one such progeny plant is then selfed or crossed with a different plant to form a subsequent progeny generation. The breeding program can repeat the steps of selfing or outcrossing for an additional 0 to 5 generations as appropriate in order to achieve the desired uniformity and stability in the resulting plant line, which retains the polymorphic allele. In most breeding programs, analysis for the particular polymorphic allele will be carried out in each generation, although analysis can be carried out in alternate generations if desired. [0162] In some cases, selection for other useful traits is also carried out, e.g., selection for fungal resistance or bacterial resistance. Selection for such other traits can be carried out before, during or after identification of individual plants that possess the desired polymorphic allele.
VI. Articles of Manufacture
[0163] Transgenic plants provided herein have various uses in the agricultural and energy production industries. For example, transgenic plants described herein can be used to make animal feed and food products. Such plants, however, are often particularly useful as a feedstock for energy production.
[0164] Transgenic plants described herein often produce higher yields of grain and/or biomass per hectare, relative to control plants that lack the exogenous nucleic acid. In some embodiments, such transgenic plants provide equivalent or even increased yields of grain and/or biomass per hectare relative to control plants when grown under conditions of reduced inputs such as fertilizer and/or water. Thus, such transgenic plants can be used to provide yield stability at a lower input cost and/or under environmentally stressful conditions such as drought. In some embodiments, plants described herein have a composition that permits more efficient processing into free sugars, and subsequently ethanol, for energy production. In some embodiments, such plants provide higher yields of ethanol, butanol, other biofuel molecules, and/or sugar-derived co-products per kilogram of plant material, relative to control plants. By providing higher yields at an equivalent or even decreased cost of production relative to controls, the transgenic plants described herein improve profitability for farmers and processors as well as decrease costs to consumers.
[0165] Seeds from transgenic plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture. Packaging material such as paper and cloth are well known in the art. A package of seed can have a label, e.g., a tag or label secured to the packaging material, a label printed on the packaging material, or a label inserted within the package, that describes the nature of the seeds therein.
[0166] Enhanced oxidative stress tolerance gives the opportunity to grow crops in oxidative stress conditions without stunted growth and diminished yields due to ion imbalance, disruption of water homeostasis, inhibition of metabolism, damage to membranes, and/or cell death. The ability to grow plants in oxidative stress conditions would result in an overall expansion of arable land and increased output of land currently marginally productive due to elevated oxidative stress conditions. [0167] Seed or seedling vigor is an important characteristic that can greatly influence successful growth of a plant, such as crop plants. Adverse environmental conditions, such as oxidative conditions, can affect a plant growth cycle, germination of seeds and seedling vigor (i.e. vitality and strength under such conditions can differentiate between successful and failed plant growth). Seedling vigor has often been defined to comprise the seed properties that determine "the potential for rapid, uniform emergence and development of normal seedlings under a wide range of field conditions". Hence, it would be advantageous to develop plant seeds with increased vigor, particularly in oxidative stress conditions. [0168] For example, increased seedling vigor would be advantageous for cereal plants such as rice, maize, wheat, etc. production. For these crops, germination and growth can often be slowed or stopped by oxidation. Genes associated with increased seed vigor under oxidative stress conditions have therefore been sought for producing improved plant varieties. (Walia et al. (2005) Plant Physiology 139:822-835).
[0169] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
VII. Examples
General Protocols
Agrobacterium-Mediated Transformation of Arabidopsis
[0170] Host Plants and Transgenes: Wild-type Arabidopsis thaliana Wassilewskija (WS) plants are transformed with Ti plasmids containing nucleic acid sequences to be expressed, as noted in the respective examples, in the sense orientation relative to the 35S promoter in a Ti plasmid. A Ti plasmid vector useful for these constructs, CRS 338, contains the Ceres- constructed, plant selectable marker gene phosphinothricin acetyltransferase (PAT), which confers herbicide resistance to transformed plants.
[0171] Ten independently transformed events are typically selected and evaluated for their qualitative phenotype in the T1 generation.
