US20160237447A1

US20160237447A1 - Transgenic Plants With Enhanced Traits

Info

Publication number: US20160237447A1
Application number: US15/027,777
Authority: US
Inventors: Mark Scott Abad; Edwards M Allen; Veena S Anil; Alice Clara Augustine; Isaac R Banks; Prasanna R Bhat; Jaishree M Chittoor-Vijayanath; Paul S Chomet; Molian Deng; Stephen Duff; Barry S Goldman; Sergey Ivashuta; Balasulojini Karunanandaa; Linda Lutfiyya; Sivalinganna Manjunath; Anil Neelam; Monnanda S Rajani; G Ramamohan; Chitresh Sharma; Char Shobha
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 2013-10-07
Filing date: 2014-10-06
Publication date: 2016-08-18
Also published as: WO2015054106A1; US11261457B2; US20220145314A1; US20190367935A1

Abstract

This disclosure provides transgenic plants having enhanced traits such as increased yield, increased nitrogen use efficiency and enhanced drought tolerance; propagules, progeny and field crops of such transgenic plants; and methods of making and using such transgenic plants. This disclosure also provides methods of producing hybrid seed from such transgenic plants, growing such seed and selecting progeny plants with enhanced traits. Also disclosed are transgenic plants with altered phenotypes which are useful for screening and selecting transgenic events for the desired enhanced trait.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35USC §119(e) of U.S. provisional application Ser. No. 61/932,899, filed on Jaunary 29, 2014, and Ser. No. 61/887,545, filed on Oct. 7, 2013 herein incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing file named “60226PCT0000_ST25.txt”, which is 364 kilobytes (measured in MS-WINDOWS) and was created on Oct. 6, 2014, is filed herewith and incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Disclosed herein are plants having enhanced traits such as increased yield, increased nitrogen use efficiency and increased water use efficiency; propagules, progenies and field crops of such plants; and methods of making and using such plants. Also disclosed are methods of producing seed from such plants, growing such seed and/or selecting progeny plants with enhanced traits.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides a plant comprising a recombinant DNA molecule comprising a polynucleotide encoding a polypeptide, wherein the nucleotide sequence of the polynucleotide is selected from the group consisting of: a) a nucleotide sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 115; b) a nucleotide sequence encoding a protein, said protein having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59-92 or 116; c) a nucleotide sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 115; and d) a nucleotide sequence encoding a protein with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59-92 or 116; wherein the plant has an enhanced trait as compared to a control plant.
In another aspect, the disclosure provides a plant comprising a recombinant DNA molecule of the disclosure, wherein the enhanced trait is selected from the group consisting of increased yield, increased nitrogen use efficiency, and increased water use efficiency.
In another aspect, the disclosure provides a plant comprising a recombinant DNA molecule of the disclosure, wherein the plant has at least one phenotype selected from the group consisting of anthocyanin, biomass, canopy area, chlorophyll score, plant height, water applied, water content and water use efficiency that is altered for said plant as compared to a control plant.
In another aspect, the disclosure provides a plant comprising a recombinant DNA molecule of the disclosure, wherein the promoter is selected from the group consisting of a constitutive, inducible, tissue specific, diurnally regulated, tissue enhanced, and cell specific promoter.
In another aspect, the disclosure provides a plant comprising a recombinant DNA molecule of the disclosure, wherein the plant is a progeny, a propagule, or a field crop.
In another aspect, the disclosure provides a plant comprising a recombinant DNA molecule of the disclosure, wherein the field crop is selected from the group consisting of corn, soybean, cotton, canola, rice, barley, oat, wheat, turf grass, alfalfa, sugar beet, sunflower, quinoa and sugar cane.
In another aspect, the disclosure provides a plant comprising a recombinant DNA molecule of the disclosure, wherein the propagule is selected from the group consisting of a cell, pollen, ovule, flower, embryo, leaf, root, stem, shoot, meristem, grain and seed.
In another aspect, the disclosure provides a plant comprising a recombinant DNA molecule of the disclosure, wherein the plant is a monocot plant or is a member of the family Poaceae, wheat plant, maize plant, sweet corn plant, rice plant, wild rice plant, barley plant, rye, millet plant, sorghum plant, sugar cane plant, turfgrass plant, bamboo plant, oat plant, brome-grass plant, Miscanthus plant, pampas grass plant, switchgrass (Panicum) plant, and/or teosinte plant, or is a member of the family Alliaceae, onion plant, leek plant, garlic plant; or wherein the plant is a dicot plant or is a member of the family Amaranthaceae, spinach plant, quinoa plant, a member of the family Anacardiaceae, mango plant, a member of the family Asteraceae, sunflower plant, endive plant, lettuce plant, artichoke plant, a member of the family Brassicaceae, Arabidopsis thaliana plant, rape plant, oilseed rape plant, broccoli plant, Brussels sprouts plant, cabbage plant, canola plant, cauliflower plant, kohlrabi plant, turnip plant, radish plant, a member of the family Bromeliaceae, pineapple plant, a member of the family Caricaceae, papaya plant, a member of the family Chenopodiaceae, beet plant, a member of the family Curcurbitaceae, melon plant, cantaloupe plant, squash plant, watermelon plant, honeydew plant, cucumber plant, pumpkin plant, a member of the family Dioscoreaceae, yam plant, a member of the family Ericaceae, blueberry plant, a member of the family Euphorbiaceae, cassava plant, a member of the family Fabaceae, alfalfa plant, clover plant, peanut plant, a member of the family Grossulariaceae, currant plant, a member of the family Juglandaceae, walnut plant, a member of the family Lamiaceae, mint plant, a member of the family Lauraceae, avocado plant, a member of the family Leguminosae, soybean plant, bean plant, pea plant, a member of the family Malvaceae, cotton plant, a member of the family Marantaceae, arrowroot plant, a member of the family Myrtaceae, guava plant, eucalyptus plant, a member of the family Rosaceae, peach plant, apple plant, cherry plant, plum plant, pear plant, prune plant, blackberry plant, raspberry plant, strawberry plant, a member of the family Rubiaceae, coffee plant, a member of the family Rutaceae, citrus plant, orange plant, lemon plant, grapefruit plant, tangerine plant, a member of the family Salicaceae, poplar plant, willow plant, a member of the family Solanaceae, potato plant, sweet potato plant, tomato plant, Capsicum plant, tobacco plant, tomatillo plant, eggplant plant, Atropa belladona plant, Datura stramonium plant, a member of the family Vitaceae, grape plant, a member of the family Umbelliferae, carrot plant, or a member of the family Musaceae, banana plant; or wherein the plant is a member of the family Pinaceae, cedar plant, fir plant, hemlock plant, larch plant, pine plant, or spruce plant.
In another aspect, the disclosure provides a method for producing a plant comprising: introducing into a plant cell a recombinant DNA molecule comprising a polynucleotide encoding a polypeptide and growing a plant from the plant cell. In this aspect, the polynucleotide comprises a polynucleotide sequence selected from the group consisting of: a) a nucleotide sequence as set forth as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 115; b) a nucleotide sequence encoding a protein comprising an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59-92 or 116; c) a nucleotide sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 115; and d) a nucleotide sequence encoding a protein with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59-92 or 116. In another aspect, the method further comprises the step of selecting a plant with an enhanced trait as compared to a control plant, wherein the enhanced trait is selected from increased yield, increased nitrogen use efficiency, and increased water use efficiency as compared to a control plant.
In another aspect, the disclosure provides a method for producing a plant comprising: introducing into a plant cell a recombinant DNA molecule of the present disclosure, growing a plant from the plant cell and selecting a plant with a phenotype selected from the group consisting of anthocyanin, biomass, canopy area, chlorophyll score, plant height, water applied, water content and water use efficiency, wherein the phenotype for the plant is altered as compared to a control plant.
In another aspect, the disclosure provides a method for increasing yield, increasing nitrogen use efficiency, or increasing water use efficiency in a plant comprising: crossing a plant comprising a recombinant DNA molecule of the present invention with itself, a second plant from the same plant line, a wild type plant, or a second plant from a different line of plants to produce a seed; growing the seed to produce a plurality of progeny plants; and selecting a progeny plant with increased yield, increased nitrogen use efficiency, or increased water use efficiency.
In another aspect, the disclosure provides a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule. In this aspect, the non-coding miRNA molecule is selected from the group consisting of a) a non-coding miRNA molecule which, when present in a plant, provides an altered level of a protein as compared to a control plant, wherein said protein is selected from the group consisting of: a phosphate transporter protein, an APETALA2-like transcription factor, an ANR1 MADS-box protein, an E3 ligase SCF complex F-box protein, a HOS1 protein, a bHLH transcription factor, a diphenol oxidase protein, and the polypeptides set forth in SEQ ID NO: 108, 110, 112 and 114; b) a non-coding miRNA molecule which, when present in a plant, interferes with the functioning of one or more species of an endogenous miRNA selected from the group consisting of: miR399, miR172, miR166, miR166, miR444, miR393 and miR397; c) a non-coding miRNA molecule comprising a miRNA binding site sequence as set forth in SEQ ID NOs: 100-106; and d) a non-coding miRNA molecule comprising a miRNA polynucleotide sequence as set forth in SEQ ID NOs: 93-99. Also in this aspect, the promoter is selected from the group consisting of a constitutive promoter, a developmental promoter, a tissue enhanced promoter, a tissue preferred promoter, a tissue specific promoter, a cell type-specific promoter, an inducible promoter and a diurnal promoter. In a further aspect, when the recombinant DNA molecule is present in a plant, the plant exhibits an altered phenotype or an enhanced trait as compared to a control plant.
In another aspect, the disclosure provides a plant comprising a recombinant DNA molecule comprising a heterologous promoter functional in plant cells operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule. In this aspect, the non-coding miRNA molecule is selected from the group consisting of: a) a non-coding miRNA molecule wherein, when present in a plant, the plant exhibits an altered level of a target protein, wherein the target protein is selected from the group consisting of: a phosphate transporter protein, an APETALA2-like transcription factor, an ANR1 MADS-box protein, an E3 ligase SCF complex F-box protein, a HOS1 protein, a bHLH transcription factor, a diphenol oxidase proteinligase, and the polypeptides set forth in SEQ ID NO: 108, 110, 112 and 114; b) a non-coding miRNA molecule which, when present in a plant, interferes with the functioning of one or more species of an endogenous miRNA selected from the group consisting of: miR399, miR172, miR166, miR166, miR444, miR393 and miR397; c) a non-coding miRNA molecule comprising a miRNA binding site sequence as set forth in SEQ ID NOs: 100-106; and d) a non-coding miRNA molecule comprising a miRNA polynucleotide sequence as set forth in SEQ ID NOs: 93-99. Also in this aspect, the heterologous promoter is selected from the group consisting of a constitutive promoter, a developmental promoter, a tissue enhanced promoter, a tissue preferred promoter, a tissue specific promoter, a cell type-specific promoter, an inducible promoter and a diurnal promoter. Also in this aspect, the plant has an enhanced trait as compared to a control plant.
In another aspect, the disclosure provides a method for producing a plant comprising: introducing into a plant cell a recombinant DNA molecule comprising a heterologous promoter functional in plant cells and operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule as provided by the disclosure, and growing a plant from the plant cell, wherein the plants exhibit an altered level of a target protein selected from the group consisting of: a phosphate transporter protein, an APETALA2-like transcription factor, an ANR1 MADS-box protein, an E3 ligase SCF complex F-box protein, a HOS1 protein, a bHLH transcription factor, a diphenol oxidase proteinligase, and the polypeptides set forth in SEQ ID NO: 108, 110, 112 and 114.
In another aspect, the disclosure provides a method for producing a plant comprising: introducing into a plant cell a recombinant DNA molecule comprising a heterologous promoter functional in plant cells operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule as provided in the disclosure, and growing a plant from the plant cell, wherein the non-coding miRNA molecule interferes with the functioning of one or more endogenous miRNA molecules selected from the group consisting of: miR399, miR172, miR166, miR166, miR444, miR393 and miR397.
In another aspect, the disclosure provides a method for producing a plant comprising: introducing into a plant cell a recombinant DNA molecule comprising a heterologous promoter functional in plant cells operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule, and growing a plant from the plant cell, wherein the non-coding miRNA molecule comprises a miRNA binding site polynucleotide sequence as set forth in SEQ ID NOs: 100-106.
In another aspect, the disclosure provides a method for producing a plant comprising: introducing into a plant cell a recombinant DNA molecule comprising a heterologous promoter functional in plant cells operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule, and growing a plant from the plant cell, wherein the non-coding miRNA molecule comprises a non-coding miRNA polynucleotide sequence as set forth in SEQ ID NOs: 93-99.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:
SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 115 are nucleotide sequences of the coding strand of the DNA constructs used in the recombinant DNA imparting an enhanced trait in plants, each representing a coding sequence for a protein.
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 116 are amino acid sequences of the cognate proteins of the DNA molecules with nucleotide sequences 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 115.
SEQ ID NOs: 59-92 are amino acid sequences of proteins homologous to the proteins with amino acid sequences 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 116.
SEQ ID NOs: 93, 94, 95, 96, 97, 98 and 99 are nucleotide sequences of the coding strand of DNA molecules used in the recombinant DNA imparting an enhanced trait or altered phenotype in plants, each representing a miRNA decoy.
SEQ ID NOs: 100, 101, 102, 103, 104, 105 and 106 are nucleotide sequences corresponding to the miRNA binding sites of the miRNA decoys with nucleotide sequences 93, 94, 95, 96, 97, 98 and 99.
SEQ ID NOs: 108, 110, 112 and 114 are amino acid sequences corresponding to proteins that are down-regulated by endogenous miR172, miR166, miR444 and miR397, respectively. SEQ ID NOs: 107, 109, 111 and 113 are polynucleotides sequences encoding those proteins.
As used herein, the term “expression” refers to the production of a polynucleotide or a protein by a plant, plant cell or plant tissue which can give rise to an altered phenotype or enhanced trait. Expression can also refer to the process by which information from a gene is used in the synthesis of functional gene products, which may include but are not limited to other polynucleotides or proteins which may serve, e.g., an enzymatic, structural or regulatory function. Gene products having a regulatory function include but are not limited to elements that affect the occurrence or level of transcription or translation of a target protein. In some cases, the expression product is a non-coding functional RNA.
“Modulation” of expression refers to the process of effecting either overexpression or suppression of a polynucleotide or a protein.
The term “suppression” as used herein refers to a lower expression level of a target polynucleotide or target protein in a plant, plant cell or plant tissue, as compared to the expression in a wild-type or control plant, cell or tissue, at any developmental or temporal stage for the gene. The term “target protein” as used in the context of suppression refers to a protein which is suppressed; similarly, “target mRNA” refers to a polynucleotide which can be suppressed or, once expressed, degraded so as to result in suppression of the target protein it encodes. In alternate non-limiting embodiments, the target protein or target polynucleotide is one the suppression of which can give rise to an enhanced trait or altered phenotype directly or indirectly. In one exemplary embodiment, the target protein is one which can indirectly increase or decrease the expression of one or more other proteins, the increased or decreased expression, respectively, of which is associated with an enhanced trait or an altered phenotype. In another exemplary embodiment, the target protein can bind to one or more other proteins associated with an altered phenotype or enhanced trait to enhance or inhibit their function and thereby effect the altered phenotype or enhanced trait indirectly.
Suppression can be applied using numerous approaches. Non limiting examples include: suppressing an endogenous gene(s) or a subset of genes in a pathway, suppressing one or more mutation that has resulted in decreased activity of a protein, suppressing the production of an inhibitory agent, to elevate, reducing or eliminating the level of substrate that an enzyme requires for activity, producing a new protein, activating a normally silent gene; or accumulating a product that does not normally increase under natural conditions.
Conversely, the term “overexpression” as used herein refers to a greater expression level of a polynucleotide or a protein in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present polypeptides. Overexpression can also occur in plant cells where endogenous expression of the present polypeptides or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or “overproduction” of the polypeptide in the plant, cell or tissue.
The term “target protein” as used herein in the context of overexpression refers to a protein which is overexpressed; “target mRNA” refers to an mRNA which encodes and is translated to produce the target protein, which can also be overexpressed. In alternative embodiments, the target protein can effect an enhanced trait or altered phenotype directly or indirectly. In the latter case it may do so, for example, by affecting the expression, function or substrate available to one or more other proteins. In an exemplary embodiment, the target protein can bind to one or more other proteins associated with an altered phenotype or enhanced trait to enhance or inhibit their function.
