WO2014122607A2 - Plantes transgéniques pour lnk - Google Patents

Plantes transgéniques pour lnk Download PDF

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Publication number
WO2014122607A2
WO2014122607A2 PCT/IB2014/058836 IB2014058836W WO2014122607A2 WO 2014122607 A2 WO2014122607 A2 WO 2014122607A2 IB 2014058836 W IB2014058836 W IB 2014058836W WO 2014122607 A2 WO2014122607 A2 WO 2014122607A2
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Prior art keywords
plant
nucleotide sequence
seq
sequence
amino acid
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WO2014122607A9 (fr
Inventor
Sabrina E. SANCHEZ
Matias L. RUGNONE
Ruben G. SCHLAEN
Ana FAIGON SOVERNA
Carlos E. HERNANDO
Andres ROMANOWSKI
Marcelo J. YANOVSKY
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Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Fundacion Instituto Leloir
Inis Biotech LLC
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Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Fundacion Instituto Leloir
Inis Biotech LLC
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Publication of WO2014122607A9 publication Critical patent/WO2014122607A9/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates to the field of plant molecular biology, more particularly to methods of expressing nucleic acids in plants.
  • Light signaling pathways and the circadian clock interact to help organisms synchronize physiological and developmental processes with periodic environmental cycles.
  • the interaction between light signaling pathways and the circadian clock plays a critical role in the process of day-length measurement, which contributes to adjust the floral transition to the most appropriate season of the year.
  • the ability to genetically manipulate flowering time in order to optimize the timing of developmental processes in crop plants is critical to maximize crop yield under different growing conditions and in different geographic regions.
  • LNK1 and LNK2 represent a new family of light and clock regulated morning genes that control both the pace of circadian rhythms and the photoperiodic regulation of flowering time.
  • the invention relates in part to manipulating the expression of members of a novel plant specific gene family that plays a key role regulating floral transition and biomass production.
  • the invention relates to nucleic acids, expression cassettes, vectors and transgenic plants containing nucleic acids corresponding to LINK, hereinafter referred to as LNK genes (night-light inducible and clock regulated genes) such as, the homologous LNK1 and LNK2 genes of A.
  • the invention also relates to the introduction and/or manipulation of these nucleic acids in plants to regulate plant growth and development and the transgenic plants generated thereby.
  • the invention provides an isolated LNK1 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: l; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: l or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK1 amino acid sequence of SEQ ID NO:2; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In further embodiments, the nucleic acids are transcribed to express a functional RNA.
  • the invention also encompasses expression cassettes and vectors comprising nucleic acids of the invention.
  • the invention provides an isolated LNK2 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 3; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In further embodiments, the nucleic acids are transcribed to express a functional RNA.
  • the invention also encompasses expression cassettes and vectors comprising nucleic acids of the invention.
  • the invention provides a vector or expression cassette comprising a LNK promoter sequence operably associated with a nucleotide sequence of interest.
  • the LNK1 promoter sequence is a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 7; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 7 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 7 or the complementary strand thereof; (d) a nucleotide sequence comprising nucleotides 1 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250; 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 to 550; 550 to 600,
  • the nucleotide sequence of interest is in sense orientation relative to the promoter. In alternative embodiments, the nucleotide sequence of interest is in antisense orientation relative to the promoter. In further embodiments, the nucleotide sequence of interest is transcribed to form a functional RNA. In particular embodiments, the functional RNA is a LNK1 RNA antagonist.
  • the he invention provides a vector or expression cassette comprising a LNK2 promoter sequence operably associated with a nucleotide sequence of interest wherein the LNK2 promoter sequence is a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 12; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 12 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 12 or the complementary strand thereof; (d) a nucleotide sequence comprising nucleotides 1 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250; 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 to 550; 550 to 600, 600
  • nucleotide sequence of interest is in sense orientation relative to the promoter. In alternative embodiments, the nucleotide sequence of interest is in antisense orientation relative to the promoter. In further embodiments, nucleotide sequence of interest is transcribed to form a functional RNA.
  • the functional RNA is a LNK RNA antagonist (e.g., a LNK1 and/or LNK2 antagonist).
  • the invention provides a transgenic plant stably transformed with an isolated nucleic acid comprising a LNK1 nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: l; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK1 amino acid sequence of SEQ ID NO:2; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In further embodiments, the nucleic acids are transcribed to express a functional RNA.
  • the invention also encompasses expression cassettes and vectors comprising nucleic acids of the invention.
  • the invention provides a transgenic plant stably transformed with an isolated nucleic acid encoding a LNK1 polypeptide selected from the group consisting of: (a) a polypeptide comprising the LNK1 amino acid sequence of SEQ ID NO:2; (b) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2; and (c) an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2.
  • the invention provides a transgenic plant stably transformed with an isolated LNK2 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO:3; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO:3 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO:3 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical
  • the invention provides a transgenic plant stably transformed with an isolated nucleic acid encoding a LNK2 polypeptide selected from the group consisting of: (a) a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (b) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4; and (c) an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO:4.
  • the invention also encompasses a transgenic plant stably transformed with both an isolated LNK1 and LNK2 nucleic acid of the invention.
  • the invention provides a transgenic plant stably transformed with a vector or expression cassette comprising a LNK promoter sequence operably associated with a nucleotide sequence of interest.
  • the LNK1 promoter sequence is a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 7; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 7 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 7 or the complementary strand thereof; (d) a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of (a), (b) or (c); (d) a nucleotide sequence that hybridizes to the complete complement
  • the LNK2 promoter sequence is a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 12; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 12 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 12 or the complementary strand thereof; (d) a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of (a), (b) or (c); (d) a nucleotide sequence that hybridizes to the complete complement of the nucleotide sequence of (a), (b) or (c) under stringent hybridization conditions.
  • the nucleotide sequence of interest is in sense orientation relative to the promote
  • the transgenic plants of the invention display altered growth and/or development compared to a wild-type control plant grown under the same conditions. In some embodiments, the transgenic plants display increased vegetative growth and/or biomass compared to wild-type control plants. In additional embodiments, the transgenic plants produce biomass for a longer period of time than wild-type control plants. In the case of seed plants, in some embodiments, the transgenic plants display delayed flowering compared to wild-type plants. In additional embodiments, the transgenic plants of the invention have larger seed yield than wild-type plants.
  • the transgenic plants of the invention have an increased tolerance to abiotic stress (e.g., high intensity light, low intensity light, drought (dehydration), high or low temperature and/or salinity (e.g., salt) compared to a wild-type plant grown under the same conditions.
  • abiotic stress e.g., high intensity light, low intensity light, drought (dehydration), high or low temperature and/or salinity (e.g., salt) compared to a wild-type plant grown under the same conditions.
  • transgenic plants of the invention display decreased vegetative growth and/or biomass compared to wild-type plants.
  • the transgenic plants display shortened (i. e. , accelerated) flowering time compared to wild-type plants.
  • the invention provides a method of modulating a circadian response of a plant, the method comprising stably transforming a plant cell with a LNK nucleic acid.
  • One embodiment provides a method of modulating a circadian response of a plan comprising, stably transforming a plant cell with a LNK1 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: l; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: l or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: l or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In additional embodiments, the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the invention provides a method of modulating a circadian response of a plant, comprising stably transforming a plant cell with for example, a nucleic acid encoding a LNK1 polypeptide selected from the group consisting of: (a) a polypeptide comprising the LNK1 amino acid sequence of SEQ ID NO:2; (b) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2; and (c) an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2.
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In additional embodiments, the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the invention provides a method of modulating a circadian response of a plant, comprising stably transforming a plant cell with a LNK2 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 3; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%,
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In additional embodiments, the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the invention provides a method of modulating a circadian response of a plant, comprising stably transforming a plant cell with for example, a nucleic acid encoding a LNK2 polypeptide selected from the group consisting of: (a) a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (b) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4; and (c) an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO:4.
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In additional embodiments, the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the invention provides a method of modulating a circadian response of a plant, comprising stably transforming a plant cell with both a LNKl and LNK2 nucleic acid of the invention.
  • the invention provides a method of modulating the flowering time of a plant comprising, stably transforming a plant cell with a LNK nucleic acid. In additional embodiments, the invention provides a method of modulating the flowering time of a plant comprising, stably transforming a plant cell with a LNKl and/or LNK2 nucleic acid of the of the invention such as, a nucleic acid described above or elsewhere herein.
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the method produces a transgenic plant in which the flowering time of the plant is accelerated. In other embodiments, the method produces a transgenic plant in which the flowering time of the plant is delayed.
  • the invention provides a method of modulating the biomass of a plant, the method comprising stably transforming a plant cell with a LNK nucleic acid.
  • the invention provides a method of modulating the biomass of a plant, the method comprising stably transforming a plant cell with a LNKl and/or LNK2 nucleic acid of the of the invention such as, a nucleic acid described above or elsewhere herein.
  • the nucleic acids are operably associated with a promoter.
  • the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the method produces a transgenic plant in which the biomass of the plant is increased. In other embodiments, the method produces a transgenic plant in which the biomass of the plant is decreased.
  • the invention provides a method of increasing yield of a plant comprising, stably transforming a plant cell with a LNK1 and/or LNK2 nucleic acid of the invention such as, a nucleic acid described above or elsewhere herein.
  • the nucleic acids are operably associated with a promoter.
  • the nucleic acids are in sense orientation relative to the promoter.
  • the nucleic acids are in antisense orientation relative to the promoter.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the method produces a transgenic plant in which the biomass of the plant is increased.
  • the method produces a transgenic plant in which the biomass of the plant is decreased.
  • the invention provides a method of increasing yield of a plant comprising, stably transforming a plant cell with a LNK nucleic acid.
  • the invention provides a method of increasing yield of a plant comprising, stably transforming a plant cell with a LNK1 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK1 amino acid sequence of SEQ ID NO:
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In additional embodiments, the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the invention provides a method of increasing yield of a plant comprising, stably transforming a plant cell with a LNK2 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 3; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%,
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In additional embodiments, the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the invention provides a method of increasing tolerance of a plant to an abiotic stress, the method comprising: stably transforming a plant cell with a LNK nucleic acid.
  • the invention provides a method of increasing tolerance of a plant to an abiotic stress, the method comprising: stably transforming a plant cell with a LNK1 and/or LNK2 nucleic acid of the invention such as, a nucleic acid described above or elsewhere herein.
  • the nucleic acids are operably associated with a promoter.
  • the nucleic acids are in sense orientation relative to the promoter.
  • the nucleic acids are in antisense orientation relative to the promoter.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the abiotic stress is a member selected from: high intensity light, low intensity light, drought (dehydration), high temperature, low temperature and high salinity (salt).
  • the method of the invention is applied to increase tolerance of a plant to a severe abiotic stress.
  • the method of the invention is applied to increase tolerance of a plant to a mild abiotic stress.
  • the invention encompasses a method of increasing the yield and/or tolerance of a plant to an abiotic stress comprising: stably transforming a plant cell with a LNK nucleic acid.
  • the invention provides a method of increasing the yield and/or tolerance of a plant to an abiotic stress comprising: stably transforming a plant cell with a LNKl and/or LNK2 nucleic acid of the invention such as, a nucleic acid described above or elsewhere herein.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the invention also provided are expression cassettes, vectors, cells, plants and plant parts comprising the isolated nucleic acids of the invention operably associated with a LNK nucleotide sequence.
  • the invention encompasses a vector or expression cassette comprising an isolated LNK polynucleotide.
  • the invention encompasses a vector or expression cassette comprising a LNKl and/or LNK2 polynucleotide sequence of the invention operably associated with a promoter sequence.
  • the expression cassette contains a sequence that encodes a selectable marker.
  • Cells, plants and plant parts transformed with these nucleic acids, vectors, and expression cassettes are also encompassed by the invention.
  • the transformed (i.e., transgenic) plants are monocots.
  • the transformed plants are dicots.
  • the cells, plants and/or plant parts correspond to sunflower, wheat, maize, soybean, rice, alfalfa or Arabidopsis .
  • the invention encompasses a vector or expression cassette comprising an isolated LNK promoter sequence.
  • the invention encompasses a vector or expression cassette comprising a LNKl and/or LNK2 promoter sequence operably associated with a nucleotide sequence of interest.
  • the nucleotide sequence of interest encodes a protein.
  • the protein encoded by the polynucleotides sequence of interest is a LNK polypeptide, such as LNKl or LNK2 or a biologically active fragment or variant thereof.
  • the nucleotide sequence of interest encodes a protein.
  • the expression cassette contains a polynucleotide sequence that is transcribed to form a functional RNA.
  • the functional RNA is an antagonist of LNK, such as, an antagonist of LNKl or LNK2 expression.
  • the expression cassette contains a sequence that encodes a selectable marker.
  • the transformed (i.e., transgenic) plants are monocots.
  • the transformed plants are dicots.
  • the cells, plants and/or plant parts correspond to sunflower, wheat, maize, soybean, rice, alfalfa or Arabidopsis .
  • the invention also encompasses products harvested from the transgenic plants of the invention and processed products produced therefrom.
  • the invention also provides seed produced from the plants of the invention and seed comprising the isolated nucleic acids, vectors and expression cassettes of the invention.
  • the invention provides a method of introducing a nucleic acid into a plant, plant part or plant cell, the method comprising transforming the plant, plant part or plant cell with an isolated nucleic acid, expression cassette or vector of the invention.
  • the invention encompasses a method of expressing a nucleotide sequence of interest in a plant, the method comprising: (a) stably transforming a plant cell with an expression cassette or vector of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence of interest in the plant.
  • the nucleotide sequence is a LNK protein coding sequence.
  • the nucleotide sequence of interest encodes LNK1, LNK2, or a biologically active fragment thereof.
  • the nucleotide sequence of interest is transcribed to form a functional RNA.
  • the functional RNA is a LNK1 and/or LNK2 antagonist.
  • Figure 1 Describes the genomic approach used to characterize light and clock interactions in the control of gene expression, which resulted in the identification of LNK genes.
  • A Experimental design. Plants were grown under 12h light/ 12h dark cycles for 14 days and then exposed or not to a lh light pulse in the middle of the night or subjective day on the 15 th day.
  • B Comparative genome- wide expression analysis of the effect of a light pulse given during subjective daytime (x-axis) vs night-time (y-axis).
  • C Overlap between 87 genes that are rhythmically expressed under multiple conditions, and 65 genes that showed a stronger induction by light during night-time compared to subjective day-time.
  • (F) Circadian expression of LNKl and LNK2 genes. Expression was determined by qRT-PCR during the 2nd and 3rd day under free running conditions. n 4. Data are average ⁇ SEM.
  • FIG. 1 LNKl and LNK2 control hypocotyl elongation, photoperiodic dependent flowering and circadian rhythms.
  • B Unkllink2 control the floral transition in plants grown under long day (LD, 16h light/8h dark) conditions.
  • C and D Flowering time measured as the number of rosette leaves at bolting in LD (C) and short day (D) conditions (SD, 8h light/ 16h dark). ANOVA followed by a Tukey ' s multiple comparison test was used to evaluate the statistical significance of differences observed between genotypes.
  • Error bars indicate +SEM ( *** . ⁇ 0.001, *P ⁇ 0.05).
  • FIG. 3 LNKl, a nuclear protein, positively regulates expression of circadian genes with an afternoon phase.
  • B Phase enrichment of circadian-regulated genes whose expression was down- or up- regulated in linkl ;link2 mutant compared to WT plants, according to RNA-seq data of plants grown under continuous light conditions. The phase over- representation analysis was conducted with Phaser (http://phaser.mocklerlab.org/), and was based on the phases of gene expression estimated from data obtained using WT plants grown under LD conditions.
  • LNK1 and LNK2 are necessary for the proper function of the circadian clock.
  • CCA1 (A), LHY (B), PRR9 (C), PRR7 (D), PRR5 (E) and TOC1 (F) mRNA expression measured by qRT-PCR in plants grown under 12h light/ 12h dark cycles and then transferred to continuous light. Values are expressed relative to PP2A and normalized to the maximum value of each gene. Data represent average + SEM (n 4). Open and lined boxes indicate subjective day and subjective night periods, respectively.