[0172] Preparation of Soil Mixture: 24L Sunshine Mix #5 soil (Sun Gro Horticulture, Ltd., Bellevue, WA) is mixed with 16L Therm-O-Rock vermiculite (Therm-O-Rock West, Inc., Chandler, AZ) in a cement mixer to make a 60:40 soil mixture. To the soil mixture is added 2 Tbsp Marathon 1% granules (Hummert, Earth City, MO), 3 Tbsp OSMOCOTE® 14-14-14 (Hummert, Earth City, MO) and 1 Tbsp Peters fertilizer 20-20-20 (J.R. Peters, Inc., Allentown, PA), which are first added to 3 gallons of water and then added to the soil and mixed thoroughly. Generally, 4-inch diameter pots are filled with soil mixture. Pots are then covered with 8-inch squares of nylon netting.
[0173] Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated. 25 drops are added to each pot. Clear propagation domes are placed on top of the pots that are then placed under 55% shade cloth and subirrigated by adding 1 inch of water. [0174] Plant Maintenance: 3 to 4 days after planting, lids and shade cloth are removed. Plants are watered as needed. After 7-10 days, pots are thinned to 20 plants per pot using forceps. After 2 weeks, all plants are subirrigated with Peters fertilizer at a rate of 1 Tsp per gallon of water. When bolts are about 5-10 cm long, they are clipped between the first node and the base of stem to induce secondary bolts. Dipping infiltration is performed 6 to 7 days after clipping.
[0175] Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL each of carbenicillin, spectinomycin and rifampicin (each at 100 mg/mL stock concentration). Agrobacterium starter blocks are obtained (96-well block with Agrobacterium cultures grown to an ODδoo of approximately 1.0) and inoculated one culture vessel per construct by transferring 1 mL from appropriate well in the starter block. Cultures are then incubated with shaking at 270C. Cultures are spun down after attaining an ODβoo of approximately 1.0 (about 24 hours). 200 mL infiltration media is added to resuspend Agrobacterium pellets. Infiltration media is prepared by adding 2.2 g MS salts, 50 g sucrose, and 5 μL 2 mg/mL benzylaminopurine to 900 mL water.
[0176] Dipping Infiltration: The pots are inverted and submerged for 5 minutes so that the aerial portion of the plant is in the Agrobacterium suspension. Plants are allowed to grow normally and seed is collected.
[0177] High-throughput Phenotypic Screening of Misexpression Mutants: Seed is evenly dispersed into water-saturated soil in pots and placed into a dark 40C cooler for two nights to promote uniform germination. Pots are then removed from the cooler and covered with 55% shade cloth for 4-5 days. Cotyledons are fully expanded at this stage. FINALE® (Sanofi Aventis, Paris, France) is sprayed on plants (3 mL FINALE® diluted into 48 oz. water) and repeated every 3-4 days until only transformants remain.
[0178] Salicylic Acid Screening: Screening is routinely performed by agar plate assay using 100 μM or 150 μM exogenous sodium salicylate. Media contains 1/2X MS (Sigma), 150 μL 1 M sodium salicylate (Sigma), 0.5 g MES hydrate (Sigma) and 0.7% phytagar (EM Science), adjusted to pH 5.7 using ION KOH.
[0179] To screen superpools, seeds are surface sterilized in 30% bleach solution for 5 minutes and then rinsed repeatedly with sterile water. Approximately 2500 seeds are sown on media plates in a monolayer at a density of 850 seeds per plate, including wild-type and positive controls. Plates are wrapped with vent tape and placed at 4°C in the dark for three days to stratify. At the end of this time, plates are transferred to a Conviron growth chamber set at 22 C, 16:8 hour light:dark cycle, 70% humidity with a combination of incandescent and fluorescent lamps emitting a light intensity of -100 μEinsteins. [0180] Seedlings are screened daily starting at 6 days. Seedlings that grow larger and stay greener compared to WS control plants are selected as positive candidates and transferred to soil for recovery and seed set.
[0181] Candidate plants are re-screened by placing 36 seeds from each candidate together with a WS control on the same sodium salicylate plate. Plates are treated as described above and seedling screening begun after 4 days as described. Leaf tissue is harvested from confirmed tolerant candidates for DNA extraction and amplification of the transgene by PCR. [0182] Alternatively, superpool seeds are sown directly on soil and sprayed with 10 mM SA. Leaf tissue is harvested from tolerant candidate plants to isolate DNA for PCR amplification of the transgene and subsequent sequencing of the PCR product. [0183] Traits assessed under sodium salicylate conditions include: seedling area, photosynthesis efficiency, salicylic acid growth index (SAG) and regeneration ability.
o Seedling area: the total leaf area of a young plant about 2 weeks old. o Photosynthesis efficiency (Fv/Fm): Seedling photosynthetic efficiency, or electron transport via photosystem II, is estimated by the relationship between Fm, the maximum fluorescence signal and the variable fluorescence, Fv. Here, a reduction in the optimum quantum yield (Fv/Fm) indicates stress, and so can be used to monitor the performance of transgenic plants compared to non-transgenic plants under oxidative stress conditions, o Salicylic Acid Growth (SAG) Index = seedling area (cm2) x photosynthesis efficiency (Fv/Fm).