Overexpression can be achieved using numerous approaches. In one embodiment, overexpression can be achieved by placing the DNA sequence encoding one or more polynucleotides or polypeptides under the control of a promoter, examples of which include but are not limited to endogenous promoters, heterologous promoters, inducible promoters and tissue specific promoters. In one exemplary embodiment, the promoter is a constitutive promoter, for example, the cauliflower mosaic virus 35S transcription initiation region. Thus, depending on the promoter used, overexpression can occur throughout a plant, in specific tissues of the plant, or in the presence or absence of different inducing or inducible agents, such as hormones or environmental signals.
Small RNAs that regulate protein expression include miRNAs and ta-siRNAs. A miRNA is a small (typically about 21 nucleotide) RNA that has the ability to modulate the expression of a target gene by binding to a messenger RNA encoding a target protein at specific “miRNA binding sites” to form a RNA duplex, leading to destabilization of the target protein messenger RNA or to translational inhibition of the target protein messenger RNA, and ultimately resulting in suppression of the target protein.
Recombinant DNA constructs can be used to modify the activity of native miRNAs by a variety of means. By increasing the expression of a miRNA, e.g. temporally or spatially, the modulation of expression of a native target gene can be enhanced. An alternative gene suppression approach for suppressing the expression of a target protein can include the use of a recombinant DNA construct that produces a synthetic miRNA that is designed to bind to a native or synthetic miRNA recognition site on messenger RNA for the target protein. By reducing the expression of a miRNA, the modulation of a native target gene can be diminished resulting in enhanced expression of the target protein. More specifically, the expression of a target protein can be enhanced by suppression of the activity of the miRNA that binds to a recognition site in the messenger RNA that is transcribed from the native gene for the target protein. Several types of recombinant DNA constructs can be designed to suppress the activity of a miRNA.
For example, a recombinant DNA construct that produces an abundance of RNA with the miRNA recognition site can be used as a decoy for the native miRNA allowing endogenous messenger RNA with the miRNA recognition site to be translated to the target protein without interference from native miRNA. A recombinant DNA construct that produces RNA with a modified miRNA recognition site, e.g. with nucleotides at positions 10 and/or 11 in a 21mer miRNA recognition site which are unpaired with respect to the native miRNA, can be used to sequester natively expressed miRNA thereby reducing the cleavage that normally occurs when miRNA binds to a recognition site. The unpaired nucleotides can be produced e.g. through additional nucleotides between positions 10 and 11 or through substitutions of the nucleotides at positions 10 and 11.
Naturally occurring or endogenous decoys exist which include one or more miRNA binding sites. A “miRNA decoy” is defined herein as a sequence that can be recognized and bound by an endogenous mature miRNA resulting in base-pairing between the miRNA decoy sequence and the endogenous mature miRNA, thereby forming a stable RNA duplex that is not cleaved because of the presence of mismatches between the miRNA decoy sequence and the mature miRNA.
Rules for predicting endogenous microRNA decoy sequences have been developed. In general, these rules define (1) mismatches that are required, and (2) mismatches that are permitted but not required. Mismatches include canonical mismatches (e.g., G-A, C-U, C-A) as well as G::U wobble pairs and indels (nucleotide insertions or deletions). These rules may be applied to design synthetic decoys.
With respect to constructing synthetic decoys, required mismatches include: (a) at least 1 mismatch between the miRNA decoy sequence and the endogenous mature miRNA at positions 9, 10, or 11 of the endogenous mature miRNA, or alternatively, (b) 1, 2, 3, 4, or 5 insertions (i.e., extra nucleotides) at a position in the miRNA decoy sequence corresponding to positions 9, 10, or 11 of the endogenous mature miRNA. In preferred embodiments, there exists either (a) at least 1 mismatch between the miRNA decoy sequence and the endogenous mature miRNA at positions 10 and/or 11 of the endogenous mature miRNA, or (b) at least 1 insertion in the miRNA decoy sequence between the nucleotides at positions corresponding to positions 10 and/or 11 of the endogenous mature miRNA. In exemplary embodiments, there can be 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions.
Mismatches that are permitted, but not required, include: (a) 0, 1, or 2 mismatches between the miRNA decoy sequence and the endogenous mature miRNA at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 of the endogenous mature miRNA, and (b) 0, 1, 2, or 3 mismatches between the miRNA decoy sequence and the endogenous mature miRNA at positions 12 through the last position of the endogenous mature miRNA (i.e., at position 21 of a 21-nucleotide mature miRNA), wherein each of the mismatches at positions 12 through the last position of the endogenous mature miRNA is adjacent to at least one complementary base-pair (i.e., so that there is not more than 2 contiguous mismatches at positions 12 through the last position of the endogenous mature miRNA). In preferred embodiments, there exist no mismatches (i.e., there are all complementary base-pairs) at positions 1, 2, 3, 4, 5, 6, 7, and 8 of the endogenous mature miRNA.
Decoy constructs may include but are not limited to DNA sequences that are transcribed into (i) naturally occurring decoys containing one or more naturally occurring miRNA binding sites specific for miRNA families, (ii) synthetic decoys constructed using a naturally occurring non-coding RNA “scaffolding” and one or more binding sites, i.e., naturally occurring decoys in which the one or more naturally occurring binding sites are substituted or supplemented with one or more synthetic miRNA binding sites, (iii) synthetic decoys wherein a miRNA binding site, which may be naturally occurring or synthetic, is introduced into the 3′-untranslated region of a coding mRNA, or (iv) decoys which are chimeras of any of the preceding decoys.
The construction and description of recombinant DNA constructs to modulate small non-coding RNA activities are disclosed in US Patent Application Publication US 2009/0070898 A1, and US Patent Application Publication US2011/0296555 A1, both of which are incorporated herein by reference. In particular, with respect to US 2009/0070898 A1, see e.g. paragraphs 182 to 186 and Example 11 at paragraphs 290 to 297.
Combinatorial regulation is a further feature of miRNA-mediated overexpression. Multiple families of miRNA molecules are known, each of which can encompass multiple species of miRNA molecules. A given miRNA family may have multiple different mRNA targets; conversely, a given target can be targeted by multiple miRNA families. Another feature of endogenous miRNA-mediated overexpression is that expression of individual miRNA families is in some cases cell-type or tissue-specific, or varies temporally, developmentally or in response to environmental stimuli. Hence expression of particular miRNA families can overlap in time, spatially, developmentally or according to growth conditions, resulting in varied patterns of target protein expression.
Hence, it is possible to produce plants with desirable altered phenotypes or enhanced traits by selectively interfering with the expression pattern or function of specific miRNA families that suppress target proteins associated with those phenotypes or traits.
Thus, in one embodiment, a target protein can be overexpressed by interfering with the endogenous activities of these small inhibitory RNA molecules, which can be accomplished by several ways. One non-limiting example is to reduce the expression of a miRNA, by which the modulation of a native target gene can be diminished and consequently enhancing expression of the target protein. Yet another embodiment of the present invention for overexpressing a target protein is, to modify the native miRNA recognition site in the mRNA encoding the protein to render it resistant to the binding of cognate miRNA which regulates the native gene. Yet another non-limiting example is to express a miRNA “decoy” constructed as described above.
Thus exemplary embodiments include plants transformed with a recombinant DNA molecule encoding at least one polynucleotide sequence transcribed into a mature miRNA decoy that recognizes and binds at least one miRNA species, wherein the polynucleotide has a sequence selected from the group consisting of SEQ ID NOs 93-99, and wherein the plants exhibit an altered phenotype or an enhanced trait or both.
Further exemplary embodiments of the disclosure include a plant transformed with a recombinant DNA molecule encoding as least one polynucleotide sequence transcribed into a mature miRNA decoy that recognizes and binds at least one miRNA species, wherein the polynucleotide comprises at least one miRNA binding site having a sequence selected from the group consisting of SEQ ID NOs: 100-106 and wherein the plants exhibit an altered phenotype or an enhanced trait or both.
Further exemplary embodiments include plants transformed with a recombinant DNA molecule encoding a heterologous promoter operably linked to at least one polynucleotide sequence transcribed into a mature miRNA decoy that recognizes and binds at least one miRNA species, wherein the promoter is a constitutive promoter, a developmental promoter, a tissue enhanced promoter, a tissue preferred promoter, a tissue specific promoter, a cell type-specific promoter, an inducible promoter or a diurnal promoter.
Further exemplary embodiments include a method of producing plants exhibiting an altered phenotype or an enhanced trait or both by transforming the plants with recombinant DNA constructs encoding a least one polynucleotide sequence transcribed into a mature miRNA decoy that recognizes and binds at least one miRNA species, wherein the polynucleotide has a sequence selected from the group consisting of SEQ ID NOs: 93-99, and wherein the plants exhibit an altered phenotype or an enhanced trait or both.
Further exemplary embodiments of the disclosure include a method of producing plants exhibiting an altered phenotype or an enhanced trait or both by transforming the plants with recombinant DNA constructs encoding a least one polynucleotide sequence transcribed into a mature miRNA decoy that recognizes and binds at least one miRNA species, wherein the polynucleotide comprises at least one miRNA binding site having sequence selected from the group consisting of SEQ ID NOs: 100-106.
Further exemplary embodiments include a method of producing plants exhibiting an altered phenotype or an enhanced trait or both by transforming the plants with a recombinant DNA molecule encoding a heterologous promoter operably linked to at least one polynucleotide sequence transcribed into a mature miRNA decoy that recognizes and binds at least one miRNA species, wherein the promoter is a constitutive promoter, a developmental promoter, a tissue enhanced promoter, a tissue preferred promoter, a tissue specific promoter, a cell type-specific promoter, an inducible promoter or a diurnal promoter.
As used herein a “plant” includes a whole plant, a transgenic plant, meristematic tissue, a shoot organ/structure (for example, leaf, stem and tuber), a root, a flower, a floral organ/structure (for example, a bract, a sepal, a petal, a stamen, a carpel, an anther and an ovule), a seed (including an embryo, endosperm, and a seed coat) and a fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and a cell (for example, guard cell, egg cell, pollen, mesophyll cell, and the like), and progeny of same. The classes of plants that can be used in the disclosed methods are generally as broad as the classes of higher and lower plants amenable to transformation and breeding techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae.
As used herein a “transgenic plant cell” means a plant cell that is transformed with stably-integrated, recombinant DNA, for example, by Agrobacterium-mediated transformation or by bombardment using microparticles coated with recombinant DNA or by other means. A plant cell of this disclosure can be an originally-transformed plant cell that exists as a microorganism or as a progeny plant cell that is regenerated into differentiated tissue, for example, into a transgenic plant with stably-integrated, recombinant DNA, or seed or pollen derived from a progeny transgenic plant.
As used herein a “control plant” means a plant that does not contain the recombinant DNA of the present disclosure that imparts an enhanced trait or altered phenotype. A control plant is used to identify and select a transgenic plant that has an enhanced trait or altered phenotype. A suitable control plant can be a non-transgenic plant of the parental line used to generate a transgenic plant, for example, a wild type plant devoid of a recombinant DNA. A suitable control plant can also be a transgenic plant that contains recombinant DNA that imparts other traits, for example, a transgenic plant having enhanced herbicide tolerance. A suitable control plant can in some cases be a progeny of a hemizygous transgenic plant line that does not contain the recombinant DNA, known as a negative segregant, or a negative isogenic line.
As used herein a “propagule” includes all products of meiosis and mitosis, including but not limited to, plant, seed and part of a plant able to propagate a new plant. Propagules include whole plants, cells, pollen, ovules, flowers, embryos, leaves, roots, stems, shoots, meristems, grains or seeds, or any plant part that is capable of growing into an entire plant. Propagule also includes graft where one portion of a plant is grafted to another portion of a different plant (even one of a different species) to create a living organism. Propagule also includes all plants and seeds produced by cloning or by bringing together meiotic products, or allowing meiotic products to come together to form an embryo or a fertilized egg (naturally or with human intervention).
As used herein a “progeny” includes any plant, seed, plant cell, and/or regenerable plant part comprising a recombinant DNA of the present disclosure derived from an ancestor plant. A progeny can be homozygous or heterozygous for the transgene. Progeny can be grown from seeds produced by a transgenic plant comprising a recombinant DNA of the present disclosure, and/or from seeds produced by a plant fertilized with pollen or ovule from a transgenic plant comprising a recombinant DNA of the present disclosure.
As used herein a “trait” is a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, certain metabolites, or oil content of seed or leaves, or by observation of a metabolic or physiological process, for example, by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the measurement of the expression level of a gene or genes, for example, by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as hyperosmotic stress tolerance or yield. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.
As used herein an “enhanced trait” means a characteristic of a transgenic plant as a result of stable integration and expression of a recombinant DNA in the transgenic plant. Such traits include, but are not limited to, an enhanced agronomic trait characterized by enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In some specific aspects of this disclosure an enhanced trait is selected from the group consisting of drought tolerance, increased water use efficiency, cold tolerance, increased nitrogen use efficiency and increased yield as shown in Tables 9-16, and altered phenotypes as shown in Tables 4-8. In another aspect of the disclosure the trait is increased yield under non-stress conditions or increased yield under environmental stress conditions. Stress conditions can include both biotic and abiotic stress, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. “Yield” can be affected by many properties including without limitation, plant height, plant biomass, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Yield can also be affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.
Also used herein, the term “trait modification” encompasses altering the naturally occurring trait by producing a detectable difference in a characteristic in a plant comprising a recombinant DNA of the present disclosure relative to a plant not comprising the recombinant DNA, such as a wild-type plant, or a negative segregant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail an increase or decrease, in an observed trait as compared to a control plant. It is known that there can be natural variations in a modified trait. Therefore, the trait modification observed entails a change of the normal distribution and magnitude of the trait in the plants as compared to a control plant.
The present disclosure relates to a plant with improved economically important characteristics, more specifically increased yield. More specifically the present disclosure relates to a plant comprising a polynucleotide of this disclosure, wherein the plant has increased yield as compared to a control plant. Many plants of this disclosure exhibited increased yield as compared to a control plant. In an embodiment, a plant of the present disclosure exhibited an improved trait that is related to yield, including but not limited to increased nitrogen use efficiency, increased nitrogen stress tolerance, increased water use efficiency and increased drought tolerance, as defined and discussed infra.
Yield can be defined as the measurable produce of economic value from a crop. Yield can be defined in the scope of quantity and/or quality. Yield can be directly dependent on several factors, for example, the number and size of organs, plant architecture (such as the number of branches, plant biomass, etc.), seed production and more. Root development, photosynthetic efficiency, nutrient uptake, stress tolerance, early vigor, delayed senescence and functional stay green phenotypes can be important factors in determining yield. Optimizing the above mentioned factors can therefore contribute to increasing crop yield.
Reference herein to an increase in yield-related traits can also be taken to mean an increase in biomass (weight) of one or more parts of a plant, which can include above ground and/or below ground (harvestable) plant parts. In particular, such harvestable parts are seeds, and performance of the methods of the disclosure results in plants with increased yield and in particular increased seed yield relative to the seed yield of suitable control plants. The term “yield” of a plant can relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant.
Increased yield of a plant of the present disclosure can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (for example, seeds, or weight of seeds, per acre), bushels per acre, tons per acre, or kilo per hectare. For example, corn yield can be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare. This is often also reported on a moisture adjusted basis, for example at 15.5 percent moisture. Increased yield can result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, shade, high plant density, and attack by pests or pathogens. This disclosure can also be used to provide plants with improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways. Also of interest is the generation of plants that demonstrate increased yield with respect to a seed component that may or may not correspond to an increase in overall plant yield.
In an embodiment, “alfalfa yield” can also be measured in forage yield, the amount of above ground biomass at harvest. Factors leading contributing to increased biomass include increased vegetative growth, branches, nodes and internodes, leaf area, and leaf area index.
