Method and compositions for conferring abiotic stress resistance on plants by modulating activity of an f-box protein family

Abstract

The present disclosure pertains to methods of enhancing abiotic stress resistance in a plant or seed by altering one or more KMD family of genes of the plant or seed. The altered KMD family of genes includes, without limitation, KMD1, KMD2, KMD3, KMD4, a gene encoding a protein with at least 40% amino-acid sequence identity with any one of KMD 1-4, or combinations thereof. The present disclosure also pertains to plants or seeds that include one or more of the aforementioned altered KMD family of genes. The present disclosure also pertains to methods of growing a plant or a seed in a field by applying a plant or seed that includes one or more of the aforementioned altered KMD family of genes to the field.

Description

TITLEMETHOD AND COMPOSITIONS FOR CONFERRING ABIOTIC STRESS RESISTANCE ON PLANTS BY MODULATING ACTIVITY OF AN F-BOX PROTEIN FAMILYSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under MCB- 1856248, awarded by the National Science Foundation; IOS-0618286, awarded by the National Science Foundation; 2019- 67013-29191, awarded by the U.S. Department of Agriculture; and 2023-67013-39413, awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 558,082, filed on February 26, 2024. The entirety of the aforementioned application is incorporated herein by reference.SEQUENCE DISCLOSURE STATEMENT

[0003] Pursuant to 37 C.F.R. § 1.834, Applicants have submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF60544.P054WO.xml”. The date of the creation of the Sequence Listing is February 25, 2025. The size of the Sequence Listing is 63,000 bytes. Applicants hereby incorporate by reference the material in the Sequence Listing.BACKGROUND

[0004] A need exists for more effective methods of developing plants and seeds that are resistant to abiotic stress. A need also exists for such plants and seeds. Numerous embodiments of the present disclosure aim to address the aforementioned needs.SUMMARY

[0005] In some embodiments, the present disclosure pertains to methods of enhancing abiotic stress resistance in a plant or seed. In some embodiments, the methods of the present disclosure include altering one or more KMD family of genes of the plant or seed (i.e., KMD family of genes that encode F-box proteins). In some embodiments, the altered KMD family of genes includes, without limitation, KMD1, KMD2, KMD3, KMD4, a gene encoding a protein with at least 40% amino-acid sequence identity with any one of KMD 1-4, or combinations thereof.

[0006] Additional embodiments of the present disclosure pertain to plants or seeds that include one or more of the aforementioned altered KMD family of genes. Further embodiments of the present disclosure pertain to methods of growing a plant or a seed in a field by applying a plant or seed that includes one or more of the aforementioned altered KMD family of genes to the field.DRAWINGS

[0007] FIGS. 1A-1B show phylogenetic analyses of KMD F-box proteins. FIG. 1A shows representative KMD F-box proteins from the eudicot Arabidopsis thaliana (At; green highlight), the rice monocot Oryza saliva. (Os; orange highlight), and the mosses Physcomitrella patens (Pp) and Selaginella moellendorffii (Sm). FIG. IB shows KMD family members from the eudicot Arabidopsis (At) and the monocots rice (Os), maize (Z. mays), wheat (T. Asetivum), barley (H. vulgare), and biscuit grass (P. vaginatum).

[0008] FIG. 2 shows regulation of cytokinin activity in plants. Shown are elements of the cytokinin biosynthesis (green box), degradation (brown box) and signal transduction pathways (yellow box). The number of genes for each signaling element in rice is listed in parentheses.

[0009] FIGS. 3A-3B show that the KMD family regulates the pre-anthesis drought stress response in rice. Drought stress was applied by withholding water for five days to wild-type (wt) and event 5 kmd mutants (kmd2 / 3 / 4-E5 and kmdl / 2 / 3 / 4-E5'). FIG. 3A shows shoots of drought-stressed wild type and event 5 kmd mutants following 14 days of watering in the recovery period after drought stress. FIG. 3B shows seed set per panicle (%) for comparing control unstressed plants to those subjected to drought stress (n=9 panicles, three plants; error bars are standard error).

[0010] FIGS. 4A-4C show that the KMD family regulates the post-anthesis drought stress response in rice. FIG. 4A shows maximum quantum yield (Fv / Fm) for photosynthesis in kmd mutants from event E5 (kmd2,3,4-E5 and kmdl ,2,3,4-E5) compared to the wild type (Kt-wt) under drought stress (d) or control (c) watered conditions at the post-anthesis stage. Two rounds of drought stress were applied, each for three days (VWC < 10%). Note the ‘priming’ that occurs following an initial drought exposure in the kmd mutant lines. FIG. 4B shows leaves of wild type and kmd mutants on day 4 of drought stress. Note that the wild type still exhibits curling and drying compared to the kmd mutant lines, which uncurled following the initial drought stress treatment. FIG. 4C shows shoots of drought- stressed wild type and kmd mutants following 12 days of watering in the recovery period after drought stress (% seed set per panicle; n=9 panicles, three plants).

[0011] FIGS. 5A-5D show that independent CRISPR-Cas9 generated mutations of the rice KMD family similarly regulate the drought stress response. FIGS. 5A-B show maximum quantum yield (Fv / Fm) for photosynthesis in kmd mutants from event E5 (kmd2 / 3 / 4-E5 and kmdl / 2 / 3 / 4-E ) and event E6 (kmdl / 2 / 3 / 4-E6) compared to the wild type (Kt-wt) under drought stress conditions at the pre-anthesis (FIG.5A) and post-anthesis stage (FIG. 5B). Two rounds of drought stress were applied, each for three days (VWC < 10%). FIGS. 5C-5D show representative shoots of drought-stressed wild type and kmd mutants following 14 days of watering in the recovery period after drought stress at the pre-anthesis (FIG. 5C) and post-anthesis stages (FIG. 5D).DETAILED DESCRIPTION

[0012] It is to be understood that both the foregoing general description and the following detailed description arc illustrative and explanatory, and arc not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components that includes one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0013] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, arc hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0014] Abiotic stresses, in particular drought and salinity stress, severely limit crop productivity worldwide. For instance, over 30% of all arable land is affected by drought stress. Likewise, over 20% of all arable land is affected by salt stress.