PCR was used to amplify the cDNA insert in one randomly chosen T2 plant. This PCR product was then sequenced to confirm the sequence in the plants.
[0184] Assessing Tolerance to Oxidative Stress: Initially, between four and ten independently transformed plant lines are selected and qualitatively evaluated for their tolerance to SA in the T2 generation. Two or three of the transformed lines that qualitatively show the strongest tolerance to oxidative stress in the T2 generation are selected for further evaluation in the T2 and T3 generations. This evaluation involves sowing seeds from the selected transformed plant lines on MS agar plates containing 100 μM or 150 μM sodium salicylate and incubating the seeds for at least 4 days to allow for germination and growth. [0185] Calculating SAG: After germination and growth, seedling area and photosynthesis efficiency of transformed lines and a wild-type control are determined. From these measurements, the Salicylic Acid Growth Index (SAG) is calculated and compared between wild-type and transformed seedlings. The SAG calculation is made by averaging seedling area and photosynthesis efficiency measurements taken from two replicates of 36 seedlings for each transformed line and a wild-type control and performing a t-test.
[0186] Determining Transgene Copy Number: T2 generation transformed plants are tested on BASTA® plates in order to determine the transgene copy number of each transformed line.
A BASTA® resistant:BASTA® sensitive segregation ratio of 15: 1 generally indicates two copies of the transgene, and such a segregation ratio of 3: 1 generally indicates one copy of the transgene.
[0187] L-Arginine Screening: Screening is routinely performed by agar plate assay using
10 mM L-arginine, pH 9. Media contains 1/2X MS (Sigma), 10 mM L-arginine (Sigma) and
0.8% phytagar (EM Science), adjusted to pH 9 using ION KOH.
[0188] To screen superpools, seeds are surface sterilized in 30% bleach solution for 5 minutes and then rinsed repeatedly with sterile water. Approximately 2500 seeds are sown on media plates in a monolayer at a density of 850 seeds per plate, including wild-type and positive controls. Plates are wrapped with vent tape and placed at 4°C in the dark for three days to stratify. At the end of this time, plates are transferred to a Conviron growth chamber set at 22°C, 16:8 hour light:dark cycle, 70% humidity with a combination of incandescent and fluorescent lamps emitting a light intensity of -100 μEinsteins.
[0189] Seedlings are screened daily starting at 5 days. Seedlings that grow larger and stay greener compared to WS control plants are selected as positive candidates and transferred to soil for recovery and seed set.
[0190] Candidate plants are re-screened by placing 36 seeds from each candidate together with a WS control on the same L-arginine plate. Plates are treated as described above and seedling screening begun after 5 days as described. Leaf tissue is harvested from confirmed tolerant candidates for DNA extraction, amplification of the transgene by PCR and sequencing of the PCR product.
[0191] Traits assessed under L-arginine conditions include: seedling area, photosynthesis efficiency and regeneration ability. o Seedling area: the total leaf area of a young plant about 2 weeks old. o Photosynthesis efficiency (Fv/Fm): Seedling photosynthetic efficiency, or electron transport via photosystem II, is estimated by the relationship between Fm, the maximum fluorescence signal and the variable fluorescence, Fv. Here, a reduction in the optimum quantum yield (Fv/Fm) indicates stress, and so can be used to monitor the performance of transgenic plants compared to non-transgenic plants under oxidative stress conditions.
[0192] PCR is used to amplify the cDNA insert in one randomly chosen T2 plant. This PCR product is then sequenced to confirm the sequence in the plants.
[0193] Validation is performed as described above using 60 seeds of each event except that the media is supplemented with 0.5 g/1 MES-hydrate (M8250-Sigma) and the pH adjusted to 5.7. [0194] In some cases, validation is performed using media that is further supplemented with 100 uM SNP.