In another embodiment, “canola yield” can also be measured in pod number, number of pods per plant, number of pods per node, number of internodes, incidence of pod shatter, seeds per silique, seed weight per silique, improved seed, oil, or protein composition.
Additionally, “corn or maize yield” can also be measured as production of shelled corn kernels per unit of production area, ears per acre, number of kernel rows per ear, weight per kernel, ear number, fresh or dry ear biomass (weight), kernel rows per ear and kernels per row.
In yet another embodiment, “cotton yield” can be measured as bolls per plant, size of bolls, fiber quality, seed cotton yield in g/plant, seed cotton yield in lb/acre, lint yield in lb/acre, and number of bales.
Specific embodiment for “rice yield” can also include panicles per hill, grain per hill, and filled grains per panicle.
Still further embodiment for “soybean yield” can also include pods per plant, pods per acre, seeds per plant, seeds per pod, weight per seed, weight per pod, pods per node, number of nodes, and the number of internodes per plant.
In still further embodiment, “sugarcane yield” can be measured as cane yield (tons per acre; kg/hectare), total recoverable sugar (pounds per ton), and sugar yield (tons/acre).
In yet still further embodiment, “wheat yield” can include: cereal per unit area, grain number, grain weight, grain size, grains per head, seeds per head, seeds per plant, heads per acre, number of viable tillers per plant, composition of seed (for example, carbohydrates, starch, oil, and protein) and characteristics of seed fill.
The terms “yield”, “seed yield” are defined above for a number of core crops. The terms “increased”, “improved”, “enhanced” are interchangeable and are defined herein.
In another embodiment, the present disclosure provides a method for the production of plants having increased yield; performance of the method gives plants increased yield. “Increased yield” can manifest as one or more of the following: (i) increased plant biomass (weight) of one or more parts of a plant, particularly aboveground (harvestable) parts, of a plant, increased root biomass (increased number of roots, increased root thickness, increased root length) or increased biomass of any other harvestable part; or (ii) increased early vigor, defined herein as an improved seedling aboveground area approximately three weeks post-germination. “Early vigor” refers to active healthy plant growth especially during early stages of plant growth, and can result from increased plant fitness due to, for example, the plants being better adapted to their environment (for example, optimizing the use of energy resources, uptake of nutrients and partitioning carbon allocation between shoot and root). Early vigor in corn, for example, is a combination of the ability of corn seeds to germinate and emerge after planting and the ability of the young corn plants to grow and develop after emergence. Plants having early vigor also show increased seedling survival and better establishment of the crop, which often results in highly uniform fields with the majority of the plants reaching the various stages of development at substantially the same time, which often results in increased yield. Therefore early vigor can be determined by measuring various factors, such as kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass, canopy size and color and others.
Further, increased yield can also manifest as (iii) increased total seed yield, which may result from one or more of an increase in seed biomass (seed weight) due to an increase in the seed weight on a per plant and/or on an individual seed basis an increased number of panicles per plant; an increased number of pods; an increased number of nodes; an increased number of flowers (“florets”) per panicle/plant; increased seed fill rate; an increased number of filled seeds; increased seed size (length, width, area, perimeter), which can also influence the composition of seeds; and/or increased seed volume, which can also influence the composition of seeds.
Increased yield can also (iv) result in modified architecture, or can occur because of modified plant architecture.
Increased yield can also manifest as (v) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass
Increased yield can also manifest as (vi) increased kernel weight, which is extrapolated from the number of filled seeds counted and their total weight. An increased kernel weight can result from an increased seed size and/or seed weight, an increase in embryo size, increased endosperm size, aleurone and/or scutellum, or an increase with respect to other parts of the seed that result in increased kernel weight.
Increased yield can also manifest as (vii) increased ear biomass, which is the weight of the ear and can be represented on a per ear, per plant or per plot basis.
In one embodiment, increased yield can be increased seed yield, and is selected from one of the following: (i) increased seed weight; (ii) increased number of filled seeds; and (iii) increased harvest index.
The disclosure also extends to harvestable parts of a plant such as, but not limited to, seeds, leaves, fruits, flowers, bolls, stems, rhizomes, tubers and bulbs. The disclosure furthermore relates to products derived from a harvestable part of such a plant, such as dry pellets, powders, oil, fat and fatty acids, starch or proteins.
The present disclosure provides a method for increasing “yield” of a plant or “broad acre yield” of a plant or plant part defined as the harvestable plant parts per unit area, for example seeds, or weight of seeds, per acre, pounds per acre, bushels per acre, tones per acre, tons per acre, kilo per hectare.
This disclosure further provides a method of increasing yield in a plant by producing a plant comprising a polynucleic acid sequence of this disclosure where the plant can be crossed with itself, a second plant from the same plant line, a wild type plant, or a plant from a different line of plants to produce a seed. The seed of the resultant plant can be harvested from fertile plants and be used to grow progeny generations of plant(s) of this disclosure. In addition to direct transformation of a plant with a recombinant DNA molecule, transgenic plants can be prepared by crossing a first plant having a stably integrated recombinant DNA molecule with a second plant lacking the DNA. For example, recombinant DNA can be introduced into a first plant line that is amenable to transformation to produce a transgenic plant which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line.
A transgenic plant transformed with a recombinant DNA molecule and having the polynucleotide of this disclosure provides the enhanced trait of increased yield compared to a control plant. Genetic markers associated with recombinant DNA can produce transgenic progeny that is homozygous for the desired recombinant DNA. Progeny plants carrying DNA for both parental traits can be back crossed into a parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as the one original transgenic parental line but having the recombinant DNA of the other transgenic parental line. The term “progeny” denotes the offspring of any generation of a parent plant prepared by the methods of this disclosure containing the recombinant polynucleotides as described herein.
As used herein “nitrogen use efficiency” refers to the processes which lead to an increase in the plant's yield, biomass, vigor, and growth rate per nitrogen unit applied. The processes can include the uptake, assimilation, accumulation, signaling, sensing, retranslocation (within the plant) and use of nitrogen by the plant.
As used herein “nitrogen limiting conditions” refers to growth conditions or environments that provide less than optimal amounts of nitrogen needed for adequate or successful plant metabolism, growth, reproductive success and/or viability.
As used herein the “increased nitrogen stress tolerance” refers to the ability of plants to grow, develop, or yield normally, or grow, develop, or yield faster or better when subjected to less than optimal amounts of available/applied nitrogen, or under nitrogen limiting conditions.
As used herein “increased nitrogen use efficiency” refers to the ability of plants to grow, develop, or yield faster or better than normal when subjected to the same amount of available/applied nitrogen as under normal or standard conditions; ability of plants to grow, develop, or yield normally, or grow, develop, or yield faster or better when subjected to less than optimal amounts of available/applied nitrogen, or under nitrogen limiting conditions.
Increased plant nitrogen use efficiency can be translated in the field into either harvesting similar quantities of yield, while supplying less nitrogen, or increased yield gained by supplying optimal/sufficient amounts of nitrogen. The increased nitrogen use efficiency can improve plant nitrogen stress tolerance, and can also improve crop quality and biochemical constituents of the seed such as protein yield and oil yield. The terms “increased nitrogen use efficiency”, “enhanced nitrogen use efficiency”, and “nitrogen stress tolerance” are used inter-changeably in the present disclosure to refer to plants with improved productivity under nitrogen limiting conditions.
As used herein “water use efficiency” refers to the amount of carbon dioxide assimilated by leaves per unit of water vapor transpired. It constitutes one of the most important traits controlling plant productivity in dry environments. “Drought tolerance” refers to the degree to which a plant is adapted to arid or drought conditions. The physiological responses of plants to a deficit of water include leaf wilting, a reduction in leaf area, leaf abscission, and the stimulation of root growth by directing nutrients to the underground parts of the plants. Plants are more susceptible to drought during flowering and seed development (the reproductive stages), as plant's resources are deviated to support root growth. In addition, abscisic acid (ABA), a plant stress hormone, induces the closure of leaf stomata (microscopic pores involved in gas exchange), thereby reducing water loss through transpiration, and decreasing the rate of photosynthesis. These responses improve the water-use efficiency of the plant on the short term. The terms “increased water use efficiency”, “enhanced water use efficiency”, and “increased drought tolerance” are used inter-changeably in the present disclosure to refer to plants with improved productivity under water-limiting conditions.
As used herein “increased water use efficiency” refers to the ability of plants to grow, develop, or yield faster or better than normal when subjected to the same amount of available/applied water as under normal or standard conditions; ability of plants to grow, develop, or yield normally, or grow, develop, or yield faster or better when subjected to reduced amounts of available/applied water (water input) or under conditions of water stress or water deficit stress.
As used herein “increased drought tolerance” refers to the ability of plants to grow, develop, or yield normally, or grow, develop, or yield faster or better than normal when subjected to reduced amounts of available/applied water and/or under conditions of acute or chronic drought; ability of plants to grow, develop, or yield normally when subjected to reduced amounts of available/applied water (water input) or under conditions of water deficit stress or under conditions of acute or chronic drought.
As used herein “drought stress” refers to a period of dryness (acute or chronic/prolonged) that results in water deficit and subjects plants to stress and/or damage to plant tissues and/or negatively affects grain/crop yield; a period of dryness (acute or chronic/prolonged) that results in water deficit and/or higher temperatures and subjects plants to stress and/or damage to plant tissues and/or negatively affects grain/crop yield.
As used herein “water deficit” refers to the conditions or environments that provide less than optimal amounts of water needed for adequate/successful growth and development of plants.
As used herein “water stress” refers to the conditions or environments that provide improper (either less/insufficient or more/excessive) amounts of water than that needed for adequate/successful growth and development of plants/crops thereby subjecting the plants to stress and/or damage to plant tissues and/or negatively affecting grain/crop yield.
As used herein “water deficit stress” refers to the conditions or environments that provide less/insufficient amounts of water than that needed for adequate/successful growth and development of plants/crops thereby subjecting the plants to stress and/or damage to plant tissues and/or negatively affecting grain yield.
As used herein a “polynucleotide” is a nucleic acid molecule comprising a plurality of polymerized nucleotides. A polynucleotide may be referred to as a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. In many instances, a polynucleotide encodes a polypeptide (or protein) or a domain or fragment thereof. Additionally, a polynucleotide can comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker, a scorable marker, or the like. A polynucleotide can be single-stranded or double-stranded DNA or RNA. A polynucleotide optionally comprises modified bases or a modified backbone. A polynucleotide can be, for example, genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. A polynucleotide can be combined with carbohydrate(s), lipid(s), protein(s), or other materials to perform a particular activity such as transformation or form a composition such as a peptide nucleic acid (PNA). A polynucleotide can comprise a sequence in either sense or antisense orientations. “Oligonucleotide” is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single-stranded.
As used herein a “recombinant polynucleotide” or “recombinant DNA” is a polynucleotide that is not in its native state, for example, a polynucleotide comprises a series of nucleotides (represented as a nucleotide sequence) not found in nature, or a polynucleotide is in a context other than that in which it is naturally found; for example, separated from polynucleotides with which it typically is in proximity in nature, or adjacent (or contiguous with) polynucleotides with which it typically is not in proximity. The “recombinant polynucleotide” or “recombinant DNA” refers to polynucleotide or DNA which has been genetically engineered and constructed outside of a cell including DNA containing naturally occurring DNA or cDNA or synthetic DNA. For example, the polynucleotide at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acids.
As used herein a “polypeptide” comprises a plurality of consecutive polymerized amino acid residues for example, at least about 15 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a series of polymerized amino acid residues that is a transcriptional regulator or a domain or portion or fragment thereof. Additionally, the polypeptide can comprise: (i) a localization domain; (ii) an activation domain; (iii) a repression domain; (iv) an oligomerization domain; (v) a protein-protein interaction domain; (vi) a DNA-binding domain; or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.
As used herein “protein” refers to a series of amino acids, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
As used herein a “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide.
A “synthetic polypeptide” is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods known in the art.
An “isolated polypeptide”, whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, for example, more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, for example, alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components, with which it is typically associated, for example, by any of the various protein purification methods.
As used herein, a “functional fragment” refers to a portion of a polypeptide provided herein which retains full or partial molecular, physiological or biochemical function of the full length polypeptide. A functional fragment often contains the domain(s), such as Pfam domains (see below), identified in the polypeptide provided in the sequence listing.
A “DNA construct” as used in the present disclosure comprises at least one expression cassette having a promoter operable in plant cells and a polynucleotide of the present disclosure. DNA constructs can be used as a means of delivering recombinant DNA molecules to a plant cell in order to effect stable integration of the recombinant molecule into the plant cell genome. In one embodiment, the polynucleotide can encode a protein or variant of a protein or fragment of a protein that is functionally defined to maintain activity in transgenic host cells including plant cells, plant parts, explants and whole plants. In another embodiment, the polynucleotide can encode a non-coding RNA that interferes with the functioning of endogenous classes of small RNAs that regulate expression, including but not limited to taRNAs, siRNAs and miRNAs. Recombinant DNA constructs are assembled using methods known to persons of ordinary skill in the art and typically comprise a promoter operably linked to DNA, the expression of which provides the enhanced agronomic trait.
Other construct components can include additional regulatory elements, such as 5′ leaders and introns for enhancing transcription, 3′ untranslated regions (such as polyadenylation signals and sites), and DNA for transit or targeting or signal peptides.
Percent identity describes the extent to which polynucleotides or protein segments are invariant in an alignment of sequences, for example nucleotide sequences or amino acid sequences. An alignment of sequences is created by manually aligning two sequences, for example, a stated sequence, as provided herein, as a reference, and another sequence, to produce the highest number of matching elements, for example, individual nucleotides or amino acids, while allowing for the introduction of gaps into either sequence. An “identity fraction” for a sequence aligned with a reference sequence is the number of matching elements, divided by the full length of the reference sequence, not including gaps introduced by the alignment process into the reference sequence. “Percent identity” (“% identity”) as used herein is the identity fraction times 100.
As used herein, a “homolog” or “homologues” means a protein in a group of proteins that perform the same biological function, for example, proteins that belong to the same Pfam protein family and that provide a common enhanced trait in transgenic plants of this disclosure. Homologs are expressed by homologous genes. With reference to homologous genes, homologs include orthologs, for example, genes expressed in different species that evolved from common ancestral genes by speciation and encode proteins retain the same function, but do not include paralogs, i.e., genes that are related by duplication but have evolved to encode proteins with different functions. Homologous genes include naturally occurring alleles and artificially-created variants.
Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. When optimally aligned, homolog proteins, or their corresponding nucleotide sequences, have typically at least about 60% identity, in some instances at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even at least about 99.5% identity over the full length of a protein or its corresponding nucleotide sequence identified as being associated with imparting an enhanced trait or altered phenotype when expressed in plant cells. In one aspect of the disclosure homolog proteins have at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% identity to a consensus amino acid sequence of proteins and homologs that can be built from sequences disclosed herein.
