# US 10,501,751 B2 · Isolated polynucleotides and polypeptides, construct and plants comprising same and methods of using same for increasing nitrogen use efficiency of plants

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- Number: US 10,501,751 B2
- Document ID: US10501751B2
- Status: Granted
- Kind: B2
- Application number: 15/672,374
- Filed: 2017-08-09
- Published: 2018-02-01
- Granted: 2019-12-10
- Earliest claimed: 2012-12-26
- Assignees: Evogene Ltd.
- Applicants: Evogene Ltd.
- Inventors: Adi Etzioni; Hagai Karchi
- Web page: https://patentgrep.com/patent/US10501751B2

## CPC

- C12N 15/8271: {for stress resistance, e.g. heavy metal resistance}
- C07K 14/415: from plants
- C12N 15/8247: {involving modified lipid metabolism, e.g. seed oil composition}
- C12N 15/8261: {with agronomic (input) traits, e.g. crop yield}
- C12N 15/8273: {for drought, cold, salt resistance}
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Provided are isolated polypeptides which are at least 80% homologous to SEQ ID NOs: 496-794, 2898-3645, and 3647-4855, isolated polynucleotides which are at least 80% identical to SEQ ID NOs: 1-495 and 795-2897, nucleic acid constructs comprising same, transgenic cells expressing same, transgenic plants expressing same and method of using same for increasing fertilizer use efficiency, nitrogen use efficiency, yield, growth rate, biomass, vigor, oil content, photosynthetic capacity, seed yield, fiber yield, fiber quality, fiber length, and/or abiotic stress tolerance of a plant.

## Claims

### Claim 1

A method of increasing growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant as compared to a control plant of the same species which is grown under the same growth conditions, the method comprising over-expressing within the plant a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 524 or an orthologue polypeptide thereof having an amino acid sequence at least 95% identical to SEQ ID NO: 524, wherein said orthologue polypeptide is capable of increasing growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant, thereby increasing the growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of the plant.

### Claim 2 (depends on 1)

The method of claim 1, further comprising selecting plants over-expressing said polypeptide for an increased growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant as compared to a control plant of the same species which is grown under the same growth conditions, wherein said abiotic stress comprises nitrogen deficiency.

### Claim 3 (depends on 1)

The method of claim 1, wherein said polypeptide is selected from the group consisting of SEQ ID NOs: 524, and 3055-3058.

### Claim 4 (depends on 1)

The method of claim 1, wherein said polypeptide is expressed from a polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 315, 29, and 958-961.

### Claim 5 (depends on 1)

The method of claim 1, wherein said polypeptide is expressed from a polynucleotide comprising the nucleic acid sequence set forth by SEQ ID NO: 315 or 29.

### Claim 6 (depends on 1)

The method of claim 1, further comprising growing the plant over-expressing said polypeptide under the abiotic stress, wherein said abiotic stress comprises nitrogen deficiency.

### Claim 7 (depends on 1)

The method of claim 1, wherein said abiotic stress is selected from the group consisting of salinity, drought, osmotic stress, water deprivation, flood, etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nitrogen deficiency, nutrient excess, atmospheric pollution and UV irradiation.

### Claim 8

A method of producing a crop comprising growing a crop plant over-expressing a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 524 or an orthologue polypeptide thereof comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 524 as compared to a control plant of the same species which is grown under the same growth conditions, wherein the crop plant is derived from plants selected for increased growth rate, increased biomass, increased seed yield, increased photosynthetic capacity, and/or increased abiotic stress tolerance as compared to a control plant of the same species which is grown under the same growth conditions, wherein said abiotic stress comprises nitrogen deficiency, and the crop plant having the increased growth rate, increased biomass, increased seed yield, increased photosynthetic capacity, and/or increased abiotic stress tolerance, thereby producing the crop.

### Claim 9 (depends on 8)

The method of claim 8, wherein said polypeptide is expressed from a polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 315, 29, and 958-961.

### Claim 10 (depends on 8)

The method of claim 8, wherein said over-expressing is of a polypeptide which comprises the amino acid sequence set forth by SEQ ID NO: 524.

### Claim 11 (depends on 8)

The method of claim 8, wherein said polypeptide is expressed from a polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 315 and 29.

### Claim 12

A plant cell comprising a nucleic acid construct comprising an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide which comprises the amino acid sequence set forth in SEQ ID NO: 524 or an orthologue polypeptide thereof comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 524, and a heterologous promoter for directing transcription of said nucleic acid sequence in a plant cell, wherein said amino acid sequence is capable of increasing growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant as compared to a control plant of the same species which is grown under the same growth conditions.

### Claim 13

A transgenic plant comprising a nucleic acid construct comprising an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide which comprises the amino acid sequence set forth in SEQ ID NO: 524 or an orthologue polypeptide thereof comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 524, and a heterologous promoter for directing transcription of said nucleic acid sequence in a plant cell, wherein said amino acid sequence is capable of increasing growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant as compared to a control plant of the same species which is grown under the same growth conditions.