[0195] Determining Transgene Copy Number: T2 generation transformed plants are tested on BASTA® plates in order to determine the transgene copy number of each transformed line. RESULTS:
[0196] The following Examples provide information for polynucleotides and their encoded polypeptides useful for increasing tolerance to oxidative stress. Enhanced oxidative stress tolerance provides the opportunity to grow crops under oxidative stress conditions without stunted growth and diminished yields. The ability to grow crops under oxidative stress conditions would result in an overall expansion of arable land and increased output of land that is currently marginally productive.
[0197] Example 1: ME02077( Ceres cDNA 36505846; Clone 268310; SEQ ID No.78)
Figure imgf000057_0001
Figure imgf000058_0001
[0198] SUMMARY:
Figure imgf000058_0002
[0199] Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Ti plasmid carrying the 35S promoter operatively linked to Ceres Clone 268310 (SEQ ID No. 79). Wildtype Ws seedlings showed necrotic lesions and stunted growth on plates containing 100 or 150 μM SA, whereas the transgenic plants showed significantly better growth. The transgene encodes a 301-amino-acid protein that shows similarity to an ethylene responsive element binding factor. Segregation ratios (BASTA® resistant: BASTA® sensitive) indicated that ME02077-02 and ME02077-03 each contain one copy of the transgene.
[0200] Two transformed lines, ME02077-02 and ME02077-03, showed the strongest tolerance to oxidative stress. These lines were identified from a screen that assessed the growth of six independent events of ME02077 on MS agar plates containing 100 μM SA. After 14 days, plates were scanned using an EPSON color scanner or fluorescence scanner and SAG calculated for each plant. The data is summarized in Figure 3, where a bar represents the average value +/- standard error of SAG for transgenic plants (T) or pooled non-transgenic plants. ME02077-02 and -03 showed significantly larger SAG values as compared to the pooled non-transgenic control based on t-test (α<0.05). Their tolerance to SA was further evaluated in a validation assay using the T3 and T3 generations.
[0201] When grown on MS agar plates containing 100 μM SA, ME02077-02 and -03 transgenic plants showed significantly increased seedling area and SAG relative to non- transgenic plants. As shown in Table 1-1 and Figures 1 and 2, the T2 generation SAG value for ME02077-02 and ME02077-03 seedlings increased by 935.6% and 804%, respectively. In the T3 generation, the SAG increase for ME02077-02 and ME02077-03 was 1937.4%, and 348.6%, respectively. The differences between transgenic and non-transgenic seedlings are statistically significant under the t-test, and clearly demonstrate that the enhanced tolerance to oxidative stress is a result of the ectopic expression of Ceres cDNA 36505846 in the
ME02077 transformant lines.
Table 1-1 Validation assay of ME02077 on SA tolerance in two generations
Figure imgf000059_0001
* SAG (SA Growth Index) = seedling area x Fv/Fm (photosynthesis efficiency)
[0202] In sum, ectopic expression of Ceres Clone 36505846 under the control of the 35S promoter enhances tolerance to oxidative stress that causes necrotic lesions and stunted growth in wild-type WS seedlings.
[0203] Example 2: ME06123 (Ceres cDNA 23537050; Ceres Λnnot. ID 508432; Locus At4s35180; SEQ ID No. 93)
Figure imgf000059_0002
[0204] SUMMARY:
Figure imgf000060_0001
[0205] Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Ti plasmid carrying the 35S promoter operatively linked to Ceres Annot. ID 508432 (SEQ ID No. 94). Wildtype Ws seedlings showed necrotic lesions and stunted growth on plates containing 100 or 150 μM SA, whereas the transgenic plants showed significantly better growth. The transgene encodes a 456-amino-acid protein that shows similarity to an amino acid transporter. Consequently, the mechanism of the SA tolerance in ME06123 likely involves the compartmentalization of the toxic molecule. Segregation ratios (BASTA® resistant: BASTA® sensitive) indicated that ME06123-01 and ME06123-03 each contain one copy of the transgene. [0206] Two transformed lines, ME06123-01 and ME06123-06, showed the strongest tolerance to oxidative stress. These lines were identified from a screen that assessed the growth of four independent events of ME06123 on MS agar plates containing 150 μM SA. Their tolerance to SA was further evaluated in a validation assay using the T2 and T3 generations or the T3 and T4 generations.