  • FIG. 1 and LNK2 are repressed by the TOC1/PRR1 family of circadian clock components.
  • E Model showing the proposed function of LNK1 and LNK2 in the circadian clock. Light regulates LNK1 and LNK2 expression in the morning, which then act to promote, directly or indirectly, the expression of a subset of afternoon genes, including the core clock gene PRR5. During the afternoon and early evening, PRR9, PRR7, PRR5 and TOC1 bind to the LNK promoters blocking their expression.
  • FIG. 7 Cladogram displaying LNK1 homologs in a broad range of species from embryophyta group. Arabidopsis thaliana LNK1 has three paralogs. Where multiple homologs were identified within a single species, the annotated gene model code is provided (if more than one transcript was identified, the one with higher score during the BLASTP was selected).
  • Rc Ricinus communis
  • Pp Physcomitrella patens
  • Solyc Solanum lycopersicum
  • Carubv Capsella rubella
  • Os Oryza sativa
  • Cs Cucumis sativus
  • Medtr Medicago truncatula
  • Sb Sorghum bicolor. Percentage bootstrap values are presented for each node.
  • FIG. 8 Temporal patterns of LNK1 and LNK2 expression under different conditions.
  • A DD_DDHC, plants grown under temperature cycles (12h 22°C/12h 12°C) and continuous darkness, and then transferred to constant temperature conditions. Measurements correspond to the first 24h under constant temperature and dark conditions.
  • B LL23 LDHH, plants grown under 12h light/ 12h dark cycles at constant temperature and then transferred to constant light and temperature conditions for three days. Measurements correspond to the 2 nd and 3 rd days under constant conditions.
  • C COL LDHH, plants grown and measured under 12h light/ 12h dark cycles at 22°C.
  • D COL_SD, plants grown and measured under 8h light/ 16h dark cycles at 22°C. Data was obtained from Mockler, T.
  • FIG. Circadian rhythms of leaf movement in additional mutant alleles of LNK1 and LNK2.
  • FIG. 13 Expression levels of flowering time genes in WT and linkl ;link2 mutant plants: (A) FT, (B) SOC1. Plants were grown and harvested under 16h light/8h dark cycles and data was obtained by high throughput RNA-sequencing. CPM: Counts per Million.
  • Figure 15 Phylogenetic tree for the AtLNK genes, homolog genes in rice
  • Figure 17 Molecular function of OsLNK2 in the control of the circadian clock and in the regulation of flowering time in rice.
  • the present invention is based, in part, on the discovery that LNK1 and/or
  • LNK2 represent of new family of light and clock regulated morning genes that control both the pace of circadian rhythms and the photoperiodic regulation of flowering time.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • LNK night-light inducible and clock regulated genes
  • LNK1 e.g., AT5G64170, represented by SEQ ID NOs: l-2 and 5-6
  • LNK2 ⁇ e.g., AT3G54500, represented by SEQ ID NOs:3, 4, and 8-11
  • LNK3 e.g., AT3G12320, represented by SEQ ID NOs: 13 and 14
  • LNK4 e.g., AT5G06980, represented by SEQ ID NOs: 15 and 16
  • members from other night-light inducible and clock regulated genes in other plant species including fragments and/or variants of these members, that display one or more biological activities of a LNK family member as described herein.
  • LNK agonist refers to a nucleic acid or polypeptide that, increases, enhances or up regulates one or more LNK biological activities (including phenotypes), such as, a LNK biological activity described herein.
  • a LNK agonist may lead to plants having one or more accelerated circadian responses in agonist treated plants than in WT plants, shorter hypocotyls than WT plants under continuous white light or red light, earlier flowering (i.e., accelerated flowering) in agonist treated plants than WT plants under long days (LD; 16 hr light/8 hr dark), shorter circadian rhythm leaf movement in agonist treated plants than in WT plants, increased FKF1, FT and/or SOC1 expression in agonist treated plants or plant cells compared to WT plants or plant cells, and increased PRR5 expression in agonist treated plants compared to WT plants or plant cells.
  • LNK antagonist refers to a nucleic acid or polypeptide that, reduces, inhibits or down regulates one or more LNK biological activities (including phenotypes), such as, a LNK biological activity described herein.
  • a LNK antagonist may lead to plants having an one or more delayed circadian responses in antagonist treated plants than in WT plants, longer hypocotyls than WT plants under continuous white light or red light; later flowering in antagonist treated plants than WT plants under long days (LD; 16 hr light/8 hr dark); longer circadian rhythm leaf movement than in WT plants; reduced FKF1 FT and/or SOC1 expression in antagonist treated plants or plant cells compared to WT plants or plant cells, and reduced PRR5 expression in antagonist treated plants compared to WT plants or plant cells.
  • a "circadian response” is a physiological and/or developmental plant process influenced by circadian rhythms and/or photoperiodism in plants.
  • the circadian response is selected from the group consisting of: gene transcription, leaf movement, photosynthetic ability, stomatal opening, hypocotyl elongation, and a photoperiodic response such as, the control of flowering.
  • biomass refers to the amount of a plant body or a part thereof.
  • increased biomass encompasses substances, foods, materials, fuels, resources and the like derived from these plants or plant parts.
  • increased biomass may refer to hypertrophy of a subterranean stem (rhizom, corm, tuber, bulb), a terrestrial stem, a flowering stem or a vine, hypertrophy of a seed, acceleration of elongation of stem length, plant length, culm length or ear length, or enlargement of a source plant organ such as a leaf.
  • a "long-day” plant is a plant that only flowers after having received more than 12 hours of light.
  • a "short-day” plant is a plant that only flowers after having received less than 12 hours of light.
  • An "abiotic stress” is a stressor from one or more outside, non-living factors that adversely affects the productivity and/or the survival of the organism.
  • Abiotic stressors include, but are not limited to: high light intensity, low light intensity, ozone, heat or high temperature, cold temperature, drought (dehydration), flooding stress (e.g., due to waterlogging and/or submergence), stress following the removal of a flooding stressor (e.g., dehydration in the period following removal of a flooding stress such as desubmergence stress), salt stress (e.g., high or excessive salt conditions), high winds, fire, poor pH (too alkaline and/or too acidic), soil compaction and/or high radiation.
  • abiotic stress will depend on the preferred conditions for that organism, and may well vary due to the presence of other biotic and/or abiotic stressors.
  • Parameters for abiotic stress factors are species specific and even variety specific and therefore vary widely according to the species/variety exposed to the abiotic stress. Thus, while one species may be severely impacted by a salinity level of 4.0 dS m "1 , another species may not be affected until at least a salinity level of 6.0 dS m "1 or even 10.0 dS m "1 . See, for example, Blaylock, A.D. ("Soil salinity, salt tolerance, and growth potential of horticultural and landscape plants," Univ.
  • the level of flooding e.g., at the roots and/or the aerial parts of the plant
  • the level of flooding is different from that for a plant that is more tolerant to excessive water (e.g., wet roots and/or submergence).
  • “Severe” abiotic stress results in death in control plants (e.g., plants not expressing an LNK polypeptide of the invention), for example, at least about 10%, 20%, 30%, 40%, 50% or even more control plants die, whereas the plants of the invention (e.g., expressing a LNK polypeptide of the invention) exhibit an increased survival.
  • Maid abiotic stress is defined herein as conditions in which control plants do not die but their production is very low (e.g., reduced by at least about 30%, 40%, 50% or more), whereas the plants of the invention exhibit increased production as compared with and/or are less severely affected, as compared with control plants.
  • Normal growth conditions are conditions in which the plants are not exposed to significant biotic, abiotic, toxicological or nutritional stress, e.g., conditions in which the plants are well irrigated and exposed to normal salt conditions.
  • "normal” growth conditions are conditions in which plants are exposed to no detectable (e.g., measurable) biotic, abiotic, toxicological or nutritional stress.
  • an "increased tolerance to abiotic stress” refers to the ability of a plant or part thereof exposed to abiotic stress and transformed with an isolated or recombinant nucleic acid of the invention (e.g., encoding an LNK polypeptide of the invention) to withstand a given abiotic stress better than a control plant or part thereof (e.g., a plant or part thereof that has been exposed to the same abiotic stress but has not been transformed with an isolated or recombinant nucleic acid molecule of the invention).
  • an isolated or recombinant nucleic acid of the invention e.g., encoding an LNK polypeptide of the invention
  • Increased tolerance to abiotic stress can be measured using a variety of parameters including, but not limited to, the size and/or number of plants or parts thereof (e.g., leaf number and/or size), productivity or yield (e.g., of seed), relative water content, electrolyte leakage, stomata conductance, photosynthetic rate, internal CO 2 concentration, transpiration rate, chlorophyll fluorescence.
  • a transformed plant or part thereof comprising an isolated or recombinant nucleic acid molecule of the invention thereby having increased tolerance to the abiotic stress, would have, for example, greater growth (e.g., plant height) and/or survival and/or yield as compared with a plant or part thereof exposed to the same stress, but not having been transformed with an isolated or recombinant nucleic acid molecule of the invention.
  • An "increased yield” refers to an enhanced or elevated production of a commercially and/or agriculturally important plant, plant biomass, plant part (e.g., roots, tubers, seed, leaves, fruit), plant material (e.g., an extract) and/or other product produced by the plant (e.g., a recombinant polypeptide) by a plant or part thereof transformed with a nucleic acid of the invention (e.g., encoding an LNK polypeptide of the invention) as compared with a control plant or part thereof (e.g., a plant or part thereof that has been exposed to the same growth conditions, but has not been transformed with a nucleic acid molecule of the invention).
  • plant biomass e.g., roots, tubers, seed, leaves, fruit
  • plant material e.g., an extract
  • other product produced by the plant e.g., a recombinant polypeptide
  • a nucleic acid of the invention e.g., encoding an LNK
  • modulate refers to an increase or decrease.
  • the terms “increase,” “increases,” “increased,” “increasing” and similar terms indicate an elevation of at least about 15%, 20% 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.
  • the terms “reduce,” “reduces,” “reduced,” “reduction” and similar terms such as, “decrease,” “decreases,” “decreased” and “decreasing” mean a decrease of at least about 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more.
  • the reduction results in no or essentially no (i.e. , an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
  • Wild-type refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which a transcription factor expression is altered, e.g., in that it has been knocked out, overexpressed, or ectopically expressed.
  • heterologous means foreign, exogenous, non-native and/or non-naturally occurring.
  • homologous means native.
  • a homologous nucleotide sequence or amino acid sequence is a nucleotide sequence or amino acid sequence naturally associated with a host cell into which it is introduced
  • a homologous promoter sequence is the promoter sequence that is naturally associated with a coding sequence, and the like.
  • a "chimeric nucleic acid,” “chimeric nucleotide sequence” or “chimeric polynucleotide” comprises a promoter operably linked to a nucleotide sequence of interest that is heterologous to the promoter (or vice versa).
  • the "chimeric nucleic acid,” “chimeric nucleotide sequence” or “chimeric polynucleotide” comprises a LNK coding sequence operably associated with a heterologous promoter.
  • the "chimeric nucleic acid,” “chimeric nucleotide sequence” or “chimeric polynucleotide” comprises a LNK nucleic acid sequence that is transcribed to form a functional RNA and that is operably associated with a heterologous promoter.
  • the LNK nucleic acid sequence is operably associated with a homologous LNK promoter sequence.
  • a “promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence or functional RNA) that is operatively associated with the promoter.
  • the coding sequence may encode a polypeptide and/or a functional RNA
  • a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription.
  • promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence or functional RNA.
  • the promoter region may comprise other elements that act as regulators of gene expression.
  • the promoter region typically contain between about 100 and 1000 nucleotides, but can be as long as 2 kb, 3 kb, 4 kb or longer in length. Promoters according to the present invention can function as constitutive and/or inducible regulatory elements.
  • the LNK promoter sequence is the region of nucleic acid sequence upstream (5') of a LNK (e.g., LNK1 and LNK2) coding sequence that is responsible for spatial and temporal regulation of LNK transcription.
  • LNK1 and LNK2 are induced by light and expressed rhythmically with peak expression in the morning or at noon. Transcription is circadian regulated, but with an RNA maximum that is "later" in the 24-hour period than that of other known circadian genes, such as CCAI and LHY (Wang and Tobin, 1998; Schaffer et al., 1998).
  • LNK-like circadian rhythm or cyclic transcriptional regulation refers to this type of a relatively delayed transcription maximum.
  • the LNK promoter will allow for altering the setting of the circadian clock. For instance, by operable linking a LNK1 or LNK2 promoter sequence with a coding sequence corresponding to another circadian-regulated gene, the circadian set on this heterologously expressed protein will altered toward that of the respective LNK1 or LNK2.
  • Sequences as short as 50 or 100 nucleotides from within the 5' regulatory region of LNK may also be employed as promoter sequences according to the invention.
  • the degree to which such a sequence provides for LNK-like circadian rhythm or cyclic transcriptional regulation, when included in an expression vector, can be determined using techniques known in the art.
  • biologically active LNK1 promoter or a “biologically active LNK2 promoter” refers to a 5' regulatory region of the respective LNK gene, or a part or a variant of such a region, that, when operably linked to the 5' end of a coding sequence and introduced into a plant, results in LNK-like circadian rhythm or cyclic transcript expression of the protein encoded by the coding sequence (i.e. , in the morning or at noon).
  • a "functional" RNA includes any untranslated RNA that has a biological function in a cell, e.g., regulation of gene expression.
  • Such functional RNAs include, but are not limited to, siRNA, shRNA, miRNA, antisense RNA, ribozymes, and the like.
  • functional RNAs are operably linked to a promoter in antisense orientation relative to the promoter.
  • operably linked or “operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related.
  • a promoter is operatively linked or operably associated to a coding sequence (e.g., nucleotide sequence of interest) if it controls the transcription of the sequence.
  • operatively linked or “operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated.
  • control sequences e.g., promoter
  • the control sequences need not be contiguous with the coding sequence, as long as they function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • a "heterologous nucleotide sequence” or “heterologous nucleotide sequence of interest” as used herein is a coding sequence that is heterologous to the LNK nucleotide sequence (i.e., is not the native LNK sequence).
  • the heterologous nucleotide sequence can encode a polypeptide or a functional RNA.
  • a “heterologous promoter” is a promoter that is heterologous to the nucleotide sequence with which it is operatively associated.
  • the LNK coding sequence can be operatively associated with a heterologous promoter (e.g., a promoter that is not the native LNK promoter sequence with which the LNK coding sequence is associated in its naturally occurring state).
  • a heterologous promoter e.g., a promoter that is not the native LNK promoter sequence with which the LNK coding sequence is associated in its naturally occurring state.
  • express By the term “express,” “expressing” or “expression” (or other grammatical variants) of a nucleic acid coding sequence, it is meant that the sequence is transcribed. In particular embodiments, the terms “express,” “expressing” or “expression” (or other grammatical variants) can refer to both transcription and translation to produce an encoded polypeptide.
  • Wild-type nucleotide sequence or amino acid sequence refers to a naturally occurring (“native") or endogenous nucleotide sequence (including a cDNA corresponding thereto) or amino acid sequence.
  • nucleic acid can be used interchangeably herein unless the context indicates otherwise. These terms encompass both RNA and DNA, including cDNA, genomic DNA, partially or completely synthetic (e.g., chemically synthesized) RNA and DNA, and chimeras of RNA and DI'JA.
  • the nucleic acid, polynucleotide or nucleotide sequence may be double -stranded or single-stranded, and further may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides).
  • nucleotides can be used, for example, to prepare nucleic acids, polynucleotides and nucleotide sequences that have altered base-pairing abilities or increased resistance to nucleases.
  • the present invention further provides a nucleic acid, polynucleotide or nucleotide sequence that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, polynucleotide or nucleotide sequence of the invention (e.g., encodes a nucleic acid, polynucleotide or nucleotide sequence comprising, consisting essentially of, or consisting the complement of a LNK coding sequence of the invention).
  • Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one- letter code, or the three letter code, both in accordance with 37 C.F.R. ⁇ 1.822 and established usage.
  • nucleic acids and polynucleotides of the invention are optionally isolated.