Homologs are inferred from sequence similarity, by comparison of protein sequences, for example, manually or by use of a computer-based tool using known sequence comparison algorithms such as BLAST and FASTA. A sequence search and local alignment program, for example, BLAST, can be used to search query protein sequences of a base organism against a database of protein sequences of various organisms, to find similar sequences, and the summary Expectation value (E-value) can be used to measure the level of sequence similarity. Because a protein hit with the lowest E-value for a particular organism may not necessarily be an ortholog or be the only ortholog, a reciprocal query is used to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of protein sequences of the base organism. A hit can be identified as an ortholog, when the reciprocal query's best hit is the query protein itself or a paralog of the query protein. With the reciprocal query process orthologs are further differentiated from paralogs among all the homologs, which allows for the inference of functional equivalence of genes. A further aspect of the homologs encoded by DNA useful in the transgenic plants of the invention are those proteins that differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.
Other functional homolog proteins differ in one or more amino acids from those of a trait-improving protein disclosed herein as the result of one or more of known conservative amino acid substitutions, for example, valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native sequence can be selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native protein or polypeptide can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side 30 chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alaninevaline, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the disclosure includes proteins that differ in one or more amino acids from those of a described protein sequence as the result of deletion or insertion of one or more amino acids in a native sequence.
“Pfam” is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, for example, Pfam version 27.0 (March 2013) contains alignments and models for 14381 protein families and uses UniProtKB as its reference sequence databases. See The Pfam protein families database: M. Punta, P. C. Coggill, R. Y. Eberhardt, J. Mistry, J. Tate, C. Boursnell, N. Pang, K. Forslund, G. Ceric, J. Clements, A. Heger, L. Holm, E. L. L. Sonnhammer, S. R. Eddy, A. Bateman, R. D. Finn Nucleic Acids Research (2012) Database Issue 40:D290-D30, which is incorporated herein by reference in its entirety. The Pfam database is currently maintained and updated by the Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the protein family alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low.
Protein domains are identified by querying the amino acid sequence of a protein against Hidden Markov Models which characterize protein family domains (“Pfam domains”) using HMMER software, which is available from the Pfam Consortium and is available through the Howard Hughes Medical Institute's Janelia Farm website (http://hmmer.janelia.org/software). A protein domain meeting the gathering cutoff for the alignment of a particular Pfam domain is considered to contain the Pfam domain.
A “Pfam domain module” is a representation of Pfam domains in a protein, in order from N terminus to C terminus. In a Pfam domain module individual Pfam domains are separated by double colons “::”. The order and copy number of the Pfam domains from N to C terminus are attributes of a Pfam domain module. Although the copy number of repetitive domains is important, varying copy number often enables a similar function. Thus, a Pfam domain module with multiple copies of a domain should define an equivalent Pfam domain module with variance in the number of multiple copies. A Pfam domain module is not specific for distance between adjacent domains, but contemplates natural distances and variations in distance that provide equivalent function. The Pfam database contains both narrowly- and broadly-defined domains, leading to identification of overlapping domains on some proteins. A Pfam domain module is characterized by non-overlapping domains. Where there is overlap, the domain having a function that is more closely associated with the function of the protein (based on the E value of the Pfam match) is selected.
Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins with the same Pfam domain module are identified by querying the amino acid sequence of protein encoded by the candidate DNA against the Hidden Markov Models which characterizes the Pfam domains using HMMER software. Candidate proteins meeting the same Pfam domain module are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this disclosure. Hidden Markov Model databases for the use with HMMER software in identifying DNA expressing protein with a common Pfam domain module for recombinant DNA in the plant cells of this disclosure are included in the computer program listing in this application.
In general, the term “variant” refers to molecules with some differences, generated synthetically or naturally, in their nucleotide or amino acid sequences as compared to a reference (native) polynucleotides or polypeptides, respectively. These differences include substitutions, insertions, deletions or any desired combinations of such changes in a native polynucleotide or amino acid sequence.
With regard to polynucleotide variants, differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and the latter nucleotide sequences may be silent (for example, the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide). Variant nucleotide sequences can encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similarly disclosed polynucleotide sequences. These variations can result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides.
As used herein “gene” or “gene sequence” refers to the partial or complete coding sequence of a gene, its complement, and its 5′ and/or 3′ untranslated regions. A gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter can be subjected to subsequent processing such as chemical modification or folding to obtain a functional protein or polypeptide. By way of example, a transcriptional regulator gene encodes a transcriptional regulator polypeptide, which can be functional or require processing to function as an initiator of transcription.
As used herein, the term “promoter” refers generally to a DNA molecule that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A promoter can be initially isolated from the 5′ untranslated region (5′ UTR) of a genomic copy of a gene. Alternately, promoters can be synthetically produced or manipulated DNA molecules. Promoters can also be chimeric, that is a promoter produced through the fusion of two or more heterologous DNA molecules. Plant promoters include promoter DNA obtained from plants, plant viruses, fungi and bacteria such as Agrobacterium and Bradyrhizobium bacteria.
Promoters which initiate transcription in all or most tissues of the plant are referred to as “constitutive” promoters. Promoters which initiate transcription during certain periods or stages of development are referred to as “developmental” promoters. Promoters whose expression is enhanced in certain tissues of the plant relative to other plant tissues are referred to as “tissue enhanced” or “tissue preferred” promoters. Promoters which express within a specific tissue of the plant, with little or no expression in other plant tissues are referred to as “tissue specific” promoters. A promoter that expresses in a certain cell type of the plant, for example a microspore mother cell, is referred to as a “cell type specific” promoter. An “inducible” promoter is a promoter in which transcription is initiated in response to an environmental stimulus such as cold, drought or light; or other stimuli such as wounding or chemical application. Many physiological and biochemical processes in plants exhibit endogenous rhythms with a period of about 24 hours. A “diurnal promoter” is a promoter which exhibits altered expression profiles under the control of a circadian oscillator. Diurnal regulation is subject to environmental inputs such as light and temperature and coordination by the circadian clock.
Sufficient expression in plant seed tissues is desired to affect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin as disclosed in U.S. Pat. No. 5,420,034, maize L3 oleosin as disclosed in U.S. Pat. No. 6,433,252, zein Z27 as disclosed by Russell et al. (1997) Transgenic Res. 6(2):157-166, globulin 1 as disclosed by Belanger et al (1991) Genetics 129:863-872, glutelin 1 as disclosed by Russell (1997) supra, and peroxiredoxin antioxidant (Per1) as disclosed by Stacy et al. (1996) Plant Mol Biol. 31 (6):1205-1216.
As used herein, the term “leader” refers to a DNA molecule isolated from the untranslated 5′ region (5′ UTR) of a genomic copy of a gene and is defined generally as a nucleotide segment between the transcription start site (TSS) and the protein coding sequence start site. Alternately, leaders can be synthetically produced or manipulated DNA elements. A leader can be used as a 5′ regulatory element for modulating expression of an operably linked transcribable polynucleotide molecule. As used herein, the term “intron” refers to a DNA molecule that can be isolated or identified from the genomic copy of a gene and can be defined generally as a region spliced out during mRNA processing prior to translation. Alternately, an intron can be a synthetically produced or manipulated DNA element. An intron can contain enhancer elements that effect the transcription of operably linked genes. An intron can be used as a regulatory element for modulating expression of an operably linked transcribable polynucleotide molecule. A DNA construct can comprise an intron, and the intron may or may not be with respect to the transcribable polynucleotide molecule.
As used herein, the term “enhancer” or “enhancer element” refers to a cis-acting transcriptional regulatory element, a.k.a. cis-element, which confers an aspect of the overall expression pattern, but is usually insufficient alone to drive transcription, of an operably linked polynucleotide. Unlike promoters, enhancer elements do not usually include a transcription start site (TSS) or TATA box or equivalent sequence. A promoter can naturally comprise one or more enhancer elements that affect the transcription of an operably linked polynucleotide. An isolated enhancer element can also be fused to a promoter to produce a chimeric promoter cis-element, which confers an aspect of the overall modulation of gene expression. A promoter or promoter fragment can comprise one or more enhancer elements that effect the transcription of operably linked genes. Many promoter enhancer elements are believed to bind DNA-binding proteins and/or affect DNA topology, producing local conformations that selectively allow or restrict access of RNA polymerase to the DNA template or that facilitate selective opening of the double helix at the site of transcriptional initiation. An enhancer element can function to bind transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors can interact with different affinities with more than one enhancer domain.
Expression cassettes of this disclosure can include a “transit peptide” or “targeting peptide” or “signal peptide” molecule located either 5′ or 3′ to or within the gene(s). These terms generally refer to peptide molecules that when linked to a protein of interest directs the protein to a particular tissue, cell, subcellular location, or cell organelle. Examples include, but are not limited to, chloroplast transit peptides (CTPs), chloroplast targeting peptides, mitochondrial targeting peptides, nuclear targeting signals, nuclear exporting signals, vacuolar targeting peptides, and vacuolar sorting peptides. For description of the use of chloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925. For description of the transit peptide region of an Arabidopsis EPSPS gene in the present disclosure, see Klee, H. J. Et al (MGG (1987) 210:437-442. Expression cassettes of this disclosure can also include an intron or introns. Expression cassettes of this disclosure can contain a DNA near the 3′ end of the cassette that acts as a signal to terminate transcription from a heterologous nucleic acid and that directs polyadenylation of the resultant mRNA. These are commonly referred to as “3′-untranslated regions” or “3′-non-coding sequences” or “3′-UTRs”. The “3′ non-translated sequences” means DNA sequences located downstream of a structural nucleotide sequence and include sequences encoding polyadenylation and other regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal functions in plants to cause the addition of polyadenylate nucleotides to the 3′ end of the mRNA precursor. The polyadenylation signal can be derived from a natural gene, from a variety of plant genes, or from T-DNA. An example of a polyadenylation sequence is the nopaline synthase 3′ sequence (nos 3′; Fraley et al., Proc. Natl. Acad. Sci. USA 80: 4803-4807, 1983). The use of different 3′ non-translated sequences is exemplified by Ingelbrecht et al., Plant Cell 1:671-680, 1989.
Expression cassettes of this disclosure can also contain one or more genes that encode selectable markers and confer resistance to a selective agent such as an antibiotic or an herbicide. A number of selectable marker genes are known in the art and can be used in the present disclosure: selectable marker genes conferring tolerance to antibiotics like kanamycin and paromomycin (nptII), hygromycin B (aph IV), spectinomycin (aadA), U.S. Patent Publication 2009/0138985A1 and gentamycin (aac3 and aacC4) or tolerance to herbicides like glyphosate (for example, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), U.S. Pat. No. 5,627,061; U.S. Pat. No. 5,633,435; U.S. Pat. No. 6,040,497; U.S. Pat. No. 5,094,945), sulfonyl herbicides (for example, acetohydroxyacid synthase or acetolactate synthase conferring tolerance to acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide (U.S. Pat. Nos. 6,225,105; 5,767,366; 4,761,373; 5,633,437; 6,613,963; 5,013,659; 5,141,870; 5,378,824; 5,605,011)), bialaphos or phosphinothricin or derivatives (e. g., phosphinothricin acetyltransferase (bar) tolerance to phosphinothricin or glufosinate (U.S. Pat. Nos. 5,646,024; 5,561,236; 5,276,268; 5,637,489; 5,273,894); dicamba (dicamba monooxygenase, Patent Application Publications US2003/0115626A1), or sethoxydim (modified acetyl-coenzyme A carboxylase for conferring tolerance to cyclohexanedione (sethoxydim)), and aryloxyphenoxypropionate (haloxyfop, U.S. Pat. No. 6,414,222).
Transformation vectors of this disclosure can contain one or more “expression cassettes”, each comprising a native or non-native plant promoter operably linked to a polynucleotide sequence of interest, which is operably linked to a 3′ UTR termination signal, for expression in an appropriate host cell. It also typically comprises sequences required for proper translation of the polynucleotide or transgene. As used herein, the term “transgene” refers to a polynucleotide molecule artificially incorporated into a host cell's genome. Such a transgene can be heterologous to the host cell. The term “transgenic plant” refers to a plant comprising such a transgene. The coding region usually codes for a protein of interest but can also code for a functional RNA of interest, for example an antisense RNA, a nontranslated RNA, in the sense or antisense direction, a microRNA, a noncoding RNA, or a synthetic RNA used in either suppression or over expression of target gene sequences. The expression cassette comprising the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. As used herein the term “chimeric” refers to a DNA molecule that is created from two or more genetically diverse sources, for example a first molecule from one gene or organism and a second molecule from another gene or organism.
Recombinant DNA constructs in this disclosure generally include a 3′ element that typically contains a polyadenylation signal and site. Known 3′ elements include those from Agrobacterium tumefaciens genes such as nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, for example disclosed in U.S. Pat. No. 6,090,627; 3′ elements from plant genes such as wheat (Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene, a rice lactate dehydrogenase gene and a rice beta-tubulin gene, all of which are disclosed in US Patent Application Publication 2002/0192813 A1; and the pea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3′), and 3′ elements from the genes within the host plant.
As used herein “operably linked” means the association of two or more DNA fragments in a recombinant DNA molecule so that the function of one, for example, protein-encoding DNA, is controlled by the other, for example, a promoter.
Transgenic plants can comprise a stack of one or more polynucleotides disclosed herein resulting in the production of multiple polypeptide sequences. Transgenic plants comprising stacks of polynucleotides can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, crossing individual transgenic lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a first gene disclosed herein with a second gene, and co-transformation of genes into a single plant cell. Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors.
Transgenic plants comprising or derived from plant cells of this disclosure transformed with recombinant DNA can be further enhanced with stacked traits, for example, a crop plant having an enhanced trait resulting from expression of DNA disclosed herein in combination with herbicide and/or pest resistance traits. For example, genes of the current disclosure can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects, or improved quality traits such as improved nutritional value. Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present disclosure can be applied include, but are not limited to, glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil and norflurazon herbicides. Polynucleotide molecules encoding proteins involved in herbicide tolerance known in the art and include, but are not limited to, a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S. Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 for imparting glyphosate tolerance; polynucleotide molecules encoding a glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyl transferase (GAT) disclosed in US Patent Application Publication 2003/0083480 A1 also for imparting glyphosate tolerance; dicamba monooxygenase disclosed in US Patent Application Publication 2003/0135879 A1 for imparting dicamba tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance; a polynucleotide molecule encoding phytoene desaturase (crtI) described in Misawa et al, (1993) Plant J. 4:833-840 and in Misawa et al, (1994) Plant J. 6:481-489 for norflurazon tolerance; a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for imparting tolerance to sulfonylurea herbicides; polynucleotide molecules known as bar genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for imparting glufosinate and bialaphos tolerance; polynucleotide molecules disclosed in US Patent Application Publication 2003/010609 A1 for imparting N-amino methyl phosphonic acid tolerance; polynucleotide molecules disclosed in U.S. Pat. No. 6,107,549 for imparting pyridine herbicide resistance; molecules and methods for imparting tolerance to multiple herbicides such as glyphosate, atrazine, ALS inhibitors, isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No. 6,376,754 and US Patent Application Publication 2002/0112260. Molecules and methods for imparting insect/nematode/virus resistance are disclosed in U.S. Pat. Nos. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and US Patent Application Publication 2003/0150017 A1.