[0207] When grown on MS agar plates containing 100 μM SA, ME06123-01 and ME06123-06 transgenic plants showed significantly increased seedling area and SAG relative to non-transgenic plants. As shown in Table 2-1 and Figure 4, SAG value for the T3 generation of ME06123-01 seedlings increased by 326.1% and by 459.9% for the T2 generation of ME06123-06 seedlings. In the T3 generation, the SAG increase for ME06123- 01 increased was 40.4% while the T4 generation of ME06123-06 increased by 76.9%. The differences between transgenic and non-transgenic seedlings are statistically significant under the t-test, and clearly demonstrate that the enhanced tolerance to oxidative stress is a result of the ectopic expression of Ceres cDNA 23537050 in the ME06123 transformant lines. Table 2-1 Validation assay of ME06123 on SA tolerance in two generations
Figure imgf000060_0002
Figure imgf000061_0001
[0208] In sum, ectopic expression of Ceres cDNA 23537050 under the control of the 35S promoter enhances tolerance to oxidative stress that causes necrotic lesions and stunted growth in wild-type WS seedlings.
[0209] ME18881, an idependent transgenic line with the same sequence as ME06123,, was also tested and the results below obtained.
Table 2-2. Validation assay of ME18881 on SA tolerance in T2 generation
Figure imgf000061_0002
[0210] Example 3: ME00922 (Ceres cDNΛ 23372643; clone 41610; SEQ ID No. 101)
Figure imgf000061_0003
Figure imgf000062_0001
[0211] SUMMARY:
Figure imgf000062_0002
[0212] Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Ti plasmid carrying the 32449 promoter operatively linked to Ceres cDNA 23372643 (SEQ ID No. 102). Wildtype Ws seedlings showed necrotic lesions and stunted growth on plates containing 10 mM L- arginine, whereas the transgenic plants showed significantly better growth. The transgene encodes a 516 amino-acid protein that contains a SET domain, which has been implicated in transcriptional regulation via histone methylation (Springer et al. 2003). Three transformed lines, ME00922-02, ME00922-03 and ME00922-05, showed the strongest qualitative tolerance to oxidative stress in a prevalidation assay. Their tolerance to 10 mM L-arginine was further evaluated in a validation assay for two generations. Segregation ratios (BASTA® resistant: BASTA® sensitive) indicated that ME00922-02, ME00922-03 and ME00922-05 each contain one copy of the transgene.
[0213] When grown on MS agar plates containing 10 mM L-arginine, ME00922-02, ME00922-03 and ME00922-05 transgenic plants showed significantly increased seedling area relative to non-transgenic plants. As shown in Table 3-1 and Figure 6, the T2 generation value for ME00922-02, ME00922-03 and ME00922-05 seedlings increased by 108.76%, 89.75% and 66.89%, respectively. In the T3 generation, three events for each line were tested. Here the greatest increase for ME00922-02, ME00922-03 and ME00922-05 was 29.68%, 43.95% and 81.39%, respectively. The differences between transgenic and non-transgenic seedlings are statistically significant under the t-test, and clearly demonstrate that the enhanced tolerance to oxidative stress is a result of the ectopic expression of Ceres cDNA 23372643 in the ME00922 transformant lines. Table 3-1 Validation assay of ME00922 on Arginine tolerance in two generations
Figure imgf000063_0001
[0214] T3 lines of ME00922 were also tested for oxidative stress tolerance on growth media supplemented with 100 μM SNP. Here, five individual lines derived from ME00922-03 and ME00922-04 showed significantly increased seedling area relative to non-transgenic plants. As shown in Figure 7 and Table 3-2, the T3 generation value for ME00922-03-01, -02 and -03 seedlings increased by 20.95%, 26.98% and 43.51%, respectively. The increase for ME00922-04-01 and -03 seedlings was 26.96% and 102.32%, respectively. Again, the differences between transgenic and non-transgenic seedlings are statistically significant under the t-test, and clearly demonstrate that the enhanced tolerance to oxidative stress is a result of the ectopic expression of Ceres cDNA 23372643 in the ME00922 transformant lines.
Figure imgf000064_0001
[0215] In sum, ectopic expression of Ceres cDNA 23372643 under the control of the 32449 promoter enhances tolerance to oxidative stress that causes necrotic lesions and stunted growth in wild-type WS seedlings.