  • An "isolated" nucleic acid molecule or polynucleotide is a nucleic acid molecule or polynucleotide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated nucleic acid molecule or isolated polynucleotide may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
  • the term “isolated” means that it is separated from the chromosome and/or cell in which it naturally occurs.
  • a nucleic acid or polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs and is then inserted into a genetic context, a chromosome, a chromosome location, and/or a cell in which it does not naturally occur.
  • the recombinant nucleic acid molecules and polynucleotides of the invention can be considered to be "isolated.”
  • an "isolated" nucleic acid or polynucleotide is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the "isolated" nucleic acid or polynucleotide can exist in a cell (e.g., a plant cell), optionally stably incorporated into the genome.
  • the "isolated" nucleic acid or polynucleotide can be foreign to the cell/organism into which it is introduced, or it can be native to the cell/organism (e.g., A. thaliana), but exist in a recombinant form (e.g., as a chimeric nucleic acid or polynucleotide) and/or can be an additional copy of an endogenous nucleic acid or polynucleotide.
  • an "isolated nucleic acid molecule” or “isolated polynucleotide” can also include a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., present in a different copy number, in a different genetic context and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule or polynucleotide.
  • the "isolated" nucleic acid or polynucleotide is substantially free of cellular material (including naturally associated proteins such as histones, transcription factors, and the like), viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • the isolated nucleic acid or polynucleotide is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
  • nucleic acid, polynucleotide or nucleotide sequence refers to a nucleic acid, polynucleotide or nucleotide sequence that has been constructed, altered, rearranged and/or modified by genetic engineering techniques.
  • the term “recombinant” does not refer to alterations that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis.
  • a "vector” is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell.
  • a vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence.
  • a "replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in the cell, i.e., capable of nucleic acid replication under its own control.
  • vector includes both viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo, and is optionally an expression vector.
  • viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo, and is optionally an expression vector.
  • a large number of vectors known in the art may be used to manipulate, deliver and express LNK polynucleotides of the invention.
  • Vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have integrated some or all of the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.
  • a “recombinant" vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (e.g., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences.
  • heterologous nucleotide sequences e.g., transgenes
  • Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects.
  • Plant viral vectors that can be used include, but are not limited to, Agrobacterium tumefaciens, Agrobacterium rhizogenes and geminivirus vectors.
  • Non-viral vectors include, but are not limited to, plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid- protein complexes, and biopolymers.
  • a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (e.g., delivery to specific tissues, duration of expression, etc).
  • fragment as applied to a nucleic acid or polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to the reference or full-length nucleotide sequence and comprising, consisting essentially of and/or consisting of contiguous nucleotides from the reference or full-length nucleotide sequence.
  • Such a fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 405, 410, 425, 450, 455, 460, 475, 500, 505, 510, 515 or 520 nucleotides or more from the reference or full-length nucleotide sequence, as long as the fragment is shorter than the reference or full-length nucleotide sequence.
  • the fragment is a biologically active nucleotide sequence, as that term is described herein.
  • a "biologically active" nucleotide sequence is one that substantially retains at least one biological activity normally associated with the wild-type nucleotide sequence, for example, the ability to drive transcription of an operatively associated coding sequence.
  • the "biologically active" nucleotide sequence substantially retains all of the biological activities possessed by the unmodified sequence.
  • substantially retains biological activity it is meant that the nucleotide sequence retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native nucleotide sequence (and can even have a higher level of activity than the native nucleotide sequence).
  • a biologically active promoter element is able to control, regulate and/or enhance the expression of a nucleotide sequence operably associated with the promoter.
  • Methods of measuring expression of a nucleotide sequence include Northern blots, RNA run-on assays and methods of measuring the presence of an encoded polypeptide (e.g., antibody based methods or visual inspection in the case of a reporter polypeptide).
  • Two nucleotide sequences are said to be “substantially identical” to each other when they share at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% sequence identity.
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids.
  • sequence similarity is similar to sequence identity (as described herein), but permits the substitution of conserved amino acids (e.g., amino acids whose side chains have similar structural and/or biochemical properties), which are well-known in the art.
  • sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman et al, J. Mol. Biol.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng et al., J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins et al, CABIOS 5: 151-153 (1989).
  • BLAST algorithm Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al, J. Mol. Biol. 215:403-410, (1990) and Karlin et al, PNAS 90:5873-5787 (1993).
  • a particularly useful BLAST program is the WU- BLAST-2 program which was obtained from Altschul et al, Methods in Enzymology, 266:460-480 (1996); http://blast.wustl/edu/blastl READMEhtml.
  • WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • the CLUSTAL program can also be used to determine sequence similarity.
  • the alignment may include the introduction of gaps in the sequences to be aligned.
  • sequences which contain either more or fewer nucleotides than the nucleic acids disclosed herein it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical nucleotides acids in relation to the total number of nucleotide bases.
  • sequence identity of sequences shorter than a sequence specifically disclosed herein will be determined using the number of nucleotide bases in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.
  • Two nucleotide sequences can also be considered to be substantially identical when the two sequences hybridize to each other under stringent conditions.
  • stringent hybridization conditions include conditions represented by a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42°C.
  • Stringent hybridization conditions and stringent hybridization wash conditions in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters.
  • nucleic acid probe assays Two nucleotide sequences considered to be substantially identical hybridize to each other under highly stringent conditions.
  • highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • LNK nucleotides of the invention hybridize with the
  • exemplary moderate stringency conditions include hybridization in 40-45% formamide, 1M NaCl, 1% SDS at 37°C, and a wash in 0.5 to 1XSSC at 55-50°C.
  • LNK nucleotides of the invention hybridize with the LNK nucleotide sequences of SEQ ID NO: 1 and/or SEQ ID NO: 3 or the complement thereof, under low stringency conditions.
  • a non-limiting example of "low stringency" hybridization conditions includes hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS at 37°C, and a wash in IX. to 2XSSC at 50-55°C.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity.
  • homologs When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% or more depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web-site, frequently asked questions page.
  • polypeptide encompasses both peptides and proteins (including fusion proteins), unless indicated otherwise.
  • a "fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame.
  • polypeptides of the invention are optionally "isolated.”
  • An "isolated” polypeptide is a polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
  • the recombinant polypeptides of the invention can be considered to be "isolated.”
  • an "isolated" polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • the "isolated" polypeptide is at least about 1%, 5%, 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more pure (w/w).
  • an "isolated" polypeptide indicates that at least about a 5-fold, 10-fold, 25-fold, 100- fold, 1000-fold, 10,000-fold, or more enrichment of the protein (w/w) is achieved as compared with the starting material.
  • the isolated polypeptide is a recombinant polypeptide produced using recombinant nucleic acid techniques.
  • the polypeptide is a fusion protein.
  • fragment as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or the full-length polypeptide (e.g., LNK) and comprising, consisting essentially of, and/or consisting of a sequence of contiguous amino acids from the reference or full-length polypeptide.
  • a fragment according to the invention may be, where appropriate, included as part of a fusion protein of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of polypeptides having a length of at least about 50, 75, 100, 125, 150, 160, 165, 170, 171, 172, 173 or 174 amino acids from the reference or full-length polypeptide, as long as the fragment is shorter than the reference or full-length polypeptide.
  • the fragment is biologically active, as that term is defined herein.
  • a "biologically active" polypeptide is one that substantially retains at least one biological activity normally associated with the wild-type polypeptide or alternatively, acts to reduce at least one biological activity normally associated with the wild-type LNK polypeptide.
  • a biologically active polypeptide is capable of modulating PRR5 and/or ELF4 expression in a transgenic plant cell or plant that recombinantly expresses the polypeptide.
  • a biologically active polypeptide is capable of modulating the expression of FKF1, SOC1 and/or FT1 in a transgenic plant cell or plant that recombinantly expresses the polypeptide.
  • the biologically active polypeptide is a LNK agonist and leads to an increased expression of PRR5, ELF4 and/or FKF1 compared to a wild-type plant or plant cell.
  • the biologically active polypeptide is a LNK antagonist and leads to a decreased expression of PRR5, ELF 4 and/or FKF1 compared to a wild-type plant or plant cell.
  • the "biologically active" polypeptide substantially retains all of the biological activities possessed by the unmodified (wild-type) LNK sequence.
  • substantially retains biological activity, it is meant that the polypeptide retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native LNK polypeptide (and can even have a higher level of activity than the native polypeptide).
  • "Introducing" in the context of a plant cell, plant tissue, plant part and/or plant means contacting a nucleic acid molecule with the plant cell, plant tissue, plant part, and/or plant in such a manner that the nucleic acid molecule gains access to the interior of the plant cell or a cell of the plant tissue, plant part or plant.
  • these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these nucleic acid molecules can be introduced into plant cells in a single transformation event, in separate transformation events, or, e.g., as part of a breeding protocol.
  • transformation refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient. Thus, a transgenic plant cell, plant tissue, plant part and/or plant of the invention can be stably transformed or transiently transformed. "Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.
  • stably introducing means that the introduced polynucleotide is stably integrated into the genome of the cell (e.g., into a chromosome or as a stable- extra-chromosomal element).
  • the integrated polynucleotide is capable of being inherited by progeny cells and plants.
  • Gene as used herein includes the nuclear and/or plastid genome, and therefore includes integration of a polynucleotide into, for example, the chloroplast genome.
  • Stable transformation as used herein can also refer to a polynucleotide that is maintained extrachromosomally, for example, as a minichromosome .
  • the terms “transformed” and “transgenic” refer to any plant, plant cell, plant tissue (including callus), or plant part that contains all or part of at least one recombinant or isolated LNK nucleic acid, polynucleotide or nucleotide sequence of the invention.
  • the recombinant or isolated nucleic acid, polynucleotide or nucleotide sequence is stably integrated into the genome of the plant (e.g., into a chromosome or as a stable extra- chromosomal element), so that it is passed on to subsequent generations of the cell or plant.
  • plant part includes reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, collenchyma cells, sclerenchyma cells, stomates, guard cells, cuticle, mesophyll cells; callus tissue; and cuttings.
  • reproductive tissues e.g., petals, sepals, stamens, pistil
  • Plant part also includes plant cells including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant organs plant cell tissue cultures, plant calli, plant clumps, and the like.
  • shoot refers to the above ground parts of a plant including the leaves and stems.
  • tissue culture encompasses cultures of tissue, cells, protoplasts and callus.
  • plant cell refers to a structural and physiological unit of the plant, which typically comprise a cell wall but also includes protoplasts.
  • a plant cell of the present invention can be in the form of an isolated single cell, tissue culture cell or can be a part of a higher-organized unit such as, for example, a plant tissue (including callus) or a plant organ. Any plant (or groupings of plants, for example, into a genus or higher order classification) can be employed in practicing the present invention including angiosperms or gymnosperms, monocots or dicots.
  • the plant cell is from a long -day plant.
  • the long-day plant is a member selected from the group consisting of Arabidopsis thaliana, wheat, barley, spinach and poplar.
  • the plant cell is from a short-day plant.
  • the short-day plant is a member selected from: rice, maize, soybean, chrysanthemum, morning glory and cosmos.
  • transgenic plants and cells of the invention include, but are not limited to, plants and cells belonging to Brassicaceae (e.g., Arabidopsis thaliana, cabbage, rapeseed), Gramineae (e.g., rice, maize, barley, wheat, switchgrass, sugar cane, sorghum), Solanaceae (e.g., tomato, eggplant, potato, tobacco), Leguminosae (e.g., soybean, garden pea, bush bean), Convolvulaceae (e.g., sweet potato), Compositae (e.g., sunflower), Euphorbiaceae (e.g., cassava, Jatropha), Rosaceae (e.g., strawberry) and/or Salix (e.g., poplar), as well as, gymnosperms, ferns and mosses.
  • Brassicaceae e.g., Arabidopsis thaliana, cabbage, rapeseed
  • Gramineae e.g., rice, mai
  • transgenic plants and plant cells of the invention include, but are not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp , alfalfa (Medicago saliva), rice (Oryza sativa), rape (Brassica napus), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tobacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp ), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp ), coco
  • Turfgrass and cells which may be employed in practicing the present invention, include but are not limited to zoysiagrasses, bentgrasses, fescue grasses, bluegrasses, St. Augustinegrasses, bermudagrasses, bufallograsses, ryegrasses and/or orchardgrasses.
  • the transgenic plants of the invention are a member selected from wheat (Tritium aestivum), corn (Zea mays) and rice (Oryza sativa).
  • the transgenic plants are alfalfa or sunflower.
  • the transgenic plants are soybean (Glycine max).
  • Exemplary transgenic vegetables and cells of the invention include, but are not limited to, Solanaceous species (e.g., tomatoes; Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota), cauliflower (Brassica oleracea), celery (apium graveolens), eggplant (Solarium melongena), asparagus (Asparagus officinalis), ochra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp ), members of the genus Cucurbita such as Hubbard squash (C.
  • Solanaceous species e.g., tomatoes; Lycopersicon esculentum
  • lettuce e.g., Lactuea sativa
  • carrots Ceaucus carota
  • cauliflower Brassica oleracea
  • celery
  • Transgenic ornamentals and ornamental cells of the invention include, azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (dianthus caryophyllus), poinsettia (Euphorbia pulcherima), and/or chrysanthemum.
  • Transgenic conifers and conifer cells of the invention include, for example pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and/or Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine
  • transgenic plants and cells of the invention are plants and cells that serve primarily as laboratory models, such as, Arabidopsis.
  • LNK nucleic acids promoters and functional RNA
  • LNK genes such as, LNK1 and LNK2 mediate gene expression that leads to the inhibition of hypocotyl elongation and accelerated flowering.
  • the invention encompasses nucleic acids that act as sense or anti-sense suppression of LNK (e.g., LNK1 and/or LNK2) expression and that act as a LNK antagonist to for example, down-regulate the endogenous expression of a LNK polypeptide.
  • LNK nucleic acids of the invention including LNK sense or LNK anti-sense sequences, are used to interfere with the expression of endogenous homologous LNK nucleic acids in a transgenic plant or plant cell.
  • sense and anti-sense technologies that may routinely be applied in preparing the compositions of the invention are known in the art.
  • sense or anti-sense sequences are introduced into a cell, where they are optionally amplified, e.g., by transcription.
  • Such sequences include both simple oligonucleotide sequences and catalytic sequences such as ribozymes.
  • a reduction or elimination of expression (i.e. , a "knock-down” or “knockout”) of a LNK protein or LNK homolog in a transgenic plant or plant cell can be obtained by introducing a LNK antisense expression cassette or expression vector into a plant or plant cell as a cDNA.
  • the regulatory protein or homolog cDNA is arranged in reverse orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector.
  • the introduced sequence need not be the full-length cDNA or gene, and need not be identical to the LNK message or gene found in the plant or plant cell type to be transformed.
  • the antisense sequence need only be capable of hybridizing to the target LNK gene or RNA.
  • the introduced sequence is of shorter length, a higher degree of homology to the endogenous LNK coding sequence will be needed for effective antisense suppression.
  • antisense sequences of various lengths can be utilized, preferably, the introduced antisense sequence in the vector/expression cassette will be at least 30 nucleotides in length, or at least 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous LNK gene in the plant cell.
  • RNA interference or microRNA-based methods
  • RNAi is a post- transcriptional, targeted gene-silencing technique that uses double -stranded RNA (dsRNA) to incite degradation of messenger RNA (mRNA) containing the same sequence as the dsRNA (Constans, The Engineer 16:36 (2002))
  • dsRNA double -stranded RNA
  • mRNA messenger RNA
  • siRNAs Small interfering RNAs, or siRNAs are produced in at least two steps: an endogenous ribonuclease cleaves longer dsRNA into shorter, 21-23 nucleotide-long RNAs (Plasterk Science 296: 1263-1265 (2002)).
  • siRNA segments then mediate the degradation of the target mRNA (Zamore, Nature Struct. Biol., 8:746-50 (2001)).
  • RNAi has been used for gene function determination in a manner similar to antisense oligonucleotides (Constans, The Engineer 16:36(2002)).
