# US 12,595,487 B2 · Methods and compositions for improving agronomic characteristics of C4 plants

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- Number: US 12,595,487 B2
- Document ID: US12595487B2
- Status: Granted
- Kind: B2
- Application number: 17/768,822
- Filed: 2020-10-15
- Published: 2024-11-28
- Granted: 2026-04-07
- Earliest claimed: 2019-10-15
- Applicants: Donald Danforth Plant Science Center; The Board of Trustees of the University of Illinois; The United States of America, AS Represented By the Secretary of Agriculture
- Inventors: Douglas K Allen; Jennifer J. Arp; Thomas P. Brutnell; Stephen P. Moose
- Web page: https://patentgrep.com/patent/US12595487B2

## CPC

- C12N 9/88: Lyases (4.)
- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- A01H 1/06: Processes for producing mutations, e.g. treatment with chemicals or with radiation (specific mutations prepared by genetic engineering on plant cell or plant tissues C12N15/00 {; process for producing transgenic plants C12N15/82})
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/8261: {with agronomic (input) traits, e.g. crop yield}
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- Y02P 20/582: Recycling of unreacted starting or intermediate materials
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

C4 plants comprising a genetic disruption of the PEPCK biosynthetic pathway are provided. The plants exhibit beneficial traits when compared to wild type plants cultivated under similar growth conditions. Methods of using the plants are also provided. The growth conditions can be adverse growth conditions. Adverse growth conditions can be limiting nitrogen conditions, drought conditions, waterlogged conditions, high disease load, high pest load, or combinations thereof.

## Claims

### Claim 1

A genetically modified maize plant having an increased grain biomass in limited nitrogen conditions as compared to that of a control maize plant cultivated under similar limited nitrogen conditions, wherein the modified maize plant comprises: a modified GRMZM2G001696 gene that comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 and that encodes a phosphoenolpyruvate carboxykinase (PEPCK) enzyme at a reduced level or with reduced enzymatic activity as compared to that of a wild type maize plant; and wherein the grain biomass of the modified maize plant in limited nitrogen conditions is increased by about 20% as compared to that of a control maize plant cultivated under similar limited nitrogen conditions.

### Claim 2 (depends on 1)

The modified maize plant of claim 1, wherein the modified GRMZM2G001696 gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 is characterized by having an insertion of a Dissociator (Os) element at the flanking edge of exon 8.

### Claim 3 (depends on 1)

The modified maize plant of claim 1, wherein the modified GRMZM2G001696 gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2 is characterized by having an insertion of a Os element in the 3′ untranslated region (UTR).

### Claim 4

A method of increasing the grain biomass of a maize plant cultivated under limited nitrogen conditions as compared to that of a control maize plant cultivated under similar limited nitrogen conditions, the method comprising: introducing into a maize plant, a genetic modification into an endogenous GRMZM2G001696 gene, thereby obtaining a modified maize plant comprising a modified GRMZM2G001696 gene that comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 and that encodes a PEPCK enzyme at a reduced level or with reduced enzymatic activity as compared to that of a control maize plant; and cultivating the modified maize plant in limited nitrogen conditions, wherein the grain biomass of the modified maize plant in limited nitrogen conditions is increased by about 20% as compared to that of a control maize plant cultivated under similar limited nitrogen conditions.