Plant Cell Transformation Methods

Numerous methods for transforming chromosomes in a plant cell with recombinant DNA are known in the art and are used in methods of producing a transgenic plant cell and plant. Two effective methods for such transformation are Agrobacterium-mediated transformation and microprojectile bombardment-mediated transformation. Microprojectile bombardment methods are illustrated in U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn); U.S. Pat. No. 6,153,812 (wheat) and U.S. Pat. No. 6,365,807 (rice). Agrobacterium-mediated transformation methods are described in U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,463,174 (canola); U.S. Pat. No. 5,591,616 (corn); U.S. Pat. No. 5,846,797 (cotton); U.S. Pat. No. 6,384,301 (soybean), U.S. Pat. No. 7,026,528 (wheat) and U.S. Pat. No. 6,329,571 (rice), US Patent Application Publication 2004/0087030 A1 (cotton), and US Patent Application Publication 2001/0042257 A1 (sugar beet), all of which are incorporated herein by reference in their entirety. Transformation of plant material is practiced in tissue culture on nutrient media, for example a mixture of nutrients that allow cells to grow in vitro. Recipient cell targets include, but are not limited to, meristem cells, shoot tips, hypocotyls, calli, immature or mature embryos, and gametic cells such as microspores, pollen, sperm and egg cells. Callus can be initiated from tissue sources including, but not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants.
In addition to direct transformation of a plant material with a recombinant DNA molecule, a transgenic plant can be prepared by crossing a first plant comprising a recombinant DNA with a second plant lacking the recombinant DNA. For example, recombinant DNA can be introduced into a first plant line that is amenable to transformation, which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing an enhanced trait, for example, enhanced yield, can be crossed with a transgenic plant line having other recombinant DNA that confers another trait, for example herbicide resistance or pest resistance, to produce progeny plants having recombinant DNA that confers both traits. Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA, for example, marker identification by analysis for recombinant DNA or, in the case where a selectable marker is linked to the recombinant, by application of the selecting agent such as a herbicide for use with a herbicide tolerance marker, or by selection for the enhanced trait. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as the original transgenic parental line but for the recombinant DNA of the other transgenic parental line.
For transformation, DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or an herbicide. Any of the herbicides to which plants of this disclosure can be resistant is an agent for selective markers. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aph IV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Examples of such selectable markers are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Markers which provide an ability to visually screen transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.
Plant cells that survive exposure to a selective agent, or plant cells that have been scored positive in a screening assay, may be cultured in vitro to regenerate plantlets. Developing plantlets regenerated from transformed plant cells can be transferred to plant growth mix, and hardened off, for example, in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻²s⁻¹of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue, and plant species. Plants can be pollinated using conventional plant breeding methods known to those of skill in the art to produce seeds, for example self-pollination is commonly used with transgenic corn. The regenerated transformed plant or its progeny seed or plants can be tested for expression of the recombinant DNA and selected for the presence of an enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from transgenic plant cells having a transgenic nucleus of this disclosure are grown to generate transgenic plants having an enhanced trait as compared to a control plant, and produce transgenic seed and haploid pollen of this disclosure. Such plants with enhanced traits are identified by selection of transformed plants or progeny seed for the enhanced trait. For efficiency a selection method is designed to evaluate multiple transgenic plants (events) comprising the recombinant DNA, for example multiple plants from 2 to 20 or more transgenic events. Transgenic plants grown from transgenic seeds provided herein demonstrate improved agronomic traits that contribute to increased yield or other traits that provide increased plant value, including, for example, improved seed quality. Of particular interest are plants having increased water use efficiency or drought tolerance, enhanced high temperature or cold tolerance, increased yield, and increased nitrogen use efficiency.
Table 1 provides a list of protein-encoding DNA (“genes”) as recombinant DNA for production of transgenic plants with enhanced traits, the elements of Table 1 are described by reference to:
PEP SEQ ID NO” which identifies an amino acid sequence.
“.“NUC SEQ ID NO” which identifies a DNA sequence.
“Gene ID” which refers to an arbitrary identifier.
“Protein Name” which is a common name for the protein encoded by the recombinant DNA.

TABLE 1

Protein sequences

NUC SEQ	PEP SEQ
ID NO	ID NO	Gene ID	Protein Name

1	2	TRDX3-1	Translationally controlled tumor protein homolog
3	4	TRDX3-2	Phytochrome B1 containing Histidine kinase-like
			ATPases
5	6	TRDX3-3	unknown protein
7	8	TRDX3-4	monogalactosyl-diacylglycerol (MGDG) synthase
			type C protein
9	10	TRDX3-5	putative GluRS (glutamate-tRNA ligase)
11	12	TRDX3-6	prenylyltransferase-like protein
13	14	TRDX3-7	protein kinase family protein
15	16	TRDX3-8	PLC1 (Phosphoinositide phospholipase C 1)
17	18	TRDX3-9	Non-specific lipid-transfer protein-like protein
19	20	TRDX3-10	ATFIP1 (Factor Interacting with PolyA
			polymerase)
21	22	TRDX3-11	cysteine protease inhibitor
23	24	TRDX3-12	hypothetical protein containing Cenp-O
			kinetochore centromere component domain
25	26	TRDX3-13	TOM20-2 (Translocase Outer Membrane 20-2)
27	28	TRDX3-14	3-dehydroquinate dehydratase/shikimate
			dehydrogenase
29	30	TRDX3-15	unknown protein
31	32	TRDX3-16	SOUL family putative heme binding protein 1
33	34	TRDX3-17	Nob1 containing a predicted RNA-binding protein
			(contains KH domains) domain
35	36	TRDX3-18	PCD (Pterin-4a-carbinolamine dehydratases)
37	38	TRDX3-19	WEI8 (weak ethylene insensitive 8) gene encoding
			Trpytophan (Trp) aminotransferase
39	40	TRDX3-20	RTH3 (root hairless 3) containing domain of
			COBRA-like protein
41	42	TRDX3-21	RGT2 (Restores Glucose Transport 2)
43	44	TRDX3-22	a protein of unknown function containing U1 zinc
			finger domain (zf-U1)
45	46	TRDX3-23	RING-H2 finger protein ATL4E
47	48	TRDX3-24	radical SAM domain-containing protein
49	50	TRDX3-25	senescence-associated protein
51	52	TRDX3-26	ATPARP2 (POLY(ADP-RIBOSE)
			POLYMERASE 2
53	54	TRDX3-27	MT3a Metallothionein 3a
55	56	TRDX3-28	Cytochrome B5 reductase
57	58	TRDX3-29	CUL3 (Cullin 3) containing cullin family domain
115	116	TRDX3-37	corn phyt: 2

Table 2 provides a list of miRNA decoy elements as recombinant DNA for production of transgenic plants with enhanced traits. The elements of Table 2 are described by reference to:
“Decoy Element NUC SEQ ID NO:” which identifies a decoy element nucleotide sequence.
“Decoy Element miRNA binding site NUC SEQ ID NO:” which identifies the miRNA binding site of the decoy element.
“Decoy Element ID:” which is an arbitrary identifier.
“Target miRNA Name” which identifies a target miRNA for binding by the decoy.
“Target Protein Name” which identifies the target protein which bind the target miRNA.
“Target Protein NUC SEQ ID NO:” which identifies the nucleotide sequence encoding the target protein of the target miRNA
“Target Protein PEP SEQ ID NO:” which identifies the amino acid sequence of the target protein of the target miRNA

TABLE 2

Decoy Elements

		Decoy
		Element
		miRNA			Target	Target
	Decoy	binding			Protein	Protein
	Element	site			NUC	PEP
Decoy	(NUC)	(NUC)	Target	Target	SEQ	SEQ
Element	SEQ ID	SEQ ID	miRNA	Protein	ID	ID
ID	NO:	NO:	Name	Names	NO:	NO:

TRDX3-30	93	100	miR399	Phosphate
				transporter
TRDX3-31	94	101	miR172	APETALA2-	107	108
				like
				transcription
				factor
TRDX3-32	95	102	miR166	Revoluta	109	110
TRDX3-33	96	103	miR444	ANR1	111	112
				MADS-box
				protein
TRDX3-34	97	104	miR172e	APETALA2-
				like
				transcription
				factor
TRDX3-35	98	105	miR393	HOS1;
				E3 ligase
				SCF complex
				F-box protein
				bHLH
				transcription
				factor
TRDX3-36	99	106	miR397	diphenol	113	114
				oxidase

Selection Methods for Transgenic Plants with Enhanced Traits

Within a population of transgenic plants each regenerated from a plant cell with recombinant DNA many plants that survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Selection from the population is necessary to identify one or more transgenic plants with an enhanced trait. Transgenic plants having enhanced traits are selected from populations of plants regenerated or derived from plant cells transformed as described herein by evaluating the plants in a variety of assays to detect an enhanced trait, for example, increased water use efficiency or drought tolerance, enhanced high temperature or cold tolerance, increased yield, increased nitrogen use efficiency, enhanced seed composition such as enhanced seed protein and enhanced seed oil. These assays can take many forms including, but not limited to, direct screening for the trait in a greenhouse or field trial or by screening for a surrogate trait. Such analyses can be directed to detecting changes in the chemical composition, biomass, physiological property, or morphology of the plant. Changes in chemical compositions such as nutritional composition of grain can be detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in chemical compositions can also be detected by analysis of contents in leaves, such as chlorophyll or carotenoid contents. Changes in biomass characteristics can be evaluated on greenhouse or field grown plants and can include plant height, stem diameter, root and shoot dry weights, canopy size; and, for corn plants, ear length and diameter. Changes in physiological properties can be identified by evaluating responses to stress conditions, for example assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology can be measured by visual observation of tendency of a transformed plant to appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other selection properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green or delayed senescence, stalk lodging, root lodging, plant health, bareness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain can be evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
Assays for screening for a desired trait are readily designed by those practicing in the art. The following illustrates screening assays for corn traits using hybrid corn plants. The assays can be adapted for screening other plants such as canola, wheat, cotton and soybean either as hybrids or inbreds.
Transgenic corn plants having increased nitrogen use efficiency can be identified by screening transgenic plants in the field under the same and sufficient amount of nitrogen supply as compared to control plants, where such plants provide higher yield as compared to control plants. Transgenic corn plants having increased nitrogen use efficiency can also be identified by screening transgenic plants in the field under reduced amount of nitrogen supply as compared to control plants, where such plants provide the same or similar yield as compared to control plants.
Transgenic corn plants having increased yield are identified by screening using progenies of the transgenic plants over multiple locations for several years with plants grown under optimal production management practices and maximum weed and pest control. Selection methods can be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons, to statistically distinguish yield improvement from natural environmental effects.
Transgenic corn plants having increased water use efficiency or drought tolerance are identified by screening plants in an assay where water is withheld for a period to induce stress followed by watering to revive the plants. For example, a selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment.
Although the plant cells and methods of this disclosure can be applied to any plant cell, plant, seed or pollen, for example, any fruit, vegetable, grass, tree or ornamental plant, the various aspects of the disclosure are applied to corn, soybean, cotton, canola, rice, barley, oat, wheat, turf grass, alfalfa, sugar beet, sunflower, quinoa and sugar cane plants.

Example 1

Corn Transformation

This example illustrates transformation methods in producing a transgenic corn plant cell, seed, and plant having altered phenotypes as shown in Tables 4-6, or an enhanced trait, for example, increased water use efficiency as shown in Tables 15-16, increased yield as shown in Tables 11-12, and increased nitrogen use efficiency as shown in Tables 9-10.
For Agrobacterium-mediated transformation of corn embryo cells corn plants were grown in the greenhouse and ears were harvested when the embryos were 1.5 to 2.0 mm in length. Ears were surface-sterilized by spraying or soaking the ears in 80% ethanol, followed by air drying. Immature embryos were isolated from individual kernels on surface-sterilized ears. Shortly after excision, immature maize embryos were inoculated with overnight grown Agrobacterium cells, and incubated at room temperature with Agrobacterium for 5-20 minutes. Inoculated immature embryos were then co-cultured with Agrobacterium for 1 to 3 days at 23° C. in the dark. Co-cultured embryos were transferred to selection media and cultured for approximately two weeks to allow embryogenic callus to develop. Embryogenic calli were transferred to culture medium containing glyphosate and subcultured at about two week intervals. Transformed plant cells were recovered 6 to 8 weeks after initiation of selection.
For Agrobacterium-mediated transformation of maize callus immature embryos are cultured for approximately 8-21 days after excision to allow callus to develop. Callus is then incubated for about 30 minutes at room temperature with the Agrobacterium suspension, followed by removal of the liquid by aspiration. The callus and Agrobacterium are co-cultured without selection for 3-6 days followed by selection on paromomycin for approximately 6 weeks, with biweekly transfers to fresh media. Paromomycin resistant calli are identified about 6-8 weeks after initiation of selection.
To regenerate transgenic corn plants individual transgenic calli resulting from transformation and selection were placed on media to initiate shoot and root development into plantlets. Plantlets were transferred to potting soil for initial growth in a growth chamber at 26° C. followed by a mist bench before transplanting to 5 inch pots where plants were grown to maturity. The regenerated plants were self-fertilized and seeds were harvested for use in one or more methods to select seeds, seedlings or progeny second generation transgenic plants (R2 plants) or hybrids, for example, by selecting transgenic plants exhibiting an enhanced trait as compared to a control plant.
The above process can be repeated to produce multiple events of transgenic corn plants from cells that were transformed with recombinant DNA from the genes identified in Table 1 or with recombinant DNA from Table 2 that is transcribed into a non-coding miRNA. Progeny transgenic plants and seeds of the transformed plants were screened for the presence and single copy of the inserted gene, and for increased water use efficiency, increased yield, increased nitrogen use efficiency, and altered phenotypes as shown in Tables 4-6. From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 or Table 2, the event(s) that showed increased yield, increased water use efficiency, increased nitrogen use efficiency, and altered phenotypes was (were) identified.