  • Expression vectors and expression cassettes that express siRNAs in transiently and stably transfected cells have been engineered to express small hairpin RNAs (shRNAs), which get processed in vivo into siRNAs-like molecules capable of carrying out gene-specific silencing (Brummelkamp et al., Science 296:550-553 (2002), and Paddison, et al, Genes & Dev.
  • RNA encoded by a transcription factor or transcription factor homolog cDNA is over-expressed can also be used to obtain co-suppression of a corresponding endogenous gene, e.g., in the manner described in U.S. Pat. No. 5,231,020.
  • Such co-suppression does not require that the entire regulatory protein cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly identical to the endogenous LNK gene of interest.
  • antisense suppression the suppressive efficiency will be enhanced as specificity of hybridization is increased, e.g., as the introduced sequence is lengthened, and/or as the sequence similarity between the introduced sequence and the endogenous regulatory protein gene is increased.
  • the invention includes LNK antagonist nucleic acids, vectors and expression cassettes that express an untranslatable form of the LNK protein mRNA, e.g., sequences comprising one or more stop codon, or nonsense mutation) can also be used to suppress expression of an endogenous LNK protein, thereby reducing or eliminating its activity and modulating one or more traits.
  • LNK antagonist nucleic acids, vectors and expression cassettes that express an untranslatable form of the LNK protein mRNA, e.g., sequences comprising one or more stop codon, or nonsense mutation
  • Methods for producing such constructs are known in the art, see, e.g., U.S. Pat. No. 5,583,021.
  • the constructs contain LNK nucleic acids comprising one or more premature stop codons introduced into LNK coding sequence.
  • the expression of an endogenous LNK gene is modified (i.e.
  • LNK1 double -stranded RNA
  • Another method for abolishing or reducing the expression of a LNK gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens . After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in one or more LNK genes (e.g., LNK1, LNK2 and both LNK1 and LNK2). Plants containing a single transgene insertion event at the desired gene can be crossed to generate homozygous plants for the mutation. Such methods are known in the art (see e.g., Koncz et al, (1992) Methods in Arabidopsis Research, World Scientific Publishing Co. Pte. Ltd., River Edge, N.J.).
  • the invention provides LNK polypeptides.
  • LNK polypeptide is intended to encompass the LNK polypeptides specifically described herein (e.g., SEQ ID NO:2 and SEQ ID NO:4) as well as equivalents thereof, e.g., that have substantially identical or similar amino acid sequences to the LNK polypeptides specifically described herein, and optionally biologically active equivalents that have one or more of the biological activities of the LNK polypeptides specifically described herein.
  • LNK polypeptide also encompasses fragments of full-length LNK polypeptides (e.g., SEQ ID NO:2 and SEQ ID NO: 4), and optionally biologically active fragments, and biologically active equivalents thereof that have substantially identical or similar amino acid sequences to a fragment of a full-length LNK polypeptide.
  • LNK polypeptide includes sequences from A. thaliana or can be a homolog from any other suitable plant species and also includes naturally occurring allelic variations, isoforms, splice variants and the like. The LNK polypeptide sequences can further be wholly or partially synthetic.
  • Biological activities associated with expression of LNK in a plant include, but are not limited to: modulating circadian rhythms, photoperiodic responses and plant phenotypes associated therewith; modulating the expression of key clock and clock output genes, such as PRR5, ELF 4 and/or FKF1; modulating flowering time and/or biomass production; modulating response to seasonal changes in photoperiod; and modulating tolerance to abiotic stress (e.g., high light intensity or low light intensity).
  • the biologically active polypeptide is a LNK agonist and leads to an increased expression of PRR5, ELF4 and/or FKF1 compared to a wild-type plant or plant cell.
  • the biologically active polypeptide is a LNK antagonist and leads to a decreased expression of PRR5, ELF4 and/or FKF1 compared to a wild-type control plant or plant cell.
  • the biologically active polypeptide modulates circadian rhythms in a transgenic plant that recombinantly expresses the LNK polypeptide compared to a wild-type control plant.
  • the biologically active polypeptide is a LNK agonist and leads to an accelerated flowering time and/or reduced biomass in a transgenic plant that recombinantly expresses the polypeptide compared to a wild-type control plant.
  • the biologically active polypeptide is a LNK antagonist and leads to a delayed flowering time and/or increased biomass in a transgenic plant that recombinantly expresses the polypeptide compared to a wild-type control plant.
  • the LNK polypeptide comprises, consists essentially of, or consists of an isolated LNK1 polypeptide of SEQ ID NO:2 or an equivalent thereof (including fragments and equivalents thereof).
  • the LNK polypeptide comprises, consists essentially of, or consists of an isolated LNK2 polypeptide of SEQ ID NO: 4 or an equivalent thereof (including fragments and equivalents thereof).
  • LNK polypeptides of the invention encompass those that have substantial amino acid sequence identity or similarity, for example, at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more amino acid sequence identity or similarity with the amino acid sequences specifically disclosed herein (e.g., SEQ ID NO:2 or SEQ ID NO:4) or a fragment thereof, optionally a biologically active fragment.
  • LNK polypeptides of the invention encompass those that have substantial amino acid sequence identity or similarity, for example, at least about 96%, 97%, 98%, 99% or more amino acid sequence identity or similarity with the amino acid sequence of SEQ ID NO: 10 or a fragment thereof, optionally a biologically active fragment.
  • the LNK polypeptide can be a fusion protein.
  • the LNK polypeptides may be useful to express as a fusion protein that can be recognized by a commercially available antibody (e.g., FLAG motifs) or as a fusion protein that can otherwise be more easily purified (e.g., by addition of a poly-His tail).
  • fusion proteins that enhance the stability of the protein may be produced, e.g., fusion proteins comprising maltose binding protein (MBP) or glutathione-S-transferase.
  • the fusion protein can comprise a reporter molecule.
  • LNK is a transcription factor and LNK fusion proteins can also be generated for use in yeast two-hybrid systems (e.g., GAL4- LNK fusions), as is known in the art.
  • LNK polypeptides specifically disclosed herein will typically tolerate substitutions in the amino acid sequence and substantially retain biological activity.
  • amino acid substitutions may be based on any characteristic known in the art, including the relative similarity or differences of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • conservative substitutions i.e. , substitution with an amino acid residue having similar properties are made in the amino acid sequence encoding the LNK polypeptide.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (see, Kyte et al, J. Mol. Biol. 157: 105 (1982)). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte et al, Id.), and these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (- 3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (- 3.9); and arginine (-4.5).
  • amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity.
  • the substitution of amino acids whose hydropathic indices are within +/- 2 is included.
  • those which are within +/-1 are included, and in certain embodiments, those within +/- 0.5 are included.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine ( ⁇ 3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • LNK polypeptides of the present invention also encompass LNK polypeptide fragments (optionally, biologically active LNK fragments), and equivalents thereof (optionally, biologically active equivalents).
  • the length of the LNK fragment is not critical.
  • Illustrative functional LNK protein fragments comprise at least about 50, 75, 100, 125, 150, 160, 165, 170, 171, 172, 173 or 174 amino acids of a LNK polypeptide.
  • the invention provides an isolated LNK polypeptide comprising, consisting essentially of, or consisting of a LNK1 amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:2; (b) an amino acid sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more amino acid sequence identity or similarity with the amino acid sequence of SEQ ID NO: 2, optionally wherein the LNK polypeptide is biologically active; and (c) a fragment of at least about 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 155, 160, 171, 172, 173 or 174 amino acid residues of the amino acid sequence of (a) or (b) above, optionally wherein the fragment is biologically active.
  • the invention provides an isolated LNK polypeptide comprising, consisting essentially of, or consisting of a LNK2 amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:4; (b) an amino acid sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more amino acid sequence identity or similarity with the amino acid sequence of SEQ ID NO: 4, optionally wherein the LNK polypeptide is biologically active; and (c) a fragment of at least about 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 155, 160, 171, 172, 173 or 174 amino acid residues of the amino acid sequence of (a) or (b) above, optionally wherein the fragment is biologically active.
  • a LNK2 amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:4; (b) an amino acid sequence having at least about 60%, 70%, 7
  • the invention further provides antibodies and antibody fragments that specifically bind to the LNK polypeptides of the invention.
  • Anti-LNK antibodies may be produced using standard procedures described in a number of texts, including Harlow and Lane ( 1988). The determination that a particular agent binds substantially only to a LNK protein (e.g., LNK1 and/or LNK2) may readily be made by using or adapting techniques known in the art.
  • One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane (1988)). Western blotting may be used to determine that a given LNK protein binding agent, such as an anti-LNKl or LNK2 monoclonal antibody, binds substantially only to LNK1 and/or LNK2.
  • antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
  • the antibody can be monoclonal or polyclonal and can be of any species of origin, including for example mouse, rat, rabbit, horse, goat, sheep or human, or can be a chimeric, humanized or human antibody. See, e.g., Walker et al, Molec. Immunol. 26:403-411 (1989).
  • the antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Patent No. 4,474,893 or 4,816,567.
  • the antibodies can also be chemically constructed according to the method disclosed in U.S. Patent No. 4,676,980.
  • Antibody fragments included within the scope of the present invention include, for example, Fab, F(ab')2, and Fc fragments, and the corresponding fragments obtained from antibodies other than IgG.
  • Such fragments can be produced by known techniques.
  • F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science 254: 1275-1281 (1989)).
  • Monoclonal antibodies according to the present invention can be produced in a hybridoma cell line according to the technique of Kohler et al., Nature 265:495-97 (1975).
  • a solution containing the appropriate antigen can be injected into a mouse and, after a sufficient time, the mouse sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells or with lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • the hybridoma cells are then grown in a suitable medium and the supernatant screened for monoclonal antibodies having the desired specificity.
  • Monoclonal Fab fragments can be produced in E. coli by recombinant techniques known to those skilled in the art. See, e.g., Huse, Science 246: 1275-1281 (1989).
  • Antibodies specific to a LNK polypeptide can also be obtained by phage display techniques known in the art.
  • the invention also provides nucleic acids encoding LNK polypeptides, optionally biologically active LNK polypeptides.
  • the nucleic acid can be from any plant species of origin (e.g., A. thaliana) or can be partially or completely synthetic.
  • the nucleic acid encoding the LNK polypeptide is an isolated nucleic acid.
  • Two nucleotide or amino acid sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences. LNK orthologs from other organisms, in particular other plants, can be routinely identified using methods known in the art (e.g., orthologs from species belonging to the Asteraceae family [also known as the Compositae family], such as a species of lettuce). For example, PCR and other amplification techniques and hybridization techniques can be used to identify such orthologs based on their sequence similarity to the sequences set forth herein.
  • the invention encompasses polynucleotides encoding the LNK polypeptides of the invention having substantial nucleotide sequence identity with the polynucleotides specifically disclosed herein encoding LNK (e.g., the LNK1 coding sequence in SEQ ID NO: l (i.e., nucleotides 302-2152), and the LNK2 coding sequence in SEQ ID NO:3 (i.e. , nucleotides 324- 2333)), or fragments thereof, and which encode a LNK polypeptide (including fragments), optionally a biologically active LNK polypeptide.
  • LNK e.g., the LNK1 coding sequence in SEQ ID NO: l (i.e., nucleotides 302-2152), and the LNK2 coding sequence in SEQ ID NO:3 (i.e. , nucleotides 324- 2333)
  • LNK e.g., the LNK1 coding sequence in SEQ ID NO: l (
  • the polynucleotides encoding the LNK polypeptides have substantial nucleotide sequence identity with the coding sequence in SEQ ID NO: 1 or the coding sequence in SEQ ID NO: 3, or fragments thereof, and which encode a LNK polypeptide (including fragments), optionally a biologically active LNK polypeptide.
  • the invention also provides polynucleotides encoding the LNK polypeptides of the invention, wherein the polynucleotide hybridizes to the complete complement of the LNK nucleic acid sequences specifically disclosed herein (e.g., SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 13 and SEQ ID NO: 15), or fragments thereof, under stringent hybridization conditions as known by those skilled in the art and encode a LNK polypeptide (including fragments), optionally a biologically active LNK polypeptide.
  • the polynucleotide hybridizes to the complete complement of the LNK nucleic acid sequences specifically disclosed herein (e.g., SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 13 and SEQ ID NO: 15), or fragments thereof, under stringent hybridization conditions as known by those skilled in the art and encode a LNK polypeptide (including fragments), optionally a biologically active LNK polypeptide.
  • the polynucleotide hybridizes to nucleotides 300 to 500, 500 to 1000, 1000 to 1500, or 1500-2000 of SEQ ID NO: l or SEQ ID NO: 3, or fragments thereof, under stringent hybridization conditions and encode a LNK polypeptide (including fragments), optionally a biologically active LNK polypeptide.
  • LNK polypeptide including fragments
  • the degeneracy of the genetic code which allows different nucleotide sequences to code for the same protein, is well known in the art.
  • plant or species-preferred codons can be used in the polynucleotides encoding the LNK polypeptides of the invention, as is also known in the art.
  • the invention also provides polynucleotides encoding fragments of a full-length LNK polypeptide, optionally biologically active fragments.
  • Exemplary polynucleotides encoding LNK fragments comprise at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 505, 510, 515 or 520 or more nucleotide bases of the coding polynucleotide sequence of SEQ ID NO: l (i.e. , nucleotides 302-2152), or SEQ ID NO:3 (i. e. , nucleotides 324- 2333).
  • the invention provides a nucleic acid (e.g., recombinant or isolated) comprising, consisting essentially of, or consisting of a nucleotide sequence encoding a LNK polypeptide, the nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising the coding nucleotide sequence of SEQ ID NO: l (i. e. , nucleotides 302-2152), or SEQ ID NO:3 (i.e.
  • nucleotides 324-2333 (b) a nucleotide sequence comprising at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 505, 510, 515 or 520 or more consecutive nucleotides of the coding nucleotide sequence of SEQ ID NO: l or SEQ ID NO: 3 (e.g., encoding a fragment, optionally a functional fragment of SEQ ID NO:3); (c) a nucleotide sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence of (a) or (b); (d) a nucleotide sequence that hybridizes to the complete complement of the nucleotide sequence of (a) or (b) under stringent hybridization conditions; and (e) a nucleot
  • the nucleotide sequence encodes the polypeptide of SEQ ID NO: 2, or an equivalent polypeptide having substantial amino acid sequence identity or similarity with SEQ ID NO: 2 (optionally, a biologically active equivalent).
  • the nucleotide sequence encodes an equivalent (optionally, a biologically active equivalent) of the polypeptide of SEQ ID NO: 2 and hybridizes to the complete complement of coding sequence of SEQ ID NO: 1 under stringent hybridization conditions.
  • the nucleotide sequence encodes the polypeptide of SEQ ID NO:4, or an equivalent polypeptide having substantial amino acid sequence identity or similarity with SEQ ID NO: 4 (optionally, a biologically active equivalent).
  • the nucleotide sequence encodes an equivalent (optionally, a biologically active equivalent) of the polypeptide of SEQ ID NO: 4 and hybridizes to the complete complement of coding sequence of SEQ ID NO: 3 under stringent hybridization conditions.
  • the nucleotide sequence is a nucleic acid selected from the group consisting of: (a) a nucleic acid encoding a polypeptide comprising the LNK1 amino acid sequence of SEQ ID NO:2; (b) a nucleic acid encoding a polypeptide having an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2; (c) a nucleic acid encoding a polypeptide having an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 2; (d) a nucleic acid encoding a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (e) a nucleic acid encoding a polypeptide
  • the invention provides an isolated LNK3 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 13; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 13 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 13 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK3 amino acid sequence of SEQ ID NO: 14; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In further embodiments, the nucleic acids are transcribed to express a functional RNA.
  • the invention also encompasses expression cassettes and vectors comprising LNK3 nucleic acids of the invention.
  • the invention provides an isolated LNK4 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 15; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 15 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 15 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK4 amino acid sequence of SEQ ID NO: 16; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16; (
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In further embodiments, the nucleic acids are transcribed to express a functional RNA.
  • the invention also encompasses expression cassettes and vectors comprising LNK4 nucleic acids of the invention.
  • the invention further provides expression cassettes comprising a LNK nucleic acid.
  • the expression cassettes comprise a LNK promoter sequence.