[0216] An ortholog of ME00922, ME22365, was also tested, providing the results below. Table 3-3 Validation assa of ME22365 on ar inine tolerance in T2 eneration
Figure imgf000064_0002
* AGI (Arginine Growth Index) = seedling area x Fv/Fm (photosynthesis efficiency)
[0217] Example 4: ME12485 (Ceres cDNA 23527804; Ceres Annot. 544535; Locus Atls26710; SEO ID No. 107)
Figure imgf000064_0003
Figure imgf000065_0001
[0218] SUMMARY:
Figure imgf000065_0002
[0219] Wild-type Arabidopsis thaliana Wassilewskija was transformed with a Ti plasmid carrying the CaMV 35S promoter operatively linked to Ceres Annot. ID 544535 (SEQ ID No. 107). Wildtype Ws seedlings showed necrotic lesions and stunted growth on plates containing 100 μM SA, whereas the transgenic plants showed significantly better growth. The transgene encodes a 168 amino acid protein of unknown function. Three transformed lines, ME12485- 05, ME12485-08 and ME12485-09, showed the strongest qualitative tolerance to oxidative stress in a prevalidation assay. Their tolerance to 100 μM SA was further evaluated in a validation assay for two generations. Segregation ratios (BASTA® resistant: BASTA® sensitive) indicated that ME12485-05, ME12485-08 and ME12485-09 each contain at least one copy of the transgene.
[0220] When grown on MS agar plates containing 100 μM SA, ME12485-05, ME12485-08 and ME12485-09 transgenic plants showed significantly increased seedling area relative to non-transgenic plants. As shown in Table 4-1 and Figure 9\ the T2 generation value for ME12485-05, ME12485-08 and ME12485-09 seedlings increased by 64.96%, 86.32% and 59.83%, respectively. In the T3 generation, the increase for ME12485-05, ME12485-08 and ME12485-09 was 23.55%, 37.36% and 19.77%, respectively. The differences between transgenic and non-transgenic seedlings are statistically significant under the t-test except one line in T3 generation (ME 12485-09-02T3), and clearly demonstrate that the enhanced tolerance to oxidative stress is a result of the ectopic expression of Ceres cDNA 23524804 in the ME12485transformant lines. Table 4 Validation assay of ME12485 on SA tolerance in two generations
Figure imgf000066_0001
[0221] In sum, ectopic expression of Ceres cDNA 23524804 under the control of the CaMV 35S promoter enhances tolerance to oxidative stress that causes necrotic lesions and stunted growth in wild-type WS seedlings.
[0222] Example 5- Determination of Functional Homologs by Reciprocal BLAST [0223] A candidate sequence was considered a functional homolog of a reference sequence if the candidate and reference sequences encoded proteins having a similar function and/or activity. A process known as Reciprocal BLAST (Rivera et al., Proc. Natl. Acad. Sci. USA, 95:6239-6244 (1998)) was used to identify potential functional homolog sequences from databases consisting of all available public and proprietary peptide sequences, including NR from NCBI and peptide translations from Ceres clones.
[0224] Before starting a Reciprocal BLAST process, a specific reference polypeptide was searched against all peptides from its source species using BLAST in order to identify polypeptides having BLAST sequence identity of 80% or greater to the reference polypeptide and an alignment length of 85% or greater along the shorter sequence in the alignment. The reference polypeptide and any of the aforementioned identified polypeptides were designated as a cluster.
[0225] The BLASTP version 2.0 program from Washington University at Saint Louis, Missouri, USA was used to determine BLAST sequence identity and E-value. The BLASTP version 2.0 program includes the following parameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3) the -postsw option. The BLAST sequence identity was calculated based on the alignment of the first BLAST HSP (High-scoring Segment Pairs) of the identified potential functional homolog sequence with a specific reference polypeptide. The number of identically matched residues in the BLAST HSP alignment was divided by the HSP length, and then multiplied by 100 to get the BLAST sequence identity. The HSP length typically included gaps in the alignment, but in some cases gaps were excluded. [0226] The main Reciprocal BLAST process consists of two rounds of BLAST searches; forward search and reverse search. In the forward search step, a reference polypeptide sequence, "polypeptide A," from source species SA was BLASTed against all protein sequences from a species of interest. Top hits were determined using an E-value cutoff of 10" 5 and a sequence identity cutoff of 35%. Among the top hits, the sequence having the lowest E-value was designated as the best hit, and considered a potential functional homolog or ortholog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original reference polypeptide was considered a potential functional homolog or ortholog as well. This process was repeated for all species of interest.