Example 2

Soybean Transformation

This example illustrates plant transformation in producing a transgenic soybean plant cell, seed, and plant having altered phenotypes as shown in Tables 7-8, or an enhanced trait, for example, increased water use efficiency or drought tolerance and increased yield as shown in Tables 13-14.
For Agrobacterium mediated transformation, soybean seeds were imbibed overnight and the meristem explants excised. Soybean explants were mixed with induced Agrobacterium cells containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette no later than 14 hours from the time of initiation of seed imbibition, and wounded using sonication. Following wounding, explants were placed in co-culture for 2-5 days at which point they were transferred to selection media to allow selection and growth of transgenic shoots. Resistant shoots were harvested in approximately 6-8 weeks and placed into selective rooting media for 2-3 weeks. Shoots producing roots were transferred to the greenhouse and potted in soil. Shoots that remained healthy on selection, but did not produce roots were transferred to non-selective rooting media for an additional two weeks. Roots from any shoots that produced roots off selection were tested for expression of the plant selectable marker before they were transferred to the greenhouse and potted in soil.
The above process can be repeated to produce multiple events of transgenic soybean plants from cells that were transformed with recombinant DNA from the genes identified in Table 1 or recombinant DNA transcribed into a miRNA decoy identified in Table 2. Progeny transgenic plants and seed of the transformed plant cells were screened for the presence and single copy of the inserted gene, and for increased water use efficiency, increased yield, increased nitrogen use efficiency, and altered phenotypes as shown in Tables 6-7.

Example 3

Identification of Altered Phenotypes in Automated Greenhouse

This example illustrates screening and identification of transgenic plants for altered phenotypes in an automated greenhouse (AGH). The apparatus and the methods for automated phenotypic screening of plants are disclosed in US Patent publication No. US20110135161 (filed on Nov. 10, 2010), which is incorporated by reference herein in its entirety.

Screening and Identification of Transgenic Corn Plants for Altered Phenotypes

Corn plants were tested in 3 screens in AGH under different conditions including non-stress, nitrogen deficit and water deficit stress conditions. All screens began with a non-stress condition during day 0-5 germination phase, after which the plants were grown for 22 days under screen specific conditions as shown in Table 3.

TABLE 3

Description of the 3 AGH screens for corn plants.

			Screen specific
		Germination phase	phase
Screen	Description	(5 days)	(22 days)

Non-stress	well watered	55% VWC	55% VWC
	sufficient nitrogen	water	8 mM nitrogen
Water deficit	limited watered	55% VWC	30% VWC
	sufficient nitrogen	water	8 mM nitrogen
Nitrogen deficit	well watered	55% VWC	55% VWC
	low nitrogen	water	2 mM nitrogen

Water deficit is defined as a specific Volumetric Water Content (VWC) that is lower than the VWC of non-stress plant. For example, a non-stressed plant might be maintained at 55% VWC and the VWC for a water-deficit assay might be defined around 30% VWC as shown in Table 3. Data were collected using visible light and hyperspectral imaging as well as direct measurement of pot weight and amount of water and nutrient applied to individual plants on a daily basis.
Nitrogen deficit is defined in part as a specific mM concentration of nitrogen that is lower than the nitrogen concentration of non-stress plants. For example, a non-stress plant might be maintained at 8 mM nitrogen while the nitrogen concentration applied in a nitrogen-deficit assay might be maintained at a concentration of 2 mM.
Eight parameters were measured for each screen. The visible light color imaging based measurements are: biomass, canopy area and plant height. Biomass (B) is defined as estimated shoot fresh weight (g) of the plant obtained from images acquired from multiple angles of view. Canopy Area (Can) is defined as area of leaf as seen in top-down image (mm²). Plant Height (H) refers to the distance from the top of the pot to the highest point of the plant derived from side image (mm). Anthocyanin score, chlorophyll score and water content score are hyperspectral imaging based parameters. Anthocyanin Score (An) is an estimate of anthocyanin in the leaf canopy obtained from a top-down hyperspectral image. Chlorophyll Score (Chl) is a measurement of chlorophyll in the leaf canopy obtained from a top-down hyperspectral image. Water Content Score (WC) is a measurement of water in the leaf canopy obtained from a top-down hyperspectral image. Water Use Efficiency (WUE) is derived from the grams of plant biomass per liter of water added. Water Applied (WA) is a direct measurement of water added to a pot (pot with no hole) during the course of an experiment.
These physiological screen runs were set up so that tested transgenic lines were compared to a control line. The collected data were analyzed against the control using % delta and certain p-value cutoff. Tables 4-6 are summaries of transgenic corn plants comprising the disclosed recombinant DNA molecules with altered phenotypes under non stress, nitrogen deficit, and water deficit conditions, respectively.
“+” denotes an increase in the tested parameter at p≦0.1; whereas “−” denotes a decrease in the tested parameter at p≦0.1. The numbers in parenthesis show penetrance of the altered phenotypes, where the denominators represent total number of transgenic events tested for a given parameter in a specific screen, and the numerators represent the number of events showing a particular altered phenotype. For example, 5 transgenic plants were screened for water use efficiency score in the non-stress screen for TRDX3-3 and 1 of the 5 tested showed increased water use efficiency at p≦0.1.

TABLE 4

Summary of transgenic corn plants with altered phenotypes in AGH
non-stress screens

Non-Stress

Gene_ID	An	B	Can	Chl	H	WA	WC	WUE

TRDX3-3	—	+1/5	−1/5	—	—	+1/5	—	+1/5
TRDX3-4	—	−3/5	−4/5	—	−3/5	−5/5	—	−1/5
TRDX3-6	−4/5	−4/5	−1/5	−1/5	−3/5	−3/5	−3/5	−2/5
TRDX3-7	—	−2/5	−1/5	+1/5	−1/5	−4/5	—	−1/5
TRDX3-12	—	−1/5	—	+1/5	−1/5	—	—	−1/5
TRDX3-13	—	+1/5	—	—	—	−1/5	+1/5	+2/5
TRDX3-16	—	−1/5	—	—	—	—	—	−1/5
TRDX3-20	−1/5	—	−2/5	—	—	−1/5	—	—
TRDX3-21	−1/4	—	+2/4	—	—	+2/4	—	—
TRDX3-25	—	+1/5	—	—	—	—	—	+1/5
TRDX3-28	—	+2/5	+1/5	+1/5	+1/5	+4/5	+1/5	+1/5
TRDX3-29	—	−1/5	−2/5	+1/5	−3/5	−1/5	+1/5	−1/5
TRDX3-35	—	−1/5	—	—	—	−1/5	—	−2/5

TABLE 5

Summary of transgenic corn plants with altered phenotypes in AGH
nitrogen-deficit screens

Nitrogen Deficit

Gene_ID	An	B	Can	Chl	H	WA	WC	WUE

TRDX3-3	—	+1/5	+1/5	+1/5	−1/5	+1/5	—	—
TRDX3-4	—	−2/5	−1/5	−1/5	+1/5	−3/5	—	—
TRDX3-6	—	—	−1/5	—	—	—	—	+2/5
TRDX3-7	−2/5	−1/5	−1/5	—	+1/5	−4/5	—	—
TRDX3-12	−1/5	−2/5	—	—	−1/5	−4/5	—	−1/5
TRDX3-13	−1/5	−1/5	—	+1/5	—	—	—	−1/5
TRDX3-16	+1/5	−3/5	−1/5	+3/5	−1/5	−4/5	−4/5	—
TRDX3-20	—	−2/5	−2/5	+1/5	−1/5	−1/5	—	−1/5
TRDX3-21	−1/5	+1/5	+2/5	+1/5	−1/5	—	+1/5	+1/5
TRDX3-24	−1/5	—	+3/5	—	—	+3/5	—	—
TRDX3-28	−1/5	+4/5	+1/5	—	+1/5	−1/5	+1/5	+1/5
TRDX3-29	—	−1/1	−1/1	—	−1/1	—	—	−1/1
TRDX3-35	+1/5	−2/5	−2/5	+3/5	−2/5	−2/5	—	−2/5

TABLE 6

Summary of transgenic corn plants with altered phenotypes in AGH
water-deficit screens

Water Deficit

Gene ID	An	B	Can	Chl	H	WA	WC	WUE

TRDX3-3	—	+1/5	—	—	−2/5	−1/5	+2/5	−1/5
TRDX3-4	—	+2/5	+1/5	+1/5	+1/5	+3/5	+1/5	—
TRDX3-6	−2/5	—	—	—	−1/5	—	—	—
TRDX3-7	+2/5	−3/5	−4/5	−2/5	−3/5	−4/5	−2/5	−2/5
TRDX3-12	—	—	−1/5	−1/5	−1/5	+4/5	—	—
TRDX3-13	—	+2/5	+1/5	−1/5	+2/5	+4/5	—	+1/5
TRDX3-16	—	+2/5	—	+3/5	—	+3/5	—	—
TRDX3-20	—	—	−1/5	—	—	−1/5	—	−1/5
TRDX3-21	−1/5	—	+1/5	+1/5	—	—	−1/5	−1/5
TRDX3-24	—	—	—	−2/5	—	+1/5	−1/5	—
TRDX3-28	+2/5	+1/5	+2/5	+1/5	+1/5	+2/5	—	+1/5
TRDX3-35	—	−2/5	—	−2/5	—	−1/5	−2/5	−2/5

Screening and Identification of Transgenic Soybean Plants for Altered Phenotypes

Soybean plants were tested in 2 screens in AGH under non-stress and water deficit stress conditions. For non-stress screen, the plants were kept under constant VWC of 55% throughout the screen length of 27 days. For water deficit screen, the VWC was kept at 55% for the first 12 days after sowing, followed by gradual dry down at a rate of 0.025 VWC per day, followed by water recovery to 55% VWC at 25 days after sowing.
Water deficit is defined as a specific Volumetric Water Content (VWC) that is lower than the VWC of non-stress plant. For example, a non-stressed plant might be maintained at 55% VWC and water-deficit assay might be defined around 30% VWC as shown in Table 3. Data were collected using visible light and hyperspectral imaging as well as direct measurement of pot weight and amount of water and nutrient applied to individual plants on a daily basis.
Eight parameters were measured for each screen. The visible light color imaging based measurements are: biomass, canopy area and plant height. Biomass (B) is defined as estimated shoot fresh weight (g) of the plant obtained from images acquired from multiple angles of view. Canopy Area (Can) is defined as area of leaf as seen in top-down image (mm²). Plant Height (H) refers to the distance from the top of the pot to the highest point of the plant derived from side image (mm).—Chlorophyll score—is a hyperspectral imaging based parameter. Chlorophyll Score (Chl) is a measurement of chlorophyll in the leaf canopy obtained from a top-down hyperspectral image. Water Use Efficiency (WUE) is derived from the grams of plant biomass per liter of water added. Water Applied (WA) is a direct measurement of water added to a pot (pot with no hole) during the course of an experiment.
These physiological screen runs were set up so that tested transgenic lines were compared to a control line. The collected data were analyzed against the control using % delta and/or certain p-value cutoff. Tables 7-8 are summaries of transgenic soybean plants comprising the disclosed recombinant DNA molecules with altered phenotypes.
“+” denotes an increase in the tested parameter at p≦0.1; whereas “−” denotes a decrease in the tested parameter at p≦0.1. The numbers in parenthesis show penetrance of the altered phenotypes, where the denominators represent total number of transgenic plants tested for a given parameter in a specific screen, and the numerators represent the number of transgenic plants showing a particular phenotype. For example, 5 transgenic plants were screened for biomass in the non-stress screen for TRDX3-26. Of the 5 tested, 2 showed a decrease in biomass at p≦0.1.

TABLE 7

Summary of transgenic soybean plants with altered phenotypes in AGH
non-stress screens

Non-Stress

Gene_ID	An	B	Can	Chl	H	WA	WC	WUE

TRDX3-26	—	−2/5	−3/5	−2/5	−2/5	−2/5	—	−1/5
TRDX3-22	—	+3/5	+1/5	+2/5	+3/5	+2/5	—	+3/5
TRDX3-27	—	—	—	+1/5	—	−1/5	—	—

TABLE 8

Summary of transgenic soybean plants with altered phenotypes in AGH
water deficit screens

Water Deficit

Gene_ID	An	B	Can	Chl	H	WA	WC	WUE

TRDX3-26	—	—	—	—	−1/5	—	—	—
TRDX3-22	—	+1/5	+1/5	—	—	−1/5	—	+1/5
TRDX3-27	—	—	—	—	+1/5	—	—	—

Example 4

Phenotypic Evaluation of Transgenic Corn Plants for Increased Nitrogen Use Efficiency

Corn Nitrogen field efficacy trials were conducted to identify genes and miRNA decoy elements that can improve nitrogen use efficiency under nitrogen limiting conditions leading to increased yield performance as compared to non transgenic controls. A yield increase in corn can be manifested as one or more of the following: an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, fresh or dry ear length/diameter/biomass (weight), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. For the Nitrogen field trial results shown in Table 9, each field was planted under nitrogen limiting condition (60 lbs/acre) and the corn ear weight or yield was compared to control plants to measure the yield increases.
Table 9 provides a list of protein encoding DNA or polynucleotide sequences (“genes”) for producing transgenic corn plant with increased nitrogen use efficiency as compared to a control plant. Polynucleotide sequences in constructs with at least one event showing significant yield or ear weight increase across multiple locations at p≦0.2 are included. The elements of Table 9 are described by reference to:
“SEQ ID NO: polynucleotide” which identifies a nucleotide sequence.
“SEQ ID NO: polypeptide” which identifies an amino acid sequence.
“Gene ID” which refers to an arbitrary identifier.
“NUE results” which refers to the sequence in a construct with at least one event showing significant yield increase at p≦0.2 across locations. The first number refers to the number of events with significant yield or ear weight increase, whereas the second number refers to the total number of events tested for each sequence in the construct. The numbers are listed for each construct separately when more than one construct was used in the trials.

TABLE 9

Recombinant DNA for increased nitrogen use efficiency in corn

SEQ ID NO:	SEQ ID NO:		NUE
Polynucleotide	Polypeptide	Gene ID	Results

5	6	TRDX3-3	2/12
7	8	TRDX3-4	3/16
11	12	TRDX3-6	2/13
13	14	TRDX3-7	Construct 1: 4/14
			Construct 2: 1/7
15	16	TRDX3-8	1/8
17	18	TRDX3-9	1/13
25	26	TRDX3-13	1/5
27	28	TRDX3-14	3/13
33	34	TRDX3-17	2/13
35	36	TRDX3-18	3/14
37	38	TRDX3-19	4/13
45	46	TRDX3-23	2/13
47	48	TRDX3-24	1/8
49	50	TRDX3-25	5/20

Table 10 provides a list of miRNA decoy elements provided as recombinant DNA for production of transgenic corn plants with increased nitrogen use efficiency. The elements of Table 10 are described by reference to:
“Decoy Element (NUC) SEQ ID NO:” which identifies a decoy element nucleotide sequence from.
“Decoy Element ID:” which is an arbitrary identifier.
“Target miRNA Name” which identifies a target miRNA for binding by the decoy.
“Protein Name (PEP)” which identifies the amino acid sequence of a protein which binds miR166.
“NUE results” which refers to the sequence in a construct with at least one event showing significant yield increase at p≦0.2 across locations. The first number refers to the number of events with significant yield or ear weight increase, whereas the second number refers to the total number of events tested for each sequence in the construct.