  • the expression cassettes comprise a nucleic acid encoding a LNK polypeptide or a nucleic acid that expresses a LNK functional RNA.
  • the invention provides expression cassettes comprising a LNK nucleic acid encoding a LNK polypeptide operably associated with a promoter.
  • the LNK nucleic acid is operably associated with a heterologous promoter.
  • the LNK nucleic acid is operably associated with endogenous LNK promoter sequence.
  • the expression cassettes comprise a LNK1 nucleic acid operably associated with a promoter.
  • the LNK1 nucleic acid is in sense orientation relative to the promoter.
  • the LNK1 nucleic acid encodes a polypeptide.
  • the expression cassettes comprise a LNK1 nucleic acid in antisense orientation relative to the promoter.
  • the LNK1 nucleic acid expresses a functional RNA.
  • the expression cassettes comprise a LNK2 nucleic acid operably associated with a promoter.
  • the LNK2 nucleic acid is in sense orientation relative to the promoter.
  • the LNK2 nucleic acid encodes a polypeptide.
  • the expression cassettes comprise a LNK2 nucleic acid in antisense orientation relative to the promoter.
  • the LNK2 nucleic acid expresses a functional RNA.
  • the expression cassettes comprise a LNK promoter sequence operably associated with a nucleic acid of interest.
  • the expression cassette comprises a LNK1 promoter.
  • the expression cassette comprises a LNK2 promoter.
  • the LNK1 nucleic acid is in nucleic acid encoding the LNK polypeptide is operably associated with a LNK promoter sequence of the invention.
  • the nucleic acid encoding the LNK polypeptide is operably associated with a heterologous promoter.
  • the heterologous promoter can be any suitable promoter known in the art
  • the promoter is a promoter for expression in plants.
  • the selection of promoters useable with the present invention can be made among many different types of promoters.
  • the choice of promoter depends upon several factors, including, but not limited to, cell- or tissue-specific expression, desired expression level, efficiency, inducibility and/or selectability.
  • tissue-specific promoter can be used (e.g., a root specific promoter).
  • a promoter inducible by other stimuli or chemicals can be used.
  • a constitutive promoter can be chosen.
  • Non-limiting examples of constitutive promoters include cestrum virus promoter (cmp) (U.S. Patent No. 7, 166,770), an actin promoter (e.g., the rice actin 1 promoter; Wang et al, Mol. Cell. Biol. 12:3399-3406 (1992); as well as U.S. Patent No. 5,641,876), Cauliflower Mosaic Virus (CaMV) 35S promoter (Odell et al, Nature 313:810-812 (1985)), CaMV 19S promoter (Lawton et al, Plant Mol. Biol.
  • an opine synthetase promoter e.g., nos, mas, ocs, etc; (Ebert et al, PNAS 84:5745-5749 (1987)), Adh promoter (Walker et al, PNAS 84:6624-6629 (1987)), sucrose synthase promoter (Yang & Russell, PNAS 87:4144-4148 (1990)), and a ubiquitin promoter.
  • the expression cassettes of the invention can further comprise enhancer elements and/or tissue preferred elements in combination with the promoter.
  • the expression cassette comprises a constitutive S35 promoter operably associated with a polynucleotide sequence encoding LNK having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:4.
  • the expression cassette comprises a constitutive S35 promoter operably associated with a polynucleotide sequence encoding LNK.
  • the heterologous promoter is a promoter for expression in a monocot plant.
  • heterologous promoter is selected from: ZmUbil (Ubiquitin), Actl (Actin), OsTubAl, (Tubulin), OsCcl (Cytochrome c), rubi3 (polyubiquitin), APX (ascorbate peroxidase), SCP1, PGD1 (phosphogluconate dehydrogenase), R1G1B (early drought induced protein) and EIF5 (translation initiation factor).
  • the heterologous promoter is a promoter for expression in a dicot plant.
  • the heterologous promoter is a CsVMV (cassava vein mosaic virus) or ScBV (sugarcane bacilliform badnavirus) promoter.
  • the heterologous promoter is an CaMV 35 S promoter.
  • tissue-specific promoters useable with the present invention include those driving the expression of seed storage proteins (e.g., 13-conglycinin, cruciferin, napin phaseolin, etc), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyi- ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al, Seed Sci. Res. 1 :209-219 (1991); as well as EP Patent No. 255378).
  • seed storage proteins e.g., 13-conglycinin, cruciferin, napin phaseolin, etc
  • zein or oil body proteins such as oleosin
  • proteins involved in fatty acid biosynthesis including acyl carrier protein, stearoyi- ACP desaturase and fatty acid desaturases (fad
  • SAMS S-adenosyi-L- methionine synthetase
  • SAMS S-adenosyi-L- methionine synthetase
  • corn light harvesting complex promoter Bansal et al, PNAS 89:3654-3658 (1992)
  • corn heat shock protein promoter O'Dell et al, EMBO J. 5:451-458 (1985); and Rochester et al, EMBO J.
  • RuBP carboxylase promoter pea small subunit RuBP carboxylase promoter (Cashmore, "Nuclear genes encoding the small subunit of ribulose-l,5-bisphosphate carboxylase” pp. 29-39 In: Genetic Engineering of Plants, Hollaender ed., Plenum Press 1983; and Poulsen et al, Mol. Gen. Genet.
  • Particularly useful for seed-specific expression is the pea vicilin promoter (Czako et al, Mol. Gen. Genet. 235:33-40 (1992); as well as U.S. Patent No. 5,625, 136).
  • Other useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al, Science 270: 1986-1988 (1995)).
  • promoters functional in plastids can be used.
  • Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5' UTR and other promoters disclosed in U.S. Patent No. 7,579,516.
  • Other promoters useful with the present invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).
  • inducible promoters can be used with the present invention.
  • inducible promoters useable with the present invention include, but are not limited to, tetracycline repressor system promoters, Lac repressor system promoters, copper-inducible system promoters, salicylate- inducible system promoters (e.g., the PRla system), glucocorticoid-inducible promoters (Aoyama et al, Plant J. 11 :605-612 (1997)), and ecdysone-inducible system promoters.
  • inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwab et al, Plant J. 4:423-432 (1993)), the UDP glucose flavonoid glycosyl- transferase promoter (Ralston et al, Genetics 119: 185-197 (1988)), the IVIPI proteinase inhibitor promoter (Cordero et al, Plant J. 6: 141-150 (1994)), the glyceraldehyde-3 -phosphate dehydrogenase promoter (Kohler et al, Plant Mol. Biol. 29: 1293-1298 (1995); Martinez et al, J.
  • inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwab et al, Plant J. 4:423-432 (1993)), the UDP glucose flavonoid glycosyl
  • promoters from viruses that infect the host plant including, but not limited to, promoters isolated from Dasheen mosaic virus, Chlorella virus (e.g., the Chlorella virus adenine methyltransferase promoter; Mitra et al, Plant Molecular Biology 26:85 (1994)), tomato spotted wilt virus, tobacco rattle virus, tobacco necrosis virus, tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus, peanut stump virus, alfalfa mosaic virus, and the like.
  • promoters isolated from Dasheen mosaic virus Chlorella virus (e.g., the Chlorella virus adenine methyltransferase promoter; Mitra et al, Plant Molecular Biology 26:85 (1994)), tomato spotted wilt virus, tobacco rattle virus, tobacco necrosis virus, tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus, peanut stump virus, alfalfa mosaic virus, and the like.
  • the expression cassettes of the invention may further comprise a transcriptional termination sequence. Any suitable termination sequence known in the art may be used in accordance with the present invention.
  • the termination region may be native with the transcriptional initiation region, may be native with the nucleotide sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens , such as the octopine synthetase and nopaline synthetase termination regions. See also, Guerineau et al, Mol. Gen. Genet. 262: 141 (1991); Proudfoot, Cell 64:671 (1991); Sanfacon et al, Genes Dev.
  • nucleotide sequence of interest is the nucleotide sequence of interest
  • the translational start site can be derived from the LNK coding sequence or, alternatively, can be the native translational start site associated with a heterologous nucleotide sequence of interest, or any other suitable translational start codon.
  • the expression cassette includes in the 5' to 3' direction of transcription, a promoter, a nucleotide sequence of interest (e.g., a heterologous nucleotide sequence of interest), and a transcriptional and translational termination region functional in plants.
  • the expression cassettes of the invention can further comprise enhancer elements and/or tissue preferred elements in combination with the promoter.
  • the expression cassette comprises a promoter sequence operably associated with the first intron of Arabidopsis Cox5c2.
  • the expression cassette comprises a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds.
  • Herbicide resistance genes generally code for a modified target protein insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. See, DeBlock et al, EMBO J. 6:2513 (1987); DeBlock et al, Plant Physiol. 91 :691 (1989); Fromm et al, BioTechnology 8:833 (1990); Gordon-Kamm et al, Plant Cell 2:603 (1990).
  • EPSPS 5-enolpyruvylshikimate-3- phosphate synthase
  • ALS acetolactate synthase
  • Resistance to glufosinate ammonium, boromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterial genes encoding phosphinothricin acetyltransferase, a nitrilase, or a 2,4- dichlorophenoxyacetate monooxygenase, which detoxify the respective herbicides.
  • Selectable marker genes that can be used according to the present invention further include, but are not limited to, genes encoding: neomycin phosphotransferase II (Fraley et al, CRC Critical Reviews in Plant Science 4: 1 (1986)); cyanamide hydratase (Maier-Greiner et al, PNAS 88:4250 (1991)); aspartate kinase; dihydrodipicolinate synthase (Peri et al., BioTechnology 11 :715 (1993)); the bar gene (Toki et al, Plant Physiol. 100: 1503 (1992); Meagher et al, Crop Sci.
  • selectable marker genes include the pat gene (for bialaphos and phosphinothricin resistance), the ALS gene for imidazolinone resistance, the HPH or HYG gene for hygromycin resistance, the Hml gene for resistance to the He- toxin, and other selective agents used routinely and known to one of ordinary skill in the art. See generally, Yarranton, Curr. Opin. Biotech. 3 :506 (1992); Chistopherson et al, PNAS 89: 6314 (1992); Yao et al, Cell 71 :63 ( 1992); Reznikoff, Mol. Microbial.
  • the nucleotide sequence of interest can additionally be operably linked to a sequence that encodes a transit peptide that directs expression of an encoded polypeptide of interest to a particular cellular compartment.
  • Transit peptides that target protein accumulation in higher plant cells to the chloroplast, mitochondrion, vacuole, nucleus, and the endoplasmic reticulum (for secretion outside of the cell) are known in the art.
  • Transit peptides that target proteins to the endoplasmic reticulum are desirable for correct processing of secreted proteins.
  • Targeting protein expression to the chloroplast has been shown to result in the accumulation of very high concentrations of recombinant protein in this organelle.
  • the pea RubP carboxylase small subunit transit peptide sequence has been used to express and target mammalian genes in plants (U.S. Patent Nos. 5,717,084 and 5,728,925).
  • mammalian transit peptides can be used to target recombinant protein expression, for example, to the mitochondrion and endoplasmic reticulum. It has been demonstrated that plant cells recognize mammalian transit peptides that target endoplasmic reticulum (U.S. Patent Nos. 5,202,422 and 5,639,947).
  • the expression cassette can comprise a 5' leader sequence that acts to enhance expression (transcription, post-transcriptional processing and/or translation) of an operably associated nucleotide sequence of interest.
  • Leader sequences are known in the art and include sequences from: picornavirus leaders, e.g., EMCV leader (Encephalomyocarditis 5' noncoding region; Elroy-Stein et al., PNAS USA, 86:6126 (1989)); potyvirus leaders, e.g., TEV leader (Tobacco Etch Virus; Allison et al., Virology, 154:9 (1986)); human immunoglobulin heavy- chain binding protein (BiP; Macajak and Sarnow, Nature 353:90 (1991)); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4; Jobling and Gehrke, Nature 325: 622 (1987)); tobacco mosaic virus leader (TMV; Gallie, MOLECULAR BIOLOG
  • the heterologous nucleotide sequence(s) of interest in the expression cassette can be any nucleotide sequence(s) of interest and can be obtained from prokaryotes or eukaryotes (e.g., bacteria, fungi, yeast, viruses, plants, mammals) or the heterologous nucleotide sequence can be synthesized in whole or in part. Further, the heterologous nucleotide sequence can encode a polypeptide or can be transcribed to produce a functional RNA. In particular embodiments, the functional RNA can be expressed to improve an agronomic trait in the plant (e.g., drought resistance, heat resistance, salt resistance, disease resistance, insect and other pest resistance [e.g., a.
  • an agronomic trait in the plant e.g., drought resistance, heat resistance, salt resistance, disease resistance, insect and other pest resistance [e.g., a.
  • the nucleotide sequence may be used in the sense orientation or antisense orientation relative to the promoter to achieve suppression of endogenous plant genes, as is known by those skilled in the art (see, e.g., U.S. Patent Nos. 5,283,184; and 5,034,323).
  • the heterologous nucleotide sequence can encode a polypeptide that imparts a desirable agronomic trait to the plant (as described above), confers male sterility, improves fertility and/or improves nutritional quality.
  • polypeptides include enzymes that can degrade organic pollutants or remove heavy metals. Such plants, and the enzymes that can be isolated therefrom, are useful in methods of environmental protection and remediation.
  • the heterologous nucleotide sequence can encode a therapeutically or pharmaceutically useful polypeptide or an industrial polypeptide (e.g., an industrial enzyme).
  • an industrial polypeptide e.g., an industrial enzyme.
  • polypeptides include, but are not limited to antibodies and antibody fragments, cytokines, hormones, growth factors, receptors, enzymes and the like.
  • Heterologous nucleotide sequences of interest suitable to confer tolerance to the herbicide glyphosate include, but are not limited to the Agrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Patent No. 5,633,435 or the glyphosate oxidoreductase gene (GOX) as described in U.S. Patent No. 5,463, 175.
  • heterologous nucleotide sequences include genes conferring resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., mutant forms of the acetolactate synthase (ALS) gene that lead to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit the action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene).
  • the bar gene encodes resistance to the herbicide basta
  • the nptll gene encodes resistance to the antibiotics kanamycin and geneticin
  • the ALS gene encodes resistance to the herbicide chlorsulfuron.
  • Suitable heterologous nucleotide sequences that confer insect tolerance include those which provide resistance to pests such as rootworm, cutworm, European Corn Borer, and the like.
  • Exemplary nucleotide sequences include, but are not limited to, a Bacillus insect control protein gene (see, e.g., WO 99/31248; U.S. Patent Nos. 5,689,052; 5,500,365; 5,880,275); Bacillus thuringiensis toxic protein genes (see, e.g., U.S. Patent Nos.
  • the heterologous nucleotide sequence can encode a reporter polypeptide (e.g., an enzyme), including but not limited to Green Fluorescent Protein, beta-galactosidase, luciferase, alkaline phosphatase, the GUS gene encoding beta- glucuronidase, and chloramphenicol acetyltransferase.
  • a reporter polypeptide e.g., an enzyme
  • an enzyme including but not limited to Green Fluorescent Protein, beta-galactosidase, luciferase, alkaline phosphatase, the GUS gene encoding beta- glucuronidase, and chloramphenicol acetyltransferase.
  • the heterologous nucleic acids may be optimized for increased expression in a transformed plant, e.g., by using plant preferred codons.
  • Methods for synthetic optimization of nucleic acid sequences are available in the art.
  • the nucleotide sequence can be optimized for expression in a particular host plant or alternatively can be modified for optimal expression in monocots or dicots. See, e.g., EP 0 359 472, EP 0 385 962, WO 91/16432; Perlak et al, PNAS 88, 3324 (1991), and Murray et al, Nucl. Acids Res. 17:477 (1989), and the like.
  • Plant preferred codons can be determined from the codons of highest frequency in the proteins expressed in that plant. Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences which may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the invention further provides vectors comprising the LNK nucleic acids of the invention and expression cassettes of the invention, including expression vectors, transformation vectors and vectors for replicating and/or manipulating the nucleotide sequences in the laboratory.
  • the vector can be a plant vector, animal (e.g., insect or mammalian) vector, bacterial vector, yeast vector or fungal vector.