[0227] In the reverse search round, the top hits identified in the forward search from all species were BLASTed against all protein sequences from the source species SA. A top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit was also considered as a potential functional homolog.
[0228] Functional homologs were identified by manual inspection of potential functional homolog sequences. Representative functional homologs for SEQ ID Nos. 79, 94, 102 and 107 are shown in Figures 3, 5 and 8 and the Sequence Listing.
Example 19: Determination of Functional Homologs by Hidden Markov Models [0229] Hidden Markov Models (HMMs) were generated by the program HMMER 2.3.2. To generate each HMM, the default HMMER 2.3.2 program parameters, conFigured for glocal alignments, were used.
[0230] An HMM was generated using the sequences shown in Figure 3 as input. These sequences were input into the model and the HMM bit score for each sequence is shown in the Sequence Listing. Additional sequences were input into the model, and the HMM bit scores for the additional sequences are shown in the Sequence Listing. The results indicate that these additional sequences are functional homologs of SEQ ID NO: 79. [0231] HMMs were also generated using the sequences shown in Figures 5 and 8 as input. These sequences were input into the respective models and the corresponding HMM bit score for each sequence is shown in the Sequence Listing. Additional sequences were input into the models, and the HMM bit scores for the additional sequences are shown in the Sequence Listing. The results indicate that these additional sequences are functional homologs of the groups in Figures 5 and 8.
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Claims

1. A method of producing a plant having increased tolerance to oxidative stress, said method comprising growing a plant cell comprising an exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide, and wherein the HMM bit score of the amino acid sequence encoded by said polypeptide is greater than about 30, said HMM based on the amino acid sequences depicted in one of Figures 3, 5 and 8, and wherein said plant has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said nucleic acid.
2. The method according to claim 1, wherein the HMM bit score of the amino acid sequence encoded by said polypeptide is greater than about 40, said HMM based on the amino acid sequences depicted in Figure 3.
3. The method according to claim 1, wherein the HMM bit score of the amino acid sequence encoded by said polypeptide is greater than about 150, said HMM based on the amino acid sequences depicted in Figure 5.
4. The method according to claim 1, wherein the HMM bit score of the amino acid sequence encoded by said polypeptide is greater than about 100, said HMM based on the amino acid sequences depicted in Figure 8.
5. A method of producing a plant having increased tolerance to oxidative stress, said method comprising growing a plant cell comprising an exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 1 10, 1 1 1, 112, 1 14, 1 16, 117, 118, 119, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 311, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357, wherein said plant produced from said plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in a control plant that does not comprise said nucleic acid.
6. A method of producing a plant, said method comprising growing a plant cell comprising an exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 1 13, 1 15, 121, 123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346, 348, 350 and 355, wherein a plant produced from said plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said nucleic acid.
7. A method of modulating the level of tolerance to oxidative stress in a plant, said method comprising introducing into a plant cell an exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide, wherein the HMM bit score of the amino acid sequence of said polypeptide is greater than about 30, said HMM based on the amino acid sequences depicted in one of Figures 3, 5 and 8, and wherein a plant produced from said plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said exogenous nucleic acid.
8. A method of modulating the level of tolerance to oxidative stress in a plant, said method comprising introducing into a plant cell an exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 110, 111, 112, 114, 116, 117, 118, 119, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 211, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 311, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357, wherein a plant produced from said plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said nucleic acid.
9. The method of any one of claims 1-5, 7 or 8, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 79, 94, 10 and 107, and said plant has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said nucleic acid.
10. A method of modulating the level of tolerance to oxidative stress in a plant, said method comprising introducing into a plant cell an exogenous nucleic acid, said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 113, 1 15, 121, 123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346, 348, 350 and 355, wherein a plant produced from said plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said nucleic acid.
1 1. A plant cell comprising an exogenous nucleic acid said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide, wherein the HMM bit score of the amino acid sequence of said polypeptide is greater than about 30, said HMM based on the amino acid sequences depicted in one of Figures 3, 5 and 8, and wherein said plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said nucleic acid.