TABLE 10

Recombinant DNA for miRNA decoy elements for
increased nitrogen use efficiency in corn

Decoy
Element				Protein	Broad
(NUC)	Decoy	Target		(PEP)	Acre
SEQ	Element	miRNA	Protein	SEQ	Yield
ID NO	ID	Name	Name	ID NO	Results

95	TRDX3-32	miR166	Revoluta	110	2/16

Example 5

Phenotypic Evaluation of Transgenic Plants for Increased Yield

This example illustrates selection and identification of transgenic plants for increased yield in both dicotyledonous and monocotyledonous plants with primary examples presented for corn and soybean in Tables 11-12 and 13-14 respectively. Polynucleotide sequences in constructs with at least one event that resulted in significant yield increase across locations at p≦0.2 are included.
Selection of Transgenic Plants with Enhanced Agronomic Trait(s): Increased Yield
Effective selection of increased and/or enhanced yielding transgenic plants uses hybrid progenies of the transgenic plants for corn, cotton, and canola, or inbred progenies of transgenic plants for soybean plants plant such as corn, cotton, canola, or inbred plant such as soy, canola and cotton over multiple locations with plants grown under optimal production management practices. An exemplary target for improved yield is a 2% to 10% increase in yield as compared to yield produced by plants grown from seed of a control plant. Selection methods can be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons, to statistically distinguish yield improvement from natural environmental effects.

Increased Yield in Corn

Table 11 provides a list of protein encoding DNA or polynucleotide sequences (“genes”) in the production of transgenic corn plants with increased yield as compared to a control plant. The elements of Table 11 are described by reference to:
“Gene (NUC) SEQ ID NO:” which identifies a nucleotide sequence.
“Gene (PEP) SEQ ID NO:” polypeptide” which identifies an amino acid sequence.
“Gene identifier” which refers to an arbitrary identifier.
“Broad acre yield results” refers to the sequence in a construct with at least one event showing significant yield increase at p≦0.2 across locations. The first number refers to the number of events with significant yield increase, whereas the second number refers to the total number of events tested for each sequence in a construct.

TABLE 11

Recombinant DNA for increased yield in corn

			Broad
Gene (NUC)	Gene (PEP)		Acre Yield
SEQ ID NO:	SEQ ID NO:	Gene ID	Results

1	2	TRDX3-1	1/5
3	4	TRDX3-2	1/8
9	10	TRDX3-5	1/13
21	22	TRDX3-11	2/16
25	26	TRDX3-13	1/6
29	30	TRDX3-15	5/16
31	32	TRDX3-16	5/30
35	36	TRDX3-18	1/6
41	42	TRDX3-21	2/20
55	56	TRDX3-28	2/22
57	58	TRDX3-29	1/8

Table 12 provides a list of miRNA decoy elements provided as recombinant DNA for production of transgenic corn plants with increased yield. The elements of Table 12 are described by reference to:
“Decoy Element (NUC) SEQ ID NO:” which identifies a nucleic acid sequence.
“Decoy Element Identifier” which is an arbitrary identifier.
“Target miRNA Name” which identifies a target miRNA for binding by the decoy.
“Protein name” which identifies a gene down-regulated by the target miRNA.
“Broad acre yield results” refers to the sequence in a construct with at least one event showing significant yield increase at p≦0.2 across locations. The first number refers to the number of events with significant yield increase, whereas the second number refers to the total number of events tested for each sequence in a construct.

TABLE 12

Recombinant DNA for miRNA decoys for increased yield in corn

Decoy
Element				Broad
(NUC)	Decoy	Target		Acre
SEQ	Element	miRNA	Protein	Yield
ID NO:	Identifier	Name	Name	Results

93	TRDX3-30	miR399	Phosphate	1/18
			transporter
95	TRDX3-32	miR172	APETALA2-	1/18
			like
			transcription
			factor
97	TRDX3-34	miR172e	APETALA2-	3/18
			like
			transcription
			factor
98	TRDX3-35	miR393	HOS1;	1/22
			E3 ligase
			SCF
			complex F-
			box protein;
			bHLH
			transcription
			factor

Increased Yield in Soybean

A yield increase in soybean can be manifested as one or more of the following: an increase in pods per plant, pods per acre, seeds per plant, seeds per pod, weight per seed, weight per pod, pods per node, number of nodes, and the number of internodes per plant.
Table 13 provides a list of protein encoding DNA or polynucleotide sequences used (“genes”) in the production of transgenic soybean plants with increased yield as compared to a control plant. The elements of Table 13 are described by reference to:
“Gene (NUC) SEQ ID NO:” which identifies a nucleotide sequence
“Gene (PEP) SEQ ID NO:” which identifies an amino acid sequence.
“Gene identifier” which refers to an arbitrary identifier.
“Broad acre yield results” which refers to the sequence in a construct with at least one event showing significant yield increase at p≦0.2 across locations. The first number refers to the number of events with significant yield increase, whereas the second number refers to the total number of events tested for each sequence in a construct.

TABLE 13

Recombinant DNA for increased yield in soybean

			Broad
Gene (NUC)	Gene (PEP)	Gene	Acre Yield
SEQ ID NO:	SEQ ID NO:	Identifier	Results

43	44	TRDX3-22	3/16
51	52	TRDX3-26	3/16
53	54	TRDX3-27	3/15

Table 14 provides a list of miRNA decoy elements provided as recombinant DNA for production of transgenic corn plants with increased yield. The elements of Table 14 are described by reference to:
“Decoy Element (NUC) SEQ ID NO:” which identifies a decoy element nucleotide sequence.
“Decoy Element ID:” which identifies a decoy element sequence.
“Target miRNA Name” which identifies a target miRNA for binding by the decoy.
“Protein name” which identifies amino acid sequences of proteins which bind the target miRNA.
“Protein (PEP) SEQ ID NO” which identifies an amino acid sequence.
“Broad acre yield results” refers to the sequence in a construct with at least one event showing significant yield increase at p≦0.2 across locations. The first number refers to the number of events with significant yield increase, whereas the second number refers to the total number of events tested for each sequence in a construct.

TABLE 14

Recombinant DNA miRNA decoys for increased yield in soybean

Decoy
Element				Protein	Broad
(NUC)	Decoy	Target		(PEP)	Acre
SEQ	Element	miRNA	Protein	SEQ	Yield
ID NO	ID	Name	Name	ID NO:	Results

94	TRDX3-31	miR172	APETALA-2-like		2/12
			transcription factor
99	TRDX3-36	miR397	diphenol dioxidase	114	1/10

Example 6

Phenotypic Evaluation of Corn for Increased Water Use Efficiency

Corn field trials were conducted to identify genes that can improve water use efficiency under water limiting conditions leading to increased yield performance as compared to non transgenic controls. A yield increase in corn can be manifested as one or more of the following: an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, fresh or dry ear length/diameter/biomass (weight), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. The water use efficiency trials for results shown in Table 15 were conducted under managed water limiting conditions, and the corn ear weight or yield was compared to control plants to measure the yield increases.
Table 15 provides a list of protein encoding DNA or polynucleotide sequences (“genes”) for producing transgenic corn plant with increased water use efficiency as compared to a control plant. Polynucleotide sequences in constructs with at least one event showing significant yield or ear weight increase across multiple locations at p≦0.2 are included. The elements of Table 15 are described by reference to:
“SEQ ID NO: polynucleotide” which identifies a nucleotide sequence.
“SEQ ID NO: polypeptide” which identifies an amino acid sequence.
“Gene identifier” which refers to an arbitrary identifier.
“WUE results” which refers to the sequence in a construct with at least one event showing significant yield increase at p≦0.2 across locations. The first number refers to the number of events with significant yield or ear weight increase, whereas the second number refers to the total number of events tested for each sequence in the construct.

TABLE 15

Recombinant DNA for Increased Water Use Efficiency in Corn

SEQ ID NO:	SEQ ID NO:	Gene	WUE
Polynucleotide	Polypeptide	Identifier	Results

7	8	TRDX3-4	2/16
11	12	TRDX3-6	2/8
13	14	TRDX3-7	2/7
19	20	TRDX3-10	3/16
23	24	TRDX3-12	3/14
33	34	TRDX3-17	4/13
35	36	TRDX3-18	1/8
39	40	TRDX3-20	3/13
45	46	TRDX3-23	2/6
49	50	TRDX3-25	1/5

Table 16 provides a list of miRNA decoy elements provided as recombinant DNA for production of transgenic corn plants with increased water use efficiency. The elements of Table 16 are described by reference to:
“Decoy Element (NUC) SEQ ID NO:” which identifies a decoy element nucleotide sequence.
“Decoy Element ID:” which is an arbitrary identifier.
“Target miRNA Name” which identifies a target miRNA for binding by the decoy.
“Protein name” which identifies amino acid sequences of proteins which bind the target miRNA.
“Gene (PEP) SEQ ID NO” which identifies an amino acid sequence.

TABLE 16

Recombinant DNA miRNA Decoys for Increased
Water Use Efficiency in Corn

Decoy
Element				Protein	Broad
(NUC)	Decoy	Target		(PEP)	Acre
SEQ	Element	miRNA	Protein	SEQ	Yield
ID NO:	ID	Name	Name	ID NO	Results

95	TRDX3-32	miR166	Revoluta	110	1/8
96	TRDX3-33	miR444	ANR1	112	1/8
			MADS-box
			protein
97	TRDX3-34	miR172e	APETALA2-		4/8
			like
			transcription
			factor

Example 7

Homolog Identification

This example illustrates the identification of homologs of proteins encoded by the DNA identified in Table 1 which were used to provide transgenic seed and plants having enhanced agronomic traits. From the sequences of the homolog proteins, corresponding homologous DNA sequences can be identified for preparing additional transgenic seeds and plants with enhanced agronomic traits.
An “All Protein Database” was constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a polynucleotide sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; it is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
The All Protein Database was queried using amino acid sequences provided in Table 1 using NCBI “blastp” program with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list was kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes of the polynucleotides provided herein, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.
The Organism Protein Database was queried using polypeptide sequences provided in Table 1 using NCBI “blastp” program with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB is queried with each sequence in the Hit List using NCBI “blastp” program with E-value cutoff of 1e-8. The hit with the best E-value was compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Homologs with at least 95% identity over 95% of the length of the polypeptide sequences provided in Table 1 are reported below in Table 17 with the SEQ ID NO of the original query sequence and the identified homologs.

TABLE 17

Protein sequences and their homologs

	Polypeptide	Homolog
	SEQ ID NO	SEQ ID NOs
	(PEP)	(PEP)

	2	59
	4	61
	8	62
	10	63, 64
	12	65, 66
	14	67
	16	68, 69
	18	70, 71
	20	72, 73
	24	74
	26	75, 76
	28	77
	34	78, 79, 80
	36	81
	38	82, 83
	42	84
	44	85
	48	86, 87
	50	88
	52	89, 90
	58	91, 92
	116	60

Example 8

Identification of Protein Domains and Domain Modules by Pfam Analysis

This example illustrates the identification of domain and domain module by Pfam analysis.
The amino acid sequences of the expressed proteins that are shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software and Pfam databases (version 27.0). The Pfam protein domains and modules for the proteins of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 48, 50, 52, 54, 56 and 58 are shown in Tables 18, 19 and 20. The Hidden Markov model databases for the identified patent families are also available from the Pfam consortium (ftp.sanger.ac.uk/pub/databases/Pfam/) allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. The function of the identified Pfam domains in proteins providing an enhanced trait in plants was verified by searching identified homologs for the conservation of the identified Pfam domains. The HMM score values for the identified Pfam domains in sequences from Table 1 are reported below in Table 18.

TABLE 18

Pfam Domains and Their Locations on Proteins
Associated with Enhanced Traits

PEP
SEQ	Pfam domain			HMM
ID NO	name	Begin	Stop	Score	E-value

2	TCTP	1	165	208.1	5.30E−66
4	PAS_2	97	217	135.0	8.90E−43
4	GAF	253	432	135.0	1.00E−42
4	PHY	443	620	232.0	1.70E−72
4	PAS	654	769	93.2	5.30E−30
4	PAS	785	904	68.4	2.80E−22
4	HisKA	930	989	38.1	7.10E−13
4	HATPase_c	1039	1139	36.3	2.40E−12
6	FmdA_AmdA	9	295	363	8.40E−113
8	MGDG_synth	86	254	205.2	2.10E−64
8	Glyco_tran_28_C	312	404	35.1	3.30E−12
10	GST_C_3	75	156	38.5	4.70E−13
10	tRNA-synt_1c	213	516	324.4	1.90E−100
10	tRNA-synt_1c_C	520	697	128.7	6.90E−41
12	TPR_11	104	160	18.1	1.40E−06
12	TPR_11	167	228	24.1	1.90E−08
14	Pkinase	33	293	215.9	1.40E−67
16	EF-hand_like	25	101	59.3	7.50E−20
16	PI-PLC-X	107	249	148.5	2.20E−47
16	PI-PLC-Y	322	409	95.1	7.00E−31
16	C2	432	524	51.6	1.60E−17
18	LTP_2	28	121	54.4	2.10E−18
20	Fip1	336	379	74.4	1.90E−25
22	Cystatin	83	136	12.0	1.20E−05
24	CENP-O	123	200	59.7	1.60E−20
26	TOM20_plant	8	201	328.4	6.70E−102
28	DHquinase_I	97	315	260.5	1.60E−80
28	Shikimate_dh_N	328	408	95.2	2.40E−30
28	Shikimate_DH	449	548	61.1	1.50E−19
30	Methyltransf_11	39	134	36.2	4.60E−12
32	SOUL	10	215	196.1	2.20E−62
34	KH_1	128	173	28.9	8.40E−11
36	Pterin_4a	80	174	103.2	3.00E−34
38	Alliinase_C	25	383	565.1	1.20E−173
40	CBM_2	68	97	9.2	0.00016
40	COBRA	164	191	1.6	0.027
40	COBRA	241	422	217.4	1.30E−68
40	CBM_2	512	547	2.7	0.017
42	Sugar_tr	103	559	427.0	9.50E−132
44	zf-met	85	109	27.3	5.60E−10
48	Radical_SAM	175	338	50.3	5.90E−17
50	Senescence	177	356	168.2	9.00E−54
52	zf-PARP	11	88	83.3	4.80E−27
52	zf-PARP	117	186	68.0	3.00E−22
52	PADR1	290	343	72.8	4.90E−24
52	BRCT	399	471	21.9	6.60E−08
52	WGR	519	600	58.9	1.60E−19
52	PARP_reg	634	765	115.3	6.90E−37
52	PARP	767	979	262.0	1.40E−81
54	Metallothio	5	19	7.6	0.00044
54	Metallothio	43	62	16.1	9.60E−07
56	FAD_binding_6	62	164	79.2	3.9E−26
56	NAD_binding_1	174	279	97.0	1.80E−31
58	Cullin	29	631	640.5	5.00E−196
58	Cullin_Nedd8	659	725	92.4	2.20E−30