### Claim 14

A method of growing a crop, the method comprising seeding seeds and/or planting plantlets of a plant transformed with a nucleic acid construct comprising an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide which comprises the amino acid sequence set forth in SEQ ID NO: 524 or an orthologue polypeptide thereof comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 524, and a heterologous promoter for directing transcription of said nucleic acid sequence in a plant cell, wherein said amino acid sequence is capable of increasing growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant as compared to a control plant of the same species which is grown under the same growth conditions, wherein the plant is derived from plants selected for at least one trait selected from the group consisting of: increased abiotic stress tolerance, increased biomass, increased growth rate increased yield and increased photosynthetic capacity as compared to a non-transformed plant, thereby growing the crop, wherein said abiotic stress comprises nitrogen deficiency.

### Claim 15

A method of selecting a transformed plant having increased growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance as compared to a control plant of the same species which is grown under the same growth conditions, the method comprising:
(a) providing the transgenic plants transformed with a nucleic acid construct comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 524 or an orthologue polypeptide thereof comprising an amino acid sequence at least 95% identical to SEQ ID NO: 524 operably linked to a promoter for directing expression in a plant cell, wherein said amino acid sequence is capable of increasing growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant as compared to a control plant of the same species which is grown under the same growth conditions; and
(b) selecting from said plants a plant having increased growth rate, increased biomass, increased seed yield, increased photosynthetic capacity, and/or increased abiotic stress tolerance as compared to a control plant of the same species which is grown under the same growth conditions, wherein said abiotic stress comprises nitrogen deficiency,
thereby selecting the plant having increased, yield, growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance as compared to the control plant of the same species which is grown under the same growth conditions.

### Claim 16 (depends on 12)

The plant cell of claim 12, wherein said promoter is a constitutive promoter.

### Claim 17 (depends on 1)

The method of claim 1, wherein said orthologue polypeptide comprises a deletion, an insertion and/or a conservative substitution of amino acid(s) with respect to the polypeptide set forth by SEQ ID NO:524 while maintaining the ability to increase growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant, wherein said abiotic stress comprises nitrogen deficiency.

### Claim 18 (depends on 1)

The method of claim 1, wherein said orthologue polypeptide is a naturally occurring polypeptide.

### Claim 19 (depends on 1)

The method of claim 1, wherein said orthologue polypeptide is selected from the group consisting of SEQ ID NO: 3055-3058.

### Claim 20 (depends on 1)

The method of claim 1, wherein said over-expressing is of the polypeptide set forth by SEQ ID NO: 524.

### Claim 21 (depends on 8)

The method of claim 8, wherein said orthologue polypeptide comprises a deletion, an insertion and/or a conservative substitution of amino acid(s) with respect to the polypeptide set forth by SEQ ID NO:524 while maintaining the ability to increase growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant, wherein said abiotic stress comprises nitrogen deficiency.

### Claim 22 (depends on 8)

The method of claim 8, wherein said orthologue polypeptide is a naturally occurring polypeptide.

### Claim 23 (depends on 8)

The method of claim 8, wherein said orthologue polypeptide is selected from the group consisting of SEQ ID NO: 3055-3058.

### Claim 24 (depends on 14)

The method of claim 14, wherein said orthologue polypeptide comprises a deletion, an insertion and/or a conservative substitution of amino acid(s) with respect to the polypeptide set forth by SEQ ID NO:524 while maintaining the ability to increase growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant, wherein said abiotic stress comprises nitrogen deficiency.

### Claim 25 (depends on 14)

The method of claim 14, wherein said orthologue polypeptide is a naturally occurring polypeptide.

### Claim 26 (depends on 14)

The method of claim 14, wherein said isolated polynucleotide encodes the polypeptide set forth by SEQ ID NO: 524.

### Claim 27 (depends on 14)

The method of claim 14, wherein said orthologue polypeptide is selected from the group consisting of SEQ ID NO: 3055-3058.

### Claim 28 (depends on 15)

The method of claim 15, wherein said orthologue polypeptide comprises a deletion, an insertion and/or a conservative substitution of amino acid(s) with respect to the polypeptide set forth by SEQ ID NO:524 while maintaining the ability to increase growth rate, biomass, seed yield, photosynthetic capacity, and/or abiotic stress tolerance of a plant, wherein said abiotic stress comprises nitrogen deficiency.

### Claim 29 (depends on 15)

The method of claim 15, wherein said orthologue polypeptide is a naturally occurring polypeptide.

### Claim 30 (depends on 15)

The method of claim 15, wherein said polynucleotide encodes the polypeptide set forth by SEQ ID NO: 524.

### Claim 31 (depends on 15)

The method of claim 15, wherein said orthologue polypeptide is selected from the group consisting of SEQ ID NO: 3055-3058.

### Claim 32 (depends on 12)

The plant cell of claim 12, wherein said polynucleotide encodes the polypeptide set forth by SEQ ID NO: 524.

### Claim 33 (depends on 13)

The transgenic plant of claim 13, wherein said polynucleotide encodes the polypeptide set forth by SEQ ID NO: 524.