# US 6,294,716 B1 · Plants having modified response to ethylene by transformation with an ETR nucleic acid

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- Number: US 6,294,716 B1
- Document ID: US6294716B1
- Status: Granted
- Kind: B1
- Application number: 08/714,524
- Filed: 1996-09-16
- Granted: 2001-09-25
- Earliest claimed: 1993-07-01
- Assignees: California Institute of Technology
- Inventors: Elliott M. Meyerowitz; Caren Chang; Anthony B. Bleecker
- Web page: https://patentgrep.com/patent/US6294716B1

## CPC

- C07K 14/415: from plants
- C12N 15/8249: {involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

## Claims

### Claim 1

An isolated nucleic acid comprising a plant ETR nucleic acid encoding an ETR protein, said ETR protein having at least 50% overall similarity to the ETR protein sequence of Arabidopsis thaliana as set forth in SEQ ID NO:3, and at least 55% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana, wherein the expression of said ETR protein encoded by said ETR nucleic acid in a plant cell results in an increased or decreased response to ethylene by said cell.

### Claim 2 (depends on 1)

The isolated nucleic acid of claim 1 wherein said ETR protein has at least 70% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana.

### Claim 3 (depends on 2)

The isolated nucleic acid of claim 2 wherein said ETR protein has at least 85% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana.

### Claim 4

An isolated nucleic acid comprising a plant ETR nucleic acid encoding an ETR protein, said ETR protein having at least 50% overall similarity to the ETR protein sequence of Arabidopsis thaliana as set forth in SEQ ID NO:3, and at least 55% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana, wherein the expression of said ETR protein encoded by said ETR nucleic acid in a plant cell results in an increased response to ethylene by said cell.

### Claim 5

An isolated modified plant ETR nucleic acid comprising a precursor ETR nucleic acid which has been modified to encode a modified ETR protein comprising the substitution, insertion or deletion of an amino acid residue in the N-terminal 316 amino acids of the ETR protein encoded by said precursor ETR nucleic acid, wherein said modified ETR protein has at least 50% overall similarity to the ETR protein sequence of Arabidopsis thaliana as set forth in SEQ ID NO:3, and at least 55% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana, wherein the expression of said modified ETR protein encoded by said isolated modified plant ETR nucleic acid in a plant cell results in an increased or decreased response to ethylene by said cell.

### Claim 6 (depends on 5)

The isolated modified plant ETR nucleic acid according to claim 5 wherein said modified ETR protein comprises the substitution of a selected amino acid residue in said precursor ETR protein with a different amino acid and wherein said selected amino acid residue in said precursor ETR protein is equivalent to an amino acid residue selected from the group consisting of Ala-31, Pro-36, Ile-62, Cys-65 and Ala-102 in the ETR protein sequence of Arabidopsis thaliana.

### Claim 7 (depends on 5)

The isolated modified plant ETR nucleic acid of claim 5 wherein said ETR protein has at least 70% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana.

### Claim 8 (depends on 5)

The isolated modified plant ETR nucleic acid of claim 5 wherein said ETR protein has at least 85% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana.

### Claim 9

An isolated modified plant ETR nucleic acid comprising a precursor ETR nucleic acid which has been modified to encode a modified ETR protein comprising the substitution or deletion of an amino acid residue in the N-terminal 316 amino acids of the ETR protein encoded by said precursor ETR nucleic acid, wherein said modified ETR protein has at least 50% overall similarity to the ETR protein sequence of Arabidopsis thaliana as set forth in SEQ ID NO:3, and at least 55% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana, wherein the expression of said modified ETR protein encoded by said isolated modified plant ETR nucleic acid in a plant cell results in a decreased response to ethylene by said cell.

### Claim 10 (depends on 9)

The isolated modified plant ETR nucleic acid of claim 9 wherein the modified ETR protein has a single amino acid substitution in the N-terminal 316 amino acids.

### Claim 11 (depends on 9)

The isolated modified plant ETR nucleic acid of claim 9 wherein the modified ETR protein has a single amino acid deletion in the N-terminal 316 amino acids.

### Claim 12 (depends on 5)

A recombinant nucleic acid comprising a promoter operably linked to the modified plant ETR nucleic acid of claim 5.

### Claim 13 (depends on 12)

The recombinant nucleic acid according to claim 12 wherein said promoter is heterologous to said modified plant ETR nucleic acid and causes expression of said modified plant ETR nucleic acid in a plant cell.

### Claim 14 (depends on 13)

The recombinant nucleic acid according to claim 13 wherein said promoter is a tissue-specific promoter or temporal-specific promoter.

