# US 12,180,493 B2 · Constructs and methods for controlling stomatal closure in plants

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- Number: US 12,180,493 B2
- Document ID: US12180493B2
- Status: Granted
- Kind: B2
- Application number: 16/321,644
- Filed: 2017-08-04
- Published: 2021-09-16
- Granted: 2024-12-31
- Earliest claimed: 2016-08-05
- Assignees: Limagrain Europe S.A.; Commissariat A L'energie Atomique Et Aux Energies Alternatives
- Applicants: Biogemma; Commissariat A L'energie Atomique Et Aux Energies Alternatives
- Inventors: Nathalie Leonhardt; Ludovic Martin; Serge Chiarenza; Helene Jacquet; Laurent Nussaume; Marie Javelle
- Web page: https://patentgrep.com/patent/US12180493B2

## CPC

- C12N 15/8261: {with agronomic (input) traits, e.g. crop yield}
- A01H 1/00: Processes for modifying genotypes {; Plants characterised by associated natural traits} (A01H4/00 takes precedence)
- A01H 5/10: Seeds
- A01H 5/12: Leaves
- A01H 6/4684: {Zea mays [maize]}
- C07K 14/415: from plants
- 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)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- 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

The present invention is related to expressing an AHA5 protein in plants, and preferentially a mutated AHA5 protein leading to a constitutive activity of AHA5, to control stomatal closure and improve tolerance to drought conditions and yield in plants.

## Claims

### Claim 1

A method for decreasing water loss in a plant comprising introducing a genetic mutation into a plasma membrane H+-ATPase 5 (AHA5) gene of Arabidopsis or Zea Mays to generate a gene with a genetic mutation, wherein the gene with the genetic mutation encodes a mutated AHA5 protein, in a cell of the plant with gene editing techniques,
wherein said mutated AHA5 protein is selected from:
(a) a mutated AHA5 protein comprising the amino acid sequence of SEQ ID NO: 31;
(b) a mutated AHA5 protein comprising the amino acid sequence of SEQ ID NO: 30; and
(c) a mutated AHA5 protein comprising the amino acid sequence of SEQ ID NO: 23; and
regenerating said cell to obtain a whole plant, wherein the mutated AHA5 protein is at least expressed in guard cells and wherein said plant has decreased water loss.

### Claim 2 (depends on 1)

The method according to claim 1, comprising introducing the genetic mutation into the AHA5 gene in a cell of the plant with CRISPR/Cas9.

### Claim 3 (depends on 1)

The method according to claim 1, comprising introducing the genetic mutation into the AHA5 gene in a cell of the plant with TALENs.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the Zea mays AHA5 gene in the cell of the plant into which the mutation is introduced encodes SEQ ID NO:2.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said mutated AHA5 protein is a mutated AHA5 protein comprising the amino acid sequence of SEQ ID NO:31.

### Claim 6 (depends on 1)

The method according to claim 1, wherein said mutated AHA5 protein is a mutated AHA5 protein comprising the amino acid sequence of SEQ ID NO:30.

### Claim 7 (depends on 1)

The method according to claim 1, wherein said mutated AHA5 protein is a mutated AHA5 protein comprising the amino acid sequence of SEQ ID NO:23.