  • the vector is a plant vector, a bacterial vector, or a shuttle vector that can replicate in either host under appropriate conditions. Bacterial and plant vectors are well-known in the art.
  • Exemplary plant vectors include plasmids (e.g., pUC or the Ti plasmid), cosmids, phage, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (Y ACs) and plant viruses.
  • plasmids e.g., pUC or the Ti plasmid
  • cosmids e.g., phage
  • BACs bacterial artificial chromosomes
  • Y ACs yeast artificial chromosomes
  • the invention also provides transgenic plants, plant parts and plant cells comprising the nucleic acids, expression cassettes and vectors of the invention.
  • the invention provides a cell comprising a LNK nucleic acid, expression cassette, or vector of the invention.
  • the cell can be transiently or stably transformed with the nucleic acid, expression cassette or vector.
  • the cell can be a cultured cell, a cell obtained from a plant, plant part, or plant tissue, or a cell in situ in a plant, plant part or plant tissue.
  • Cells can be from any suitable species, including plant ⁇ e.g., Arabidopsis thaliana and Helianthus annuus), bacterial, yeast, insect and/or mammalian cells.
  • the cell is a plant cell or bacterial cell.
  • the invention also provides a plant part (including a plant tissue culture) comprising a nucleic acid, expression cassette, or vector of the invention.
  • the plant part can be transiently or stably transformed with the nucleic acid, expression cassette or vector.
  • the plant part can be in culture, can be a plant part obtained from a plant, or a plant part in situ.
  • the plant part comprises a transgenic cell of the invention, as described in the preceding paragraph.
  • Seed comprising the LNK nucleic acid, expression cassette, or vector of the invention are also provided.
  • the nucleic acid, expression cassette or vector is stably incorporated into the genome of the seed.
  • the invention also contemplates a transgenic plant comprising a LNK nucleic acid, expression cassette, or vector of the invention.
  • the plant can be transiently or stably transformed with the LNK nucleic acid, expression cassette or vector.
  • the plant comprises a cell or plant part of the invention, as described in the preceding paragraphs.
  • the transgenic plant has an altered circadian rhythm compared to a non-transgenic control wild-type plant.
  • the transgenic plant has an altered PRR5, ELF4 and/or FKF1 expression profile compared to a non-transgenic control wild-type plant.
  • the transgenic plant has an accelerated flowering time and/or decreased biomass compared to a non-transgenic control wild-type plant.
  • the transgenic plant has a delayed flowering time and/or increased biomass compared to a non-transgenic control wild-type plant.
  • the transgenic plant has increased yield compared to a non-transgenic control wild-type plant.
  • the transgenic plant has an increased tolerance to an abiotic stress (e.g., high intensity light, low intensity light, drought, high temperature, low temperature and/or high salinity (e.g., salt)) compared to a non-transgenic control wild-type plant.
  • an abiotic stress e.g., high intensity light, low intensity light, drought, high temperature, low temperature and/or high salinity (e.g., salt)
  • the invention encompasses a transgenic plant stably transformed with an isolated nucleic acid encoding a LNK polypeptide.
  • the invention encompasses a transgenic plant stably transformed with an isolated nucleic acid comprising a LNK1 nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: l ; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: l or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: l or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK1 amino acid sequence of SEQ ID NO:2; (e) a nucleotide sequence
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. [0201] In additional embodiments, the invention provides a transgenic plant stably transformed with an isolated nucleic acid encoding a LNKl polypeptide selected from the group consisting of: (a) a polypeptide comprising the LNKl amino acid sequence of SEQ ID NO:2; (b) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2; and (c) an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2.
  • the invention also encompasses a transgenic plant stably transformed with an isolated LNK2 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO:3; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNKl amino acid sequence of SEQ ID NO:4; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the group consisting
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. [0203] In additional embodiments, the invention provides a transgenic plant stably transformed with an isolated nucleic acid encoding a LNK2 polypeptide selected from the group consisting of: (a) a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (b) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4; and (c) an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO:4.
  • the invention also encompasses a transgenic plant stably transformed with both an isolated LNK1 and LNK2 nucleic acid of the invention.
  • the nucleic acid sequence encodes the LNK1 polypeptide sequence of SEQ ID NO:2 and/or the LNK2 polypeptide sequence of SEQ ID NO:4.
  • the invention encompasses transgenic plant stably transformed with an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: l; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: l or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: l or the complementary strand thereof; (d) the nucleotide sequence of SEQ ID NO:3; (e) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 3 or the complementary strand thereof; (f) a nucleotide sequence comprising at least 100
  • the transgenic plant comprises at least 10, 15,
  • the transgenic plants comprises at least 10, 15, 20, 25 or 30 consecutive nucleotides of the LNK2 nucleotide sequence of SEQ ID NO:3, or the complementary strand thereof.
  • the transgenic plants are transformed with a vector comprising a LNK nucleic acid.
  • the vector comprises a LNK1 nucleic acid such as described herein.
  • the vector comprises a LNK2 nucleic acid such as described herein.
  • the invention provides a transgenic plant stably transformed with an isolated nucleic acid comprising a LNK3 nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 13; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 13 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 13 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK3 amino acid sequence of SEQ ID NO: 14; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. In further embodiments, the nucleic acids are transcribed to express a functional RNA.
  • the invention also encompasses expression cassettes and vectors comprising nucleic acids of the invention.
  • the invention provides a transgenic plant stably transformed with an isolated nucleic acid encoding a LNK3 polypeptide selected from the group consisting of: (a) a polypeptide comprising the LNK3 amino acid sequence of SEQ ID NO: 14; (b) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14; and (c) an amino acid sequence comprising at least 50, 100, 150, 200 or 250 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 14.
  • the invention provides a transgenic plant stably transformed with an isolated LNK4 nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 15; (b) a nucleotide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 15 or the complementary strand thereof; (c) a nucleotide sequence comprising at least 100 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 15 or the complementary strand thereof; (d) a nucleotide sequence encoding a polypeptide comprising the LNK4 amino acid sequence of SEQ ID NO: 16; (e) a nucleotide sequence encoding an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical
  • the nucleic acids are operably associated with a promoter. In some embodiments, the nucleic acids are in sense orientation relative to the promoter. In alternative embodiments, the nucleic acids are in antisense orientation relative to the promoter. [0211] In one embodiment, the invention provides a transgenic plant stably transformed with an isolated nucleic acid encoding a LNK4 polypeptide selected from the group consisting of: (a) a polypeptide comprising the LNK4 amino acid sequence of SEQ ID NO: 16; (b) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16; and (c) an amino acid sequence comprising at least 50, 100, 150, 200 or 250 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 16.
  • the invention also encompasses a transgenic plant stably transformed with both an isolated LNK3 and LNK4 nucleic acid of the invention.
  • the nucleic acid sequence encodes the LNK3 polypeptide sequence of SEQ ID NO: 14 and/or the LNK4 polypeptide sequence of SEQ ID NO: 16.
  • the transgenic plants are transformed with an expression cassette comprising a LNK nucleic acid.
  • the expression cassette comprises a LNK promoter sequence operably associated with a nucleic acid sequence of interest.
  • the expression cassette comprises a LNK coding sequence or is transcribed to form a functional LNK RNA.
  • the expression cassette comprises a LNK nucleic acid coding sequence or a LNK nucleic acid that is transcribed to form a functional RNA, operably associated with a promoter.
  • the promoter is an endogenous LNK promoter sequence.
  • the promoter is a heterologous promoter sequence.
  • the expression cassette comprises a selectable marker.
  • the transgenic plants are transformed with an expression cassette comprising a LNKl nucleic acid.
  • the expression cassette comprises a LNKl promoter sequence operably associated with a nucleic acid sequence of interest.
  • the expression cassette comprises a LNKl coding sequence or is transcribed to a functional LNKl RNA.
  • the expression cassette comprises a LNKl nucleic acid coding sequence or a LNKl nucleic acid that is transcribed to a functional RNA, operably associated with a promoter.
  • the promoter is an endogenous LNKl promoter sequence.
  • the promoter is a heterologous promoter.
  • the expression cassette comprises a selectable marker.
  • the transgenic plants are transformed with an expression cassette comprising a LNK2 nucleic acid.
  • the expression cassette comprises a LNK2 promoter sequence operably associated with a nucleic acid sequence of interest.
  • the expression cassette comprises a LNK2 coding sequence or is transcribed to a functional LNK2 RNA.
  • the expression cassette comprises a LNK2 nucleic acid coding sequence or a LNK nucleic acid that is transcribed to a functional RNA, operably associated with a promoter.
  • the promoter is an endogenous LNK2 promoter sequence.
  • the promoter is a heterologous promoter.
  • the expression cassette comprises a selectable marker.
  • the invention also encompasses a crop comprising a plurality of the transgenic plants of the invention, as described herein.
  • Non-limiting examples of the types of crops comprising a plurality of transgenic plants of the invention include an agricultural field, a golf course, a residential lawn or garden, a public lawn or garden, a road side planting, an orchard, and/or a recreational field (e.g., a cultivated area comprising a plurality of the transgenic plants of the invention).
  • Products harvested from the plants of the invention are also provided.
  • a harvested product include a seed (e.g., sunflower seeds and grain), a leaf, a stem, a shoot, a fruit, flower, root, biomass (e.g., for biofuel production) and/or extract.
  • a processed product produced from the harvested product is provided.
  • a processed product include a protein (e.g., a recombinant protein), an extract, a medicinal product (e.g., artemicin as an antimalarial agent), a fiber or woven textile, a fragrance, dried fruit, a biofuel (e.g., ethanol), a tobacco product (e.g., cured tobacco, cigarettes, chewing tobacco, cigars, and the like), an oil (e.g., sunflower oil, corn oil, canola oil, and the like), a nut butter, a seed butter (e.g., sunflower butter), a flour or meal (e.g., wheat flour, corn meal) and/or any other animal feed (e.g., soy, maize, barley, rice, alfalfa) and/or human food product (e.g., a processed wheat, maize, rice or soy food product).
  • processed product can be cut, dried,
  • the invention also provides methods of delivering (i.e., introducing) a nucleic acid, expression cassette or vector of the invention to a target plant or plant cell (including callus cells or protoplasts), plant parts, seed, plant tissue (including callus), and the like.
  • the invention further comprises host plants, cells, plant parts, seeds, tissue culture (including callus) transiently or stably transformed with the nucleic acids, expression cassettes or vectors of the invention.
  • the methods of the invention comprise transforming the plant, plant part or plant cell with a nucleic acid, expression cassette, or vector comprising an LNK coding sequence operably associated with a promoter.
  • the methods of the invention comprise transforming the plant, plant part or plant cell with a nucleic acid, expression cassette, or vector comprising an LNK nucleotide sequence transcribed to form a functional RNA in operable association with a promoter.
  • seed produced from the inventive transgenic plants comprise a nucleic acid, expression cassette or vector of the invention stably incorporated into the genome.
  • nucleic acids Methods of introducing nucleic acids, transiently or stably, into plants, plant tissues, cells, protoplasts, seed, callus and the like are known in the art.
  • Stably transformed nucleic acids can be incorporated into the genome.
  • Exemplary transformation methods include biological methods using viruses and Agrobacterium, physicochemical methods such as electroporation, floral dip methods, polyethylene glycol, ballistic bombardment, microinjection, and the like.
  • Other transformation technology includes the whiskers technology that is based on mineral fibers (see 'e.g., U.S. Patent No. 5,302,523 and 5,464,765) and pollen tube transformation.
  • the vector is microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (Crossway, Mol. Gen. Genetics 202: 179 (1985)).
  • the genetic material is transferred into the plant cell using polyethylene glycol (Krens et al, Nature 296:72 (1982)).
  • protoplasts are fused with minicells, cells, lysosomes, or other fusible lipid-surfaced bodies that contain the nucleotide sequence to be transferred to the plant (Fraley et al., PNAS 79: 1859 (1982)).
  • Nucleic acids may also be introduced into the plant cells by electroporation
  • plant protoplasts are electroporated in the presence of nucleic acids comprising the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the nucleic acid. Electroporated plant protoplasts reform the cell wall, divide and regenerate.
  • electroporation is that large pieces of DNA, including artificial chromosomes, can be transformed by this method.
  • Ballistic transformation typically comprises the steps of: (a) providing a plant material as a target; (b) propelling a microprojectile carrying the heterologous nucleotide sequence at the plant target at a velocity sufficient to pierce the walls of the cells within the target and to deposit the nucleotide sequence within a cell of the target to thereby provide a transformed target.
  • the method can further include the step of culturing the transformed target with a selection agent and, optionally, regeneration of a transformed plant.
  • the technique may be carried out with the nucleotide sequence as a precipitate (wet or freeze-dried) alone, in place of the aqueous solution containing the nucleotide sequence.
  • Any ballistic cell transformation apparatus can be used in practicing the present invention.
  • Exemplary apparatus are disclosed by Sandford et al, Particulate Science and Technology 5:27 (1988)), Klein et al, Nature 327:70 (1987)), and in EP 0 270 356.
  • Such apparatus have been used to transform maize cells (Klein et al, PNAS 85:4305 (1988)), soybean callus (Christou et al, Plant Physiol.
  • This apparatus comprises a bombardment chamber, which is divided into two separate compartments by an adjustable-height stopping plate.
  • An acceleration tube is mounted on top of the bombardment chamber.
  • a macroprojectile is propelled down the acceleration tube at the stopping plate by a gunpowder charge.
  • the stopping plate has a borehole formed therein, which is smaller in diameter than the microprojectile.
  • the macroprojectile carries the microprojectile(s), and the macroprojectile is aimed and fired at the borehole. When the macroprojectile is stopped by the stopping plate, the microprojectile(s) is propelled through the borehole.
  • the target is positioned in the bombardment chamber so that a microprojectile(s) propelled through the bore hole penetrates the cell walls of the cells in the target and deposit the nucleotide sequence of interest carried thereon in the cells of the target.
  • the bombardment chamber is partially evacuated prior to use to prevent atmospheric drag from unduly slowing the microprojectiles.
  • the chamber is only partially evacuated so that the target tissue is not desiccated during bombardment.
  • a vacuum of between about 400 to about 800 millimeters of mercury is suitable.
  • an aqueous solution containing the nucleotide sequence of interest as a precipitate may be carried by the macroprojectile (e.g., by placing the aqueous solution directly on the plate-contact end of the macroprojectile without a microprojectile, where it is held by surface tension), and the solution alone propelled at the plant tissue target (e.g., by propelling the macroprojectile down the acceleration tube in the same manner as described above).
  • Other approaches include placing the nucleic acid precipitate itself ("wet" precipitate) or a freeze-dried nucleotide precipitate directly on the plate-contact end of the macroprojectile without a microprojectile.
  • the nucleotide sequence is delivered by a microprojectile.
  • the microprojectile can be formed from any material having sufficient density and cohesiveness to be propelled through the cell wall, given the particle's velocity and the distance the particle must travel.
  • materials for making microprojectiles include metal, glass, silica, ice, polyethylene, polypropylene, polycarbonate, and carbon compounds (e.g., graphite, diamond).
  • Non-limiting examples of suitable metals include tungsten, gold, and iridium.
  • the particles should be of a size sufficiently small to avoid excessive disruption of the cells they contact in the target tissue, and sufficiently large to provide the inertia required to penetrate to the cell of interest in the target tissue. Particles ranging in diameter from about one-half micrometer to about three micrometers are suitable. Particles need not be spherical, as surface irregularities on the particles may enhance their carrying capacity.
  • the nucleotide sequence may be immobilized on the particle by precipitation.
  • the precise precipitation parameters employed will vary depending upon factors such as the particle acceleration procedure employed, as is known in the art.
  • the carrier particles may optionally be coated with an encapsulating agents such as polylysine to improve the stability of nucleotide sequences immobilized thereon, as discussed in EP 270356 (column 8).
  • plants may be transformed using Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Agrobacterium- ediated nucleic acid transfer exploits the natural ability of A. tumefaciens and A. rhizogenes to transfer DNA into plant chromosomes.
  • Agrobacterium is a plant pathogen that transfers a set of genes encoded in a region called T-DNA of the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, into plant cells.