### Claim 15 (depends on 13)

The recombinant nucleic acid according to claim 13 wherein said promoter is inducible.

### Claim 16

A recombinant nucleic acid comprising a promoter operably linked to an ETR nucleic acid, wherein said ETR nucleic acid hybridizes with a probe having the sequence represented in SEQ ID NO:2 at hybridization conditions of 50° C. in 5×SSPE and washing conditions of 50° C. in 0.2×SSPE, wherein the expression of an ETR protein encoded by said ETR nucleic acid in a plant cell results in an increased or decreased response to ethylene by said cell.

### Claim 17

A method of producing a plant comprising transformed plant cells having a detectable increased or decreased response to ethylene as compared to untransformed cells of a corresponding wild-type plant, said method comprising the steps of:
a) transforming at least one plant cell with a modified ETR nucleic acid comprising a precursor ETR nucleic acid which has been modified to encode a modified ETR protein comprising the substitution, insertion or deletion of an amino acid residue in the N-terminal 316 amino acids of the ETR protein encoded by said precursor ETR nucleic acid, wherein said modified ETR protein has at least about 50% overall similarity to the ETR protein sequence of Arabidopsis thaliana as set forth in SEQ ID NO:3, and at least about 55% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana;
b) regenerating plants from one or more of the thus transformed plant cells; and
c) selecting a plant comprising said transformed plant cells having a detectable increased or decreased response to ethylene.

### Claim 18 (depends on 17)

The method according to claim 17 wherein said modified ETR nucleic acid is operably linked to a tissue-specific promoter.

### Claim 19

A method of producing a plant comprising transformed plant cells having a detectable decrease in response to ethylene as compared to untransformed cells of a corresponding wild-type plant, said method comprising the steps of:
a) transforming at least one plant cell with a modified ETR nucleic acid comprising a precursor ETR nucleic acid which has been modified to encode a modified ETR protein comprising a substitution, insertion or deletion of an amino acid residue in the N-terminal 316 amino acids of the ETR protein encoded by said precursor ETR nucleic acid, wherein said modified ETR protein has at least about 50% overall similarity to the ETR protein sequence of Arabidopsis thaliana as set forth in SEQ ID NO:3, and at least about 55% similarity to the N-terminal 316 amino acids of said ETR protein sequence of Arabidopsis thaliana;
b) regenerating plants from one or more of the thus transformed plant cells; and
c) selecting a plant comprising said transformed plant cells having a detectable decrease in response to ethylene.

### Claim 20 (depends on 19)

The method of claim 19 wherein the modified ETR protein has a single amino acid substitution in the N-terminal 316 amino acids.

### Claim 21 (depends on 19)

The method of claim 19 wherein the modified ETR protein has a single amino acid deletion in the N-terminal 316 amino acids.

### Claim 22 (depends on 13)

A plant cell transformed with the recombinant nucleic acid of claim 13.

### Claim 23 (depends on 22)

A plant comprising the plant cell of claim 22.

### Claim 24 (depends on 5)

A plant comprising plant cells transformed with the isolated modified plant ETR nucleic acid of claim 5 wherein said plant cells have an increased or decreased response to ethylene as compared to untransformed cells of a corresponding wild-type plant.

### Claim 25 (depends on 23)

The plant according to claim 23 wherein said modified ETR protein comprises the substitution of a selected amino acid residue with a different amino acid, wherein said selected amino acid residue is equivalent to an amino acid residue selected from the group consisting of Ala-31, Pro-36, Ile-62, Cys-65 and Ala-102 in the ETR protein sequence of Arabidopsis thaliana.

### Claim 26 (depends on 24)

The plant according to claim 24 wherein a tissue-specific promoter is operably linked to said modified ETR nucleic acid.

### Claim 27 (depends on 26)

The plant according to claim 26 wherein said plant is fruit-bearing and said tissue specific promoter is a fruit-specific promoter.

### Claim 28 (depends on 27)

The plant according to claim 27 wherein said plant has a decrease in the rate of fruit ripening.

### Claim 29 (depends on 28)

Fruit from the plant according to claim 28.

### Claim 30 (depends on 29)

The fruit according to claim 29 wherein said fruit is tomato.

### Claim 31 (depends on 17)

A plant produced by the method of claim 17.

### Claim 32 (depends on 9)

A plant comprising plant cells transformed with the isolated modified plant ETR nucleic acid of claim 9, said plant cells having a detectable decrease in response to ethylene as compared too untransformed cells of a corresponding wild-type plant.