12. A plant cell comprising an exogenous nucleic acid said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 85 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 80, 82, 83, 84, 85, 87, 88, 89, 90, 91, 94, 96, 97, 98, 99, 100, 102, 104, 105, 107, 109, 1 10, 111, 112, 114, 116, 117, 118, 119, 120, 122, 124, 126, 127, 128, 130, 131, 132, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 157, 158, 160, 161, 162, 163, 165, 167, 168, 169, 170, 171, 173, 175, 176, 178, 180, 181, 182, 183, 184, 186, 188, 190, 192, 194, 195, 197, 199, 201, 203, 205, 207, 208, 209, 21 1, 213, 215, 217, 219, 220, 221, 222, 223, 225, 227, 229, 230, 231, 232, 233, 235, 237, 238, 239, 240, 241, 242, 244, 245, 246, 247, 248, 249, 251, 252, 253, 255, 256, 258, 260, 261, 263, 264, 266, 267, 269, 270, 271, 272, 273, 274, 275, 277, 279, 281, 282, 284, 286, 288, 289, 290, 291, 293, 295, 296, 297, 299, 300, 301, 302, 304, 306, 308, 309, 31 1, 312, 313, 314, 315, 316, 318, 319, 321, 322, 324, 326, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 341, 343, 345, 347, 349, 351, 352, 353, 354, 356 and 357, wherein a plant produced from said plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said nucleic acid.
13. A plant cell comprising an exogenous nucleic acid said exogenous nucleic acid comprising a regulatory region operably linked to a nucleotide sequence having 85 percent or greater sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO: 78, 81, 86, 92, 93, 95, 101, 103, 106, 108, 113, 115, 121, 123, 125, 129, 133, 136, 138, 149, 154, 156, 159, 164, 166, 172, 174, 177, 179, 185, 187, 189, 191, 193, 196, 198, 200, 202, 204, 206, 210, 212, 214, 216, 218, 224, 226, 228, 234, 236, 243, 250, 254, 257, 259, 262, 265, 268, 276, 278, 280, 283, 285, 287, 292, 294, 298, 303, 305, 307, 310, 317, 320, 323, 325, 327, 334, 338, 340, 342, 344, 346, 348, 350 and 355, wherein a plant produced from said plant cell has a difference in the level of tolerance to oxidative stress as compared to the corresponding level in tolerance to oxidative stress of a control plant that does not comprise said nucleic acid.
14. A transgenic plant comprising the plant cell of any one of claims 1 1 -13.
15. The transgenic plant of claim 14, wherein said plant is a member of a species selected from the group consisting of Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearl millet).
16. A product comprising seed or vegetative tissue from a transgenic plant according to claim 11-13.
17. The product of claim 16, wherein said product is a food or feed product.
18. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide having 80% or greater sequence identity to the amino acid sequence set forth in SEQ ID NOs: 79, 94, 102 and 107.
19. A method of identifying whether a polymorphism is associated with variation in a trait, said method comprising: a) determining whether one or more genetic polymorphisms in a population of plants is associated with the locus for a polypeptide selected from the group consisting of the polypeptides depicted in Figures 3, 5 and 8 and functional homologs thereof; and b) measuring the correlation between variation in said trait in plants of said population and the presence of said one or more polymorphisms in plants of said population, thereby identifying whether or not said one or more polymorphisms are associated with variation in said trait.
20. The method of claim 19, wherein said trait is an increased tolerance to oxidative stress or increased biomass.
21. A method of making a plant line, said method comprising: a) determining whether one or more genetic polymorphisms in a population of plants is associated with the locus for a polypeptide selected from the group consisting of the polypeptides depicted in Figures 3, 5 and 8 and functional homologs thereof; b) identifying one or more plants in said population in which the presence of at least one allele at said one or more polymorphisms is associated with variation in oxidative stress tolerance; c) crossing each said one or more identified plants with itself or a different plant to produce seed; d) crossing at least one progeny plant grown from said seed with itself or a different plant; and e) repeating steps c) and d) for an additional 0-5 generations to make said plant line, wherein said at least one allele is piesent in said plant line.
22. The method of claim 20 or 21, wherein said population is a population of switchgrass plants.
PCT/US2007/081301 2003-08-18 2007-10-12 Nucleotide sequences and polypetides encoded thereby useful for increasing tolerance to oxidative stress in plants WO2008046069A2 (en)

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