  • the typical result of transfer of the Ti plasmid is a tumorous growth called a crown gall in which the T- DNA is stably integrated into a host chromosome. Integration of the Ri plasmid into the host chromosomal DNA results in a condition known as "hairy root disease".
  • the ability to cause disease in the host plant can be removed by deletion of the genes in the T-DNA without loss of DNA transfer and integration.
  • the DNA to be transferred is attached to border sequences that define the end points of an integrated T-DNA.
  • Agrobacterium mediated transformation has been achieved in several monocot species, including cereal species such as rye, maize (Rhodes et al, Science 240:204 (1988)), and rice (Hiei et al, Plant J. 6:271 (1994)).
  • A. rhizogenes Transformation using A. rhizogenes has developed analogously to that of A. tumefaciens and has been successfully utilized to transform, for example, alfalfa, Solarium nigrum L., and poplar (U.S. Patent No. 5,777,200). As described by U.S. Patent No. 5, 773,693, it is preferable to use a disarmed A. tumefaciens strain, however, the wild-type A. rhizogenes may be employed. An illustrative strain of A. rhizogenes is strain 15834.
  • the Agrobacterium strain is modified to contain the nucleotide sequences to be transferred to the plant.
  • the nucleotide sequence to be transferred is incorporated into the T-region and is typically flanked by at least one T-DNA border sequence, optionally two T-DNA border sequences.
  • a variety of Agrobacterium strains are known in the art particularly, and can be used in the methods of the invention. See, e.g., Hooykaas, Plant Mol. Biol. 13:327 (1989); Smith et al, Crop Science 35:301 (1995); Chilton, PNAS 90, 3119 (1993); Mollony et al, Monograph Theor. Appl. Genet NY 19, 148 (1993); Ishida et al, Nature Biotechnol. 14:745 (1996); and Komari et al, The Plant J. 10: 165 (1996).
  • the Ti (or Ri) plasmid contains a vir region.
  • the vir region is important for efficient transformation, and appears to be species-specific.
  • cointegrate the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the PMLJ1 shuttle vector of DeBlock et al, EMBO J. 3: 1681 (1984), and the non-oncogenic Ti plasmid pGV2850 described by Zambryski et al, EMBOJ. 2:2143 ( 1983).
  • the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation.
  • the other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector described by Bevan, Nucleic Acids Research 12: 871 1 (1984), and the non-oncogenic Ti plasmid PAL4404 described by Hoekma, et al, Nature 303 : 179 (1983).
  • Binary vector systems have been developed where the manipulated disarmed T-DNA carrying the heterologous nucleotide sequence of interest and the vir functions are present on separate plasmids. In this manner, a modified T-DNA region comprising foreign DNA (the nucleic acid to be transferred) is constructed in a small plasmid that replicates in E. coll This plasmid is transferred conjugatively in a tri-parental mating or via electroporation into A. tumefaciens that contains a compatible plasmid with virulence gene sequences. The vir functions are supplied in trans to transfer the T-DNA into the plant genome. Such binary vectors are useful in the practice of the present invention.
  • super-binary vectors are employed. See, e.g., U.S. Patent No. 5,591,615 and EP 604662.
  • Such a super- binary vector has been constructed containing a DNA region originating from the hypervirulence region of the Ti plasmid pTiBo542 (Jin et al, J. Bacterial. 169:4417 (1987)) contained in a super-virulent A. tumefaciens A281 exhibiting extremely high transformation efficiency (Hood et al, Biotechnol. 2:702 ( 1984); Hood et al, J. Bacterial. 168: 1283 (1986); Komari et al, J. Bacterial. 166: 88
  • Exemplary super-binary vectors known to those skilled in the art include pTOK162 (Japanese patent Appl. (Kokai) No. 4-222527, EP 504,869, EP 604,662, and United States Patent No. 5,591,616) and pTOK233 (Komari, Plant Cell Reports 9:303 (1990); Ishida et al, Nature Biotechnology 14:745 (1996)).
  • Other super- binary vectors may be constructed by the methods set forth in the above references.
  • Super-binary vector pTOK162 is capable of replication in both E. coli and in A. tumefaciens .
  • the vector contains the virB, virC and virG genes from the virulence region of pTiBo542.
  • the plasmid also contains an antibiotic resistance gene, a selectable marker gene, and the nucleic acid of interest to be transformed into the plant.
  • the nucleic acid to be inserted into the plant genome is typically located between the two border sequences of the T region.
  • Super-binary vectors of the invention can be constructed having the features described above for pTOK162.
  • the T-region of the super-binary vectors and other vectors for use in the invention are constructed to have restriction sites for the insertion of the genes to be delivered.
  • the DNA to be transformed can be inserted in the T- DNA region of the vector by utilizing in vivo homologous recombination. See, Herrera-Esterella et al, EMBO J. 2:987 (1983); Horch et al, Science 223:496 (1984).
  • homologous recombination relies on the fact that the super-binary vector has a region homologous with a region of bpR322 or other similar plasmids.
  • a desired gene is inserted into the super-binary vector by genetic recombination via the homologous regions.
  • the nucleotide sequence of interest is incorporated into the plant nuclear genome, typically flanked by at least one T-DNA border sequence and generally two T- DNA border sequences.
  • Plant cells may be transformed with Agrobacterici by any means known in the art, e.g., by co-cultivation with cultured isolated protoplasts, or transformation of intact cells or tissues.
  • the first uses an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cultured protoplasts. Identification of transformed cells or plants is generally accomplished by including a selectable marker in the transforming vector, or by obtaining evidence of successful bacterial infection.
  • Protoplasts which have been transformed by any method known in the art, can also be regenerated to produce intact plants using known techniques.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently root. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • the regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner.
  • the plants are grown and harvested using conventional procedures.
  • transgenic plants may be produced using the floral dip method (See, e.g., Clough et al, Plant J. 16:735-743 (1998), which avoids the need for plant tissue culture or regeneration.
  • plants are grown in soil until the primary inflorescence is about 10 cm tall.
  • the primary inflorescence is cut to induce the emergence of multiple secondary inflorescences.
  • the inflorescences of these plants are typically dipped in a suspension of Agrobacterium containing the vector of interest, a simple sugar (e.g., sucrose) and surfactant. After the dipping process, the plants are grown to maturity and the seeds are harvested.
  • Transgenic seeds from these treated plants can be selected by germination under selective pressure (e.g., using the chemical bialaphos).
  • Transgenic plants containing the selectable marker survive treatment and can be transplanted to individual pots for subsequent analysis.
  • the invention encompasses a method of expressing a
  • the method comprises transforming the plant, plant part or plant cell with a nucleic acid, expression cassette, or vector of the invention comprising a nucleotide sequence encoding the LNK polypeptide.
  • the plant can be transiently or stably transformed.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the expressed polypeptide is a LNK1 polypeptide as described herein.
  • the expressed polypeptide is a LNK2 polypeptide as described herein.
  • the nucleic acid, expression cassette, or vector expresses both a LNK1 and LNK2 polypeptide as described herein.
  • the invention additionally provides a method of expressing a LNK nucleic acid in a plant, plant part or plant cell.
  • the method comprises transforming the plant, plant part or plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK polynucleotide sequence of the invention.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the transcription of the LNK polynucleotide produces a functional RNA.
  • the functional RNA is a member selected from: siR A, shR A, miR A, antisense RNA and a ribozyme.
  • the functional RNA is a LNK antagonist.
  • the polynucleotide encodes a LNK polypeptide of the invention.
  • the LNK polypeptide is a LNK agonist.
  • the LNK polypeptide is a LNK antagonist.
  • the invention additionally provides a method of expressing a LNK1 nucleic acid in a plant, plant part or plant cell.
  • the method comprises transforming the plant, plant part or plant cell with a LNK1 nucleic acid, expression cassette, or vector operably associated with a LNK polynucleotide sequence of the invention.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the method comprises transforming the plant, plant part or plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK1 polynucleotide sequence of the invention.
  • the transcription of the LNK1 polynucleotide produces a functional RNA.
  • the functional RNA is a member selected from: siRNA, shRNA, miRNA, antisense RNA and a ribozyme.
  • the functional RNA is a LNK1 antagonist.
  • the polynucleotide encodes a LNK1 polypeptide of the invention.
  • the LNK1 polypeptide is a LNK1 agonist.
  • the LNK1 polypeptide is a LNK1 antagonist.
  • the invention additionally provides a method of expressing a LNK2 nucleic acid in a plant, plant part or plant cell.
  • the method comprises transforming the plant, plant part or plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK2 polynucleotide sequence of the invention.
  • the method further comprises the steps of (i) regenerating a stably transformed plant from the stably transformed plant cell; and (ii) expressing the nucleotide sequence in the plant.
  • the method comprises transforming the plant, plant part or plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK2 polynucleotide sequence of the invention.
  • the transcription of the LNK2 polynucleotide produces a functional RNA.
  • the functional RNA is a member selected from: siRNA, shRNA, miRNA, antisense RNA and a ribozyme.
  • the functional RNA is a LNK2 antagonist.
  • the polynucleotide encodes a LNK2 polypeptide of the invention.
  • the LNK2 polypeptide is a LNK2 agonist.
  • the LNK2 polypeptide is a LNK2 antagonist.
  • the invention provides a method of modulating a circadian response in a plant, comprising: stably transforming a plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK polynucleotide sequence of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the LNK polynucleotide sequence in the plant (e.g., in an amount effective to modulate a circadian response of the plant).
  • the resulting transgenic plant displays a modulated (i.e., altered) expression of PRR5, ELF 4 and/or FKF1.
  • the resulting transgenic plant displays and increased expression of LNK protein. In some embodiments, the resulting transgenic plant displays and increased expression of PRR5, ELF4 and/or FKF1. In alternative embodiments, the resulting transgenic plant displays a decreased (i.e. , reduced) expression of LNK protein. In other embodiments, the resulting transgenic plant displays a decreased expression of PRR5, ELF4 and/or FKF1.
  • the LNK polynucleotide is a LNK agonist. In alternative embodiments, the LNK polynucleotide is a LNK antagonist.
  • the invention provides a method of modulating a circadian response in a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector encoding an LNK polypeptide of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence of the nucleic acid in the plant (e.g., in an amount effective to modulate a circadian response of the plant).
  • the resulting transgenic plant displays a modulated expression of PRR5, ELF4 and/or FKF1.
  • the resulting transgenic plant displays and increased expression of LNK protein.
  • the resulting transgenic plant displays and increased expression o ⁇ PRR5, ELF4 and/or FKFl. In alternative embodiments, the resulting transgenic plant displays a decreased (/ ' . e. , reduced) expression of LNK protein. In other embodiments, the resulting transgenic plant displays a decreased expression of PRR5, ELF 4 and/or FKFl.
  • the LNK polypeptide is a LNK agonist. In alternative embodiments, the LNK polypeptide is a LNK antagonist.
  • the invention provides a method of modulating the adjustment of daily and/or seasonal rhythms in a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector encoding an LNK polypeptide of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence of the nucleic acid in the plant (e.g., in an amount effective to alter daily and/or seasonal rhythms in a plant).
  • the resulting transgenic plant displays a modulated (i.e. , altered) expression of PRR5, ELF4 and/or FKFl .
  • the resulting transgenic plant displays and increased expression of LNK protein. In some embodiments, the resulting transgenic plant displays and increased expression of PRR5, ELF4 and/or FKFl . In alternative embodiments, the resulting transgenic plant displays a decreased (i.e., reduced) expression of LNK protein. In other embodiments, the resulting transgenic plant displays a decreased expression of PRR5, ELF4 and/or FKFl.
  • the LNK polypeptide is a LNK agonist. In alternative embodiments, the LNK polypeptide is a LNK antagonist.
  • the invention provides a method of modulating the adjustment of daily and/or seasonal rhythms in a plant, wherein the method comprises: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK polynucleotide sequence of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective in an amount effective to alter daily and/or seasonal rhythms in a plant).
  • the resulting transgenic plant displays a modulated (i.e., altered) expression of PRR5, ELF 4 and/or FKFl .
  • the resulting transgenic plant displays and increased expression of LNK protein.
  • the resulting transgenic plant displays and increased expression ⁇ , ELF4 and/or FKF1.
  • the resulting transgenic plant displays a decreased (/ ' . e. , reduced) expression of LNK protein.
  • the resulting transgenic plant displays a decreased expression of PRR5, ELF 4 and/or FKF1.
  • the LNK polynucleotide is a LNK agonist.
  • the LNK polynucleotide is a LNK antagonist.
  • transcription of the LNK polynucleotide produces a functional RNA.
  • the functional RNA is a member selected from: siRNA, shRNA, miRNA, antisense RNA, and a ribozyme
  • the invention provides a method of increasing the yield from a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK polynucleotide sequence of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to increase the yield from the plant).
  • the invention provides a method of the prolonging (e.g., increasing) the life span and/or delaying development of a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK polynucleotide sequence of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to prolong the life span and/or delay the development of a plant).
  • transcription of the LNK polynucleotide produces a functional RNA.
  • the functional RNA is a member selected from: siRNA, shRNA, miRNA, antisense RNA, and a ribozyme.
  • the functional RNA is a LNK antagonist.
  • the invention provides a method of increasing the yield from a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector encoding an LNK polypeptide of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to increase the yield from the plant).
  • the invention provides a method of the prolonging (e.g., increasing) the life span and/or delaying development of a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector encoding an LNK polypeptide of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to prolong the life span and/or delay the development of a plant).
  • the resulting transgenic plant displays a decreased (i.e. , reduced) expression of LNK protein.
  • the resulting transgenic plant displays a decreased expression of PRR5, ELF 4 and/or FKF1.
  • the LNK polypeptide is a LNK antagonist.
  • the invention provides a method of delaying the flowering time of a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector operably associated with a LNK polynucleotide sequence of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to delay the flowering time of a plant).
  • transcription of the LNK polynucleotide produces a functional RNA.
  • the functional RNA is a member selected from: siRNA, shRNA, miRNA, antisense RNA, and a ribozyme.
  • the functional RNA is a LNK antagonist.
  • the invention provides a method of delaying the flowering time of a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector encoding an LNK polypeptide of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to delay the flowering time of the plant).
  • the LNK polypeptide is a LNK antagonist.
  • the invention provides a method of increasing the biomass of a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector encoding an LNK polypeptide of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to increase the biomass of the plant).
  • the LNK polypeptide is a LNK antagonist.
  • the invention provides a method of accelerating the flowering time of a plant, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector encoding an LNK polypeptide of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to accelerate the flowering time of the plant).
  • the LNK polypeptide is a LNK agonist.
  • the invention also encompasses a method of increasing tolerance of a plant to abiotic stress, the method comprising: (a) stably transforming a plant cell with a nucleic acid, expression cassette, or vector encoding an LNK polypeptide of the invention; (b) regenerating a stably transformed plant from the stably transformed plant cell of (a); and (c) expressing the nucleotide sequence in the plant (e.g., in an amount effective to increase the tolerance of the plant to an abiotic stress).
  • the method optionally includes the further step of exposing the plant to the abiotic stress (e.g., growing the plant under the abiotic stress conditions).
  • Abiotic stress is as described elsewhere herein.
  • the abiotic stress comprises drought, salt stress, submergence stress and/or waterlogging stress, and/or stress after removal of a submergence stressor (e.g., desubmergence stress).
  • the methods of the invention can be used to increase the yield of a plant and/or increase tolerance of a plant to abiotic stress.
  • the abiotic stress comprises drought, salt stress, waterlogging stress, submergence stress, and/or desubmergence stress.
  • the transgenic plants of the invention are grown under the abiotic stress conditions.
  • the transgenic plants are grown under normal cultivation conditions.
  • the invention provides a method of increasing yield and/or increasing tolerance of a plant to abiotic stress, the method comprising: (a) stably transforming a plant cell with an isolated nucleic acid encoding a polypeptide selected from the group consisting of (i) a polypeptide comprising the LNKl amino acid sequence of SEQ ID NO:2; (ii) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2; and (iii) an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2.
  • the invention further includes regenerating a stably transformed plant from the stably transformed plant cell; and expressing the nucleotide sequence in the plant.