TABLE 19

Pfam Domain Modules and Their Positions

PEP SEQ
ID NO	Pfam Domain Module	Position

4	PAS_2::GAF::PHY::PAS::HisKA::HATPase_c	97-217::253-432::443-620::654-769,
		785-904::930-989::1039-1139
8	MGDG_synth::Glyco_tran_28_C	86-254::312-404
10	GST_C_3::tRNA-synt_1c::tRNA-	75-156::215-516::520-697
	synt_1c_C
12	TPR_11::TPR_11	104-160, 167-228
16	EF-hand_like::PI-PLC-X::PI-PLC-	25-101::107-249::322-409:: 432-524
	Y::C2
	HMGL-like::LeuA_dimer	84-366::459-604
28	DHquinase_I::Shikimate_dh_N::Shikimate_DH	97-315::328-408::449-548
40	CBM_2::COBRA::CBM_2	68-97::164-191, 241-422::512-547
46	Lung_7-TM_R::zf-RING_2	24-64::110-153
52	zf-	11-88, 117, 186::290-343::399-
	PARP::PADR1::BRCT::WGR::PARP_reg::PARP	471::519-600::634-765::767-979
54	Metallothio:: Metallothio	5-19, 43-62
56	FAD_binding_6[1]::NAD_binding_1	62-164::174-279
58	Cullin[1]::Cullin_Nedd8	29-631::659-725

TABLE 20

Pfam Domain Properties

PEP SEQ	Pfam domain	Accession	Gathering
ID NO	name	number	cutoff	Domain description

2	TCTP	PF00838	20.8	Translationally controlled tumour
				protein
4	GAF	PF01590	20.9	GAF domain
4	HATPase_c	PF02518	21.3	Histidine kinase-, DNA gyrase B-,
				and HSP90-like ATPase
4	HisKA	PF00512	22.4	His Kinase A (phospho-acceptor)
				domain
4	PAS	PF00989	22.6	PAS fold
4	PAS_2	PF08446	14.0	PAS fold
4	PHY	PF00360	18.0	Phytochrome region
6	FmdA_AmdA	PF03069	19.5	Acetamidase/Formamidase
				family
8	Glyco_tran_28_C	PF04101	21.0	Glycosyltransferase family 28 C-
				terminal domain
8	MGDG_synth	PF06925	20.9	Monogalactosyldiacylglycerol
				(MGDG) synthase
10	GST_C_3	PF14497	27.0	Glutathione S-transferase, C-
				terminal domain
10	tRNA-synt_1c	PF00749	19.8	tRNA synthetases class I (E and
				Q), catalytic domain
10	tRNA-synt_1c_C	PF03950	21.0	tRNA synthetases class I (E and
				Q), anti-codon binding domain
12	TPR_11	PF13414	26.8	TPR repeat
14	Pkinase	PF00069	20.4	Protein kinase domain
16	C2	PF00168	4.5	C2 domain
16	EF-hand_like	PF09279	20.9	Phosphoinositide-specific
				phospholipase C, efhand-like
16	PI-PLC-X	PF00388	22.1	Phosphatidylinositol-specific
				phospholipase C, X domain
16	PI-PLC-Y	PF00387	20.2	Phosphatidylinositol-specific
				phospholipase C, Y domain
18	LTP_2	PF14368	22.0	Probable lipid transfer
20	Fip1	PF05182	22.7	Fip1 motif
22	Cystatin	PF00031	20.9	Cystatin domain
24	CENP-O	PF09496	19.6	Cenp-O kinetochore centromere
				component
26	TOM20_plant	PF06552	20.6	Plant specific mitochondrial
				import receptor subunit TOM20
28	DHquinase_I	PF01487	20.7	Type I 3-dehydroquinase
28	Shikimate_DH	PF01488	24.3	Shikimate/quinate 5-
				dehydrogenase
28	Shikimate_dh_N	PF08501	21.3	Shikimate dehydrogenase
				substrate binding domain
30	Methyltransf_11	PF08241	21.2	Methyltransferase domain
32	SOUL	PF04832	21.2	SOUL heme-binding protein
34	KH_1	PF00013	20.2	KH domain
36	Pterin_4a	PF01329	20.8	Pterin_4a
38	Allinase_C			Allinase
40	CBM_2	PF00553	21.1	Cellulose binding domain
40	COBRA	PF04833	19.0	COBRA-like protein
42	Sugar_tr	PF00083	20.7	Sugar (and other) transporter
44	zf-met	PF12874	13.3	Zinc-finger of C2H2 type
46	Lung_7-TM_R	PF06814	25.2	Lung seven transmembrane
				receptor
46	zf-RING_2	PF13639	27.0	Ring finger domain
48	Radical_SAM	PF04055	29.4	Radical SAM superfamily
50	Senescence	PF06911	21.6	Senescence-associated protein
54	Metallothio	PF00131	20.6	Metallothionein

Example 9

Construction of miRNA Decoys

This example illustrates monocot and dicot plant transformation to produce recombinant DNA molecules useful for stable integration into plant chromosomes in the nuclei of plant cells to provide transgenic plants having enhanced traits by suppressing the activity of miRNAs.
The recombinant DNA molecules of SEQ ID NOs: 94, 95, 96, 97, 98 and 99 were constructed as follows.
Synthetic miRNA binding site sequences capable of hybridizing, respectively, miR172, miR166, miR444, miR172e, miR393 and miR397 species under physiological conditions were constructed by modifying naturally occurring miRNA binding sites. Specifically, the binding site sequences were constructed by inserting three nucleotides between nucleotides 10 and 11 of the polynucleotide sequences encoding naturally occurring binding sites specific for miR172, miR166, miR444, miR172e, miR393 and miR397, and in some cases by adding additional nucleotides at either the 5′ or the 3′ end of the naturally occurring biding site sequences. These modifications gave synthetic binding sites having polynucleotide sequences as set forth in SEQ ID NOs: 101-106.
The synthetic decoys were then constructed using the naturally occurring Zea mays miR399_47862C decoy of SEQ ID NO: 93 as a scaffolding. The native miR399 binding site of SEQ ID NO: 100 was removed and substituted with one of the synthetic miRNA binding sites set forth in SEQ ID NOs101-106, to give a recombinant DNA of, respectively, SEQ ID NO: 94-99.
Transformation vectors, each comprising a heterologous promoter operably linked to a polynucleotide encoding either the decoy of naturally occurring miR399 from Zea mays as set forth in SEQ ID NO: 93 or a synthetic decoy as set forth in SEQ ID NOs: 94-99 were then constructed by methods known in the art and used to transform plant cells, from which transgenic plants were produced as described above. The transgenic plants were then tested for altered phenotypes and enhanced traits, again as described above.
Similarly, recombinant DNA molecules comprising polynucleotide sequences transcribed into non-coding miRNA decoys are constructed as above, by using other native decoy sequences as a scaffolding, and using binding site sequences which bind some or all of other miRNA families that are naturally occurring or are derived from naturally occurring sites.

Claims

We claim:

1. A plant comprising a recombinant DNA molecule comprising a polynucleotide encoding a polypeptide, wherein the nucleotide sequence of the polynucleotide is selected from the group consisting of:

a) a nucleotide sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 115;

b) a nucleotide sequence encoding a protein, said protein having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59-92 or 116;

c) a nucleotide sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 115; and

d) a nucleotide sequence encoding a protein with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59-92 or 116;

wherein said plant has an enhanced trait as compared to a control plant.

2. The plant of claim 1, wherein said enhanced trait is selected from the group consisting of increased yield, increased nitrogen use efficiency, and increased water use efficiency.

3. The plant of claim 1, wherein said plant has at least one phenotype selected from the group consisting of anthocyanin, biomass, canopy area, chlorophyll score, plant height, water applied, water content and water use efficiency that is altered for said plant as compared to a control plant.

4. The plant of claim 1, wherein the recombinant DNA molecule further comprises a heterologous promoter that is operably linked to the polynucleotide encoding a polypeptide, wherein said promoter is selected from the group consisting of a constitutive, inducible, tissue specific, diurnally regulated, tissue enhanced, and cell specific promoter.

5. The plant of claim 1, wherein said plant is a progeny, a propagule, or a field crop.

6. The plant of claim 5, wherein said field crop is selected from the group consisting of corn, soybean, cotton, canola, rice, barley, oat, wheat, turf grass, alfalfa, sugar beet, sunflower, quinoa and sugar cane.

7. The plant of claim 5, wherein said propagule is selected from the group consisting of a cell, pollen, ovule, flower, embryo, leaf, root, stem, shoot, meristem, grain and seed.

8. The plant of claim 1, wherein said plant is a monocot plant or is a member of the family Poaceae, wheat plant, maize plant, sweet corn plant, rice plant, wild rice plant, barley plant, rye, millet plant, sorghum plant, sugar cane plant, turfgrass plant, bamboo plant, oat plant, brome-grass plant, Miscanthus plant, pampas grass plant, switchgrass (Panicum) plant, and/or teosinte plant, or is a member of the family Alliaceae, onion plant, leek plant, garlic plant; or wherein the plant is a dicot plant or is a member of the family Amaranthaceae, spinach plant, quinoa plant, a member of the family Anacardiaceae, mango plant, a member of the family Asteraceae, sunflower plant, endive plant, lettuce plant, artichoke plant, a member of the family Brassicaceae, Arabidopsis thaliana plant, rape plant, oilseed rape plant, broccoli plant, Brussels sprouts plant, cabbage plant, canola plant, cauliflower plant, kohlrabi plant, turnip plant, radish plant, a member of the family Bromeliaceae, pineapple plant, a member of the family Caricaceae, papaya plant, a member of the family Chenopodiaceae, beet plant, a member of the family Curcurbitaceae, melon plant, cantaloupe plant, squash plant, watermelon plant, honeydew plant, cucumber plant, pumpkin plant, a member of the family Dioscoreaceae, yam plant, a member of the family Ericaceae, blueberry plant, a member of the family Euphorbiaceae, cassava plant, a member of the family Fabaceae, alfalfa plant, clover plant, peanut plant, a member of the family Grossulariaceae, currant plant, a member of the family Juglandaceae, walnut plant, a member of the family Lamiaceae, mint plant, a member of the family Lauraceae, avocado plant, a member of the family Leguminosae, soybean plant, bean plant, pea plant, a member of the family Malvaceae, cotton plant, a member of the family Marantaceae, arrowroot plant, a member of the family Myrtaceae, guava plant, eucalyptus plant, a member of the family Rosaceae, peach plant, apple plant, cherry plant, plum plant, pear plant, prune plant, blackberry plant, raspberry plant, strawberry plant, a member of the family Rubiaceae, coffee plant, a member of the family Rutaceae, citrus plant, orange plant, lemon plant, grapefruit plant, tangerine plant, a member of the family Salicaceae, poplar plant, willow plant, a member of the family Solanaceae, potato plant, sweet potato plant, tomato plant, Capsicum plant, tobacco plant, tomatillo plant, eggplant plant, Atropa belladona plant, Datura stramonium plant, a member of the family Vitaceae, grape plant, a member of the family Umbelliferae, carrot plant, or a member of the family Musaceae, banana plant; or wherein the plant is a member of the family Pinaceae, cedar plant, fir plant, hemlock plant, larch plant, pine plant, or spruce plant.

9. A method for producing a plant comprising:

introducing into a plant cell a recombinant DNA molecule comprising a polynucleotide encoding a polypeptide, wherein the polynucleotide comprises a polynucleotide sequence selected from the group consisting of:

a) a nucleotide sequence as set forth as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 or 115;

b) a nucleotide sequence encoding a protein comprising an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59-92 or 116;

and growing a plant from said plant cell.

10. The method of claim 9, further comprising selecting a plant with an enhanced trait as compared to a control plant, wherein said enhanced trait is selected from increased yield, increased nitrogen use efficiency, and increased water use efficiency as compared to a control plant.

11. The plant of claim 9, further comprising selecting a plant with a phenotype selected from the group consisting of anthocyanin, biomass, canopy area, chlorophyll score, plant height, water applied, water content and water use efficiency, wherein said phenotype for said plant is altered as compared to a control plant.

12. A method for increasing yield, increasing nitrogen use efficiency, or increasing water use efficiency in a plant comprising:

crossing the plant of claim 1 with itself, a second plant from the same plant line, a wild type plant, or a second plant from a different line of plants to produce a seed;

growing said seed to produce a plurality of progeny plants; and

selecting a progeny plant with increased yield, increased nitrogen use efficiency, or increased water use efficiency.

13. A recombinant DNA molecule, said molecule comprising a promoter operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule, wherein:

a) said non-coding miRNA molecule is selected from the group consisting of:

i) a non-coding miRNA molecule which, when present in a plant, provides an altered level of a protein as compared to a control plant, wherein said protein is selected from the group consisting of: a phosphate transporter protein, an APETALA2-like transcription factor, an ANR1 MADS-box protein, an E3 ligase SCF complex F-box protein, a HOS1 protein, a bHLH transcription factor, a diphenol oxidase proteinligase;

ii) a non-coding miRNA molecule which, when present in a plant, provides an altered level of a protein as compared to a control plant, wherein said protein is selected from the group consisting of: the polypeptide sequences set forth in SEQ ID NO: 108, 110, 112 and 114;

iii) a non-coding miRNA molecule which, when present in a plant, interferes with the functioning of one or more species of an endogenous miRNA selected from the group consisting of: miR399, miR172, miR166, miR166, miR444, miR393 and miR397;

iv) a non-coding miRNA molecule comprising a miRNA binding site sequence as set forth in SEQ ID NOs: 100-106;

v) a non-coding miRNA molecule comprising a miRNA polynucleotide sequence as set forth in SEQ ID NOs: 93-99; and

b) said promoter is selected from the group consisting of a constitutive promoter, a developmental promoter, a tissue enhanced promoter, a tissue preferred promoter, a tissue specific promoter, a cell type-specific promoter, an inducible promoter and a diurnal promoter;

wherein, when said recombinant DNA molecule is present in a plant, said plant exhibits an altered phenotype or an enhanced trait as compared to a control plant.

14. A plant comprising a recombinant DNA molecule, said molecule comprising a heterologous promoter functional in plant cells operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule, wherein

a) said non-coding miRNA molecule is selected from the group consisting of:

wherein said plant has an enhanced trait as compared to a control plant.

15. A method for producing a plant comprising:

introducing into a plant cell a recombinant DNA molecule comprising a heterologous promoter functional in plant cells operably linked to a polynucleotide that is transcribed into a non-coding miRNA molecule, wherein

a) said plants exhibit an altered level of a protein selected from the group consisting of: a phosphate transporter protein, an APETALA2-like transcription factor, an ANR1 MADS-box protein, an E3 ligase SCF complex F-box protein, a HOS1 protein, a bHLH transcription factor, a diphenol oxidase proteinligase; or

b) said plants exhibit an altered level of a protein selected from the group consisting of: the polypeptides set forth in SEQ ID NO: 108, 110, 112 and 114; or

c) said non-coding miRNA molecule interferes with the functioning of one or more species of an endogenous miRNA selected from the group consisting of: miR399, miR172, miR166, miR166, miR444, miR393 and miR397; or

d) said non-coding miRNA molecule comprises a miRNA binding site polynucleotide sequence as set forth in SEQ ID NOs: 100-106; or

e) said non-coding miRNA molecule comprises a miRNA polynucleotide sequence as set forth in SEQ ID NOs: 93-99; and

growing a plant from said plant cell.