  • the invention provides a method of increasing yield and/or increasing tolerance of a plant to abiotic stress, the method comprising: (a) stably transforming a plant cell with an isolated nucleic acid encoding a polypeptide selected from the group consisting of (i)a polypeptide comprising the LNK2 amino acid sequence of SEQ ID NO:4; (ii) an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4; and (iii) an amino acid sequence comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800 or more contiguous amino acid residues of the amino acid sequence of SEQ ID NO:4.
  • the invention further includes regenerating a stably transformed plant from the stably transformed plant cell; and expressing the nucleotide sequence in the plant.
  • the clock and photoreceptor mutants used in this study were prr7-3;prr9-l (31), tocl -101(42), phyA-211 ;phyB-9(43) and cryl-bl04;cry2-l(44).
  • hypocotyl length measurements wild-type, linkl, Unk2 and Hnkl;link2 seedlings were grown on 0.8% agar under complete darkness, continuous white light (LL), short day (8h light/ 16 h dark) photoperiods, continuous red (100 ⁇ rrfV 1 ) or continuous blue light (10 ⁇ m ' V 1 ), and the final length of the hypocotyls was measured after 4 days.
  • Light effects on hypocotyl elongation were calculated normalizing hypocotyl length under each light regime relative to hypocotyl length of the same genotypes under constant dark conditions.
  • the 35S:ZN ⁇ 7 :YFP construct was assembled using the coding region of the At5g64l 70.2 gene model, according to the Arabidopsis Information Resource (TAIRIO), through Gateway technology (Invitrogen) using the pEarly gate 101 as destination vector (46).
  • TAIRIO Arabidopsis Information Resource
  • the leaves were infected from the abaxial side using a syringe without needle, and 3.5 days later different discs of infected leaves were analyzed.
  • Imaging was completed using a LSM 5 Pascal Axioplan2 confocal microscope equipped with an argon ion (488nm) excitation laser system and a X20 objective lens. Image manipulation was completed with LSM image browser software. Similar localization patterns were observed in different infected leaves. qRT-PCR
  • Arabidopsis thaliana LNK1 (splice variant 2) as query in BLASTP tool (worldwideweb at phytozome.net/). Protein sequences were aligned using Clustal Omega program. A maximum likelihood phylogenetic tree was constructed using SeaView Version 4 (Gouy, M. et al , 2010). Bootstrap analysis with 1000 replicates was performed. Gouy, M., G RNA-seq reads were estimated using Illumina Pipeline version 1.3. Reads were quality-filtered using the standard Illumina process and demultiplexed with 2 allowed barcode mismatches. Sequence files were generated in FASTQ format. Table 1 provides a summary table of main read count statistics.
  • TopHat parameters were used with the exception of maximun intron length parameter, that was set to a value of 5000 nt following estimated values reported in [Hong et al, D. Mol. Biol. Evol. 23:2392-2404 (2006)] .
  • Transcript abundances were estimated using Cufflink [Trapnell et al., "Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks", Nature Protocols 7:562-578 (2012)] with default parameter setting.
  • RNA samples were prepared following the TruSeqTM RNA Sample Preparation Guide (Illumina).
  • RNA was polyA-purified, fragmented and first-strand cDNA synthesized by reverse transcriptase (Superscript II-Invitrogen) and random hexamers. This was followed by RNA degradation and second strand cDNA synthesis. End repair process and addition of a single 'A' nucleotide to the 3' ends allowed ligation of multiple indexing adapters. Then, an enrichment step of 12 cycles of PCR was performed. Library validation included quality control and quantification. Samples were pooled to create 17-multiplexed DNA libraries and they were single-end sequenced with an Illumina Genome Analyzer II kit on the Illumina GAIIx platform, providing lOObp sequences.
  • RNA-seq reads were estimated using Illumina Pipeline version 1.3. Reads were quality-filtered using the standard Illumina process and demultiplexed with 2 allowed barcode mismatches. Sequence files were generated in FASTQ format. Table 1 provides a summary table of main read count statistics. Sequence data was made available from GEO repository (accession number: GSE43865). The TopHat suite [Trapnell et al., "TopHat: discovering splice junctions with RNA-Seq". Bioinformatics doi: 10.1093/bioinformatics/btpl20] was used to map reads to the A. thaliana TAIR10 reference genome [The Arabidopsis Genome Initiative 2000] .
  • a non-specific prefiltering step was conducted in order to filter out genes with less than two counts per million reads in at least three samples resulting in 21143 (22628) - out from 33602 genes - that were considered for further analysis, in the Col vs linkllink2 (time-course) experiment. Differences in the RNA composition for each library, was taken into account through a normalization step using the trimmed mean of M-values (TMM) methodology.
  • LNK genes integrate light and clock signaling networks at core of
  • LNK night-light inducible and clock regulated genes
  • LNK1 and LNK2 are more strongly induced by light in the middle of the night, when the clock is most responsive to this signal. Further analysis revealed that the morning phased LNK1 and LNK2 genes control circadian rhythms and photomorphogenic responses. In addition, because they regulate the expression of a subset of clock and flowering time genes in the afternoon, they are required for photoperiodic dependent flowering. LNK1 and LNK2 themselves are directly repressed by members of the TIMING OF CAB1 EXPRESSION (TOCi PSEUDO RESPONSE REGULATOR (PRRI) family of core-clock genes in the afternoon and early night. Thus, LNK1 and LNK2 integrate early light signals with temporal information provided by core oscillator components to control the expression of afternoon genes, allowing plants to keep track of seasonal changes in day-length.
  • PRRI TIMING OF CAB1 EXPRESSION
  • CABII expression is acutely induced by red light pulses, but the effectiveness of this treatment oscillates during a 24-hr day, with maximal effects when photosynthetic activity is expected to be at its peak during the day, and minimal effects during the night (15-17).
  • Clock regulation of light signaling also influences physiological processes such as germination (18), stem elongation (19, 20), and the clock itself (16, 21, 22).
  • germination 18, stem elongation (19, 20), and the clock itself (16, 21, 22).
  • brief light pulses are most effective in resetting the phase of circadian rhythms during the night rather than during the subjective day (i.e. the phase that would have been illuminated if the plants were kept under light/dark cycles) (21-23). This phenomenon is shared across kingdoms suggesting that it is critical for the appropriate adjustment of circadian rhythms to the environment (24).
  • Clock entrainment is most sensitive to light pulses given during the night, a treatment that simulates seasonal changes in day-length. Consistent with this, the subset of 65 genes responding at least twice as strongly to the night-light treatment was significantly enriched in clock genes, a phenomenon that was specific for this particular class of light regulated genes (data not provided). Clock genes are also enriched among those with oscillations that are robust to different experimental conditions, such as continuous light, continuous darkness, short days, long days, temperature cycles, etc. (26). Thus, we reasoned that the list of genes that are more effectively induced by night-light and also cycle under multiple conditions should contain new candidate clock regulators.
  • LNK1 and LNK2 are of medium size, about 66 kDa, with 35% sequence similarity across their entire length.
  • LNK3 (AT3G12320) and LNK4 (AT5G06980) proteins are smaller, with 60% sequence similarity, and with a third of conserved positions also shared with LNK1/LNK2 ( Figure 6).
  • LNK homologs can be found throughout land-plants, including nonvascular plants.
  • LNK3 and LNK4 appear to be the result of a recent duplication event within the Brassicaceae. ( Figure 7). Since LNK1 responded most strongly to the night-light treatment (Table 6), we focused on LNK1 and its closest homolog, LNK2.
  • LNK1 and LNK2 regulate light signaling and biological timing
  • Unk2 mutants also had longer hypocotyls than WT plants in red light (Figure 10), whilst the differences in hypocotyl length were not statistically significant under most other light conditions ( Figures 2 A and 10).
  • the linkl ;link2 double mutant had significantly longer hypocotyls than either single mutant or WT seedlings under most light conditions tested, and the phenotype was stronger under red or white light than blue- light ( Figures 2A and 10).
  • LNK1 and LNK2 activate clock controlled genes with afternoon peak
  • LNK proteins lack known functional domains, but transiently expressed
  • LNKLYFP localized to the nuclei of Nicotiana benthamiana cells, suggesting a role in the regulation of gene expression (Figure 3A).
  • Down-regulated genes also included the flowering time genes FLAVINBINDING KELCH REPEAT F-BOX 1 (FKF1; Figure 3D), which was also present in the cluster of genes with peak expression at ZT10 ( Figure 3C), as well as FLOWERING LOCUS T (FT) and SUPPRESOR OF CONSTANS OVEXPRESSION 1 (SOCI) ( Figure 13). All three genes are positive regulators of flowering time, with FKF1 acting upstream of FT and SOCI. Therefore, the late flowering of linkl ;link2 mutants under LD is likely due to reduced FKF1 expression, which in turn leads to reduced ⁇ and SOCI mRNA levels ( Figure 13).
  • PRR5 expression is severely affected in linkl ;link2 mutants under free-running conditions
  • CCA1 and LHY also promote the expression of PRR9 and PRR7(3 ⁇ ) which, sequentially with PRR5 and TOC1/PRR1 , repress CCA1 and LHY expression throughout the remaining of the day and early night(32-34).
  • ELF4, ELF3 and LUX ARPvHYTHMO(LUX) proteins form a complex during the evening that represses TOC1 expression, allowing CCA1 and LHY mRNA levels to start rising again in the late part of the night (19).
  • PRR7 expression during the second day in continuous light was increased to eight hours on the third day, consistent with a lengthening of circadian period by approximately 2.5 hours in the linkl;link2 mutant compared to WT plants ( Figure 4 A-D).
  • the phase of CCAl expression was delayed less than four hours in nk2 single mutants on the third day ( Figure 14), consistent with an increase in period length of rhythmic leaf movement of approximately one hour (Figure 2E).
  • LNKl and LNK2 act initially as activators of a subset of genes with peak expression in the afternoon, such as PRR5 ( Figures 3F and 4E), ELF4 ( Figure 3F) and FKFl ( Figure 3D), which later affect the rhythmic expression of other core-clock and clock-output genes.
  • LNKl and LNK2 are repressed by members of the TOCl/PRRl family of clock genes
  • LNKl -4 promoters we compared the expression patterns of LNKl and LNK2 in WT, tocl, or prr9;prr7 mutant plants, entrained under light/dark cycles and then transferred to constant light conditions.
  • their mRNA levels at the trough of the circadian oscillations were increased in the prr9;prr7 double mutant ( Figure 5 A and B).
  • LNK genes a partially redundant family of plant-specific genes that control photomorphogenic and photoperiodic responses, as well as circadian rhythms.
  • LNKl and LNK2 are regulated by the phytochrome photoreceptors and predominantly affect responses to red light, pointing to an important role in phytochrome signaling. In turn, they are expressed rhythmically with peak expression in the morning or at noon, most likely as a result of their repression by members of the TOC1/PR 1 family of core clock regulators during the afternoon and early night.
  • LNKl and LNK2 link phytochrome and circadian signaling to regulate many physiological processes, including time keeping by the clock itself.
  • LNK genes A comparison of LNK genes with other morning expressed clock genes is informative. Like LNK genes, CCAl and LHY are light induced genes whose mRNAs reach peak levels in the early morning. CCAl and LHY, however, lengthen the period of circadian rhythms, while LNKl and LNK2 shorten it (36, 37). In contrast, PRR9 and PRR7 are similar to LNKl and LNK2 in that they are expressed during the morning and early afternoon, are induced by light, and that they decrease period length and promote flowering (31). However, different from LNKl and LNK2, which at least under constant light do not seem to be required for normal CCAl and LHY expression (Figure 4), PRR9 and PRR7 are repressors of CCAl and H7 (31).
  • LNKl and LNK2 are plant-specific proteins without recognizable functional domains. This is reminiscent of the clock components ELF3 and ELF4, which only very recently were shown to participate in an evening phased protein complex that represses the expression of TOC1 and PRR9 (19).
  • ELF3 and ELF4 which only very recently were shown to participate in an evening phased protein complex that represses the expression of TOC1 and PRR9 (19).
  • LNKl and LNK2 mRNAs reach maximum levels in the morning or at noon, but that their positive effects on gene expression appear to be concentrated in the afternoon. Together with the nuclear localization of LNKl, this suggests that LNKl and/or LNK2 are regulatory proteins under post-transcriptional and/or post-translational control, similar to several other clock proteins.
  • AtLNK homologs in Oryza sativa we also searched for orthologs in Sorghum bicolor and Glycine max using the BlastP algorithm of PlantGDB for sorghum (http://www.plantgdb.org/SbGDB/cgi-bin/blastGDB.pl) and soy (http://www.plantgdb.org/GmGDB/cgi-bin/blastGDB.pl). respectively.
  • Sorghum bicolor and Glycine max were aligned by MEGA6.
  • the evolutionary history of taxa was inferred by the neighbor-joining method according to Saitou N and Nei M. (Saitou N. and Nei M. (1987).
  • the neighbor-joining method A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406-425).
  • the bootstrap consensus tree inferred from 1000 replicates [Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791] is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed.
  • LNK function is conserved in dicots and monocots
  • OsLNKl and OsLNK2 rice genes display diurnal oscillations in gene expression with peak levels in the early morning ( Figure 16).
  • the expression of the rice LNK gene orthologs is regulated by the circadian clock.
  • Figure 17 we examined the molecular function of OsLNK2 in the control of the rice circadian clock as well as in the regulation of flowering time in this species using wild type rice plants and the mutant line PGF_2C-50209.L (Figure 17). Then, we grew plants from this mutant background side by side with wild-type plants under LD conditions (16 h light /8 h darkness).
  • LNK1 and LNK2 genes regulate flowering time under long day conditions promoting the expression of the flowering time gene FKF1.
  • FKF1 flowering time gene
  • OsLNK2 also functions promoting the expression of this gene.
  • LNK genes have a conserved function in the control of FKF1, a key flowering time gene in rice and Arabidopsis.
  • OsLNK2 we analyzed the role of OsLNK2 in the regulation of rice clock genes.
  • LNKl and LNK2 genes accelerate the pace of the circadian clock activating the expression of clock genes peaking in the afternoon or early evening, such as PRR5.
  • LNK1 cDNA sequence (AT5G64170.2; SEQ ID NO: 1):
  • LNKl polypeptide sequence (AT5G64170.2; SEQ ID NO:2):
  • LNKl cDNA sequence (AT5G64170.1; SEQ ID NO:5):
  • LNK1 polypeptide sequence (AT5G64170.1; SEQ IDNO:6):
  • LNK1 promoter sequence (SEQ ID NO: 7):
  • LNK2 cDNA including splice variants e.g., AT3G54500.2; AT3G54500.1 or AT3G54500.4: LNK2 cDNA sequence (AT3G54500.3; SEQ ID NO:3).
  • the start codon (atg) is double-underlined and in bold:
  • LNK2 polypeptide sequence (AT3G54500.3; SEQ ID NO:4):
  • LNK2 cDNA sequence (AT3G54500.1; SEQ ID NO:8). The start codon
  • LNK2 polypeptide sequence (AT3G54500.1; SEQ ID NO:9):
  • LNK2 cDNA sequence (AT3G54500.2; SEQ ID NO: 10). The start codon
  • LNK2 polypeptide sequence (AT3G54500.2; SEQ ID NO: 11)
  • LNK2 promoter sequence SEQ ID NO: 12
  • the start codon (atg) is double-underlined and in bold:

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Abstract

La présente invention concerne des polypeptides LNK isolés et des acides nucléiques codant pour ceux-ci. L'invention concerne également des procédés d'introduction d'un acide nucléique codant pour des polypeptides LNK et/ou des antagonistes de LNK dans une cellule végétale, une partie de plante ou une plante, par exemple pour augmenter la tolérance à un stress abiotique, pour retarder le développement et/ou prolonger la durée de vie d'une plante et/ou pour augmenter le rendement d'une plante. L'invention concerne également des plantes transformées, des tissus végétaux transformés, des cellules végétales transformées et une graine végétale transformée comprenant les acides nucléiques, cassettes d'expression et vecteurs de l'invention.
PCT/IB2014/058836 2013-02-06 2014-02-06 Plantes transgéniques pour lnk Ceased WO2014122607A2 (fr)

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