[0015] Moreover, the Poaceae grass family, which includes cereal grains such as rice, maize, wheat, and barley, are the most important caloric food sources worldwide and are all affected by these abiotic stresses. Rice and wheat are particularly sensitive. For instance, drought alone affects more than 50% of the world’s rice production.

[0016] Therefore, improving abiotic stress resistance, especially drought resistance, is a major focus of crop research as it affects an important industry. However, current methods of combating abiotic stress resistance have numerous limitations.

[0017] For instance, many of the molecular approaches to combat abiotic stress, as described in the literature, involve transgenic expression of abiotic stress regulators, resulting in genetically modified organisms (GMOs). However, GMO crops are subject to numerous regulations that may affect their full commercial potential.

[0018] Additionally, studies have proposed exogenous treatment methods to confer drought resistance. Some of these studies have focused on treatments with the plant hormone abscisic acid (ABA), nutrients, amino acids, and rhizobacteria. However, such treatment approaches can be time and resource consuming.

[0019] As such, a need exists for more effective methods of developing plants and seeds that are resistant to abiotic stress. In particular, a need exists for methods of developing abiotic stress resistant plants and seeds without GMO and chemical-based methods. A need also exists for such altered plants and seeds. Numerous embodiments of the present disclosure aim to address the aforementioned needs.

[0020] In some embodiments, the present disclosure pertains to methods of enhancing abiotic stress resistance in a plant or seed. In some embodiments, the methods of the present disclosure include altering one or more KMD family of genes of the plant or seed (i.c., KMD family of genes that encode F-box proteins). In some embodiments, the altered KMD family of genes includes, without limitation, KMD1, KMD2, KMD3, KMD4, a gene encoding a protein with at least 40% amino-acid sequence identity with any one of KMD 1-4, or combinations thereof. Additional embodiments of the present disclosure pertain to plants or seeds that include one or more of the aforementioned altered KMD family of genes. Further embodiments of the present disclosure pertain to methods of growing a plant or a seed in a field by applying a plant or seed that includes one or more of the aforementioned altered KMD family of genes to the field. In some embodiments, the plant or seed demonstrates enhanced resistance to abiotic stress. As set forth in more detail herein, the methods, plants, and seeds of the present disclosure can have numerous embodiments.

[0021] Altered KMD family of genes

[0022] The methods of the present disclosure may be utilized to alter various KMD family of genes. Additionally, the plants and seeds of the present disclosure can include various altered KMD family of genes.

[0023] For instance, in some embodiments, the altered KMD family of genes includes any one of KMD1, KMD2, KMD3, and KMD4. In some embodiments, the altered KMD family of genes includes KMD1, KMD2, KMD3, and KMD4.

[0024] In some embodiments, the altered KMD family of genes includes KMD1. In some embodiments, KMD1 includes a sequence of any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 50% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 55% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 60% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 65% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 70% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 75% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 80% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 85% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 90% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 95% nucleotide sequence identity with any one of SEQ ID NOS: 1-4. In some embodiments, KMD1 includes a sequence with at least 99% nucleotide sequence identity with any one of SEQ ID NOS: 1-4.

[0025] In some embodiments, KMD1 expresses a protein that includes any one of SEQ ID NOS: 5- 10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 45% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 50% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 55% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 60% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 65% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 70% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 75% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 80% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 85% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 90% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 95% amino-acid sequence identity with any one of SEQ ID NOS: 5-10. In some embodiments, KMD1 expresses a protein that includes a sequence with at least 99% amino-acid sequence identity with any one of SEQ ID NOS: 5-10.

[0026] In some embodiments, the altered KMD family of genes includes KMD2. In some embodiments, KMD2 includes a sequence of any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 45% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 50% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 55% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 60% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 65% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 70% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 75% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 80% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 85% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 90% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 95% nucleotide sequence identity with any one of SEQ ID NOS: 11-14. In some embodiments, KMD2 includes a sequence with at least 99% nucleotide sequence identity with any one of SEQ ID NOS: 11-14.

[0027] In some embodiments, KMD2 expresses a protein that includes any one of SEQ ID NOS: 15- 20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 45% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 50% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 55% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 60% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 65% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 70% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 75% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 80% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 85% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 90% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 95% amino-acid sequence identity with any one of SEQ ID NOS: 15-20. In some embodiments, KMD2 expresses a protein that includes a sequence with at least 99% amino-acid sequence identity with any one of SEQ ID NOS: 15-20.

[0028] In some embodiments, the altered KMD family of genes includes KMD3. In some embodiments, KMD3 includes a sequence of any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 50% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 55% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 60% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 65% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 70% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD 3 includes a sequence with at least 75% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 80% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 85% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 90% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 95% nucleotide sequence identity with any one of SEQ ID NOS: 21-24. In some embodiments, KMD3 includes a sequence with at least 99% nucleotide sequence identity with any one of SEQ ID NOS: 21-24.

[0029] In some embodiments, KMD3 expresses a protein that includes any one of SEQ ID NOS: 25- 30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 50% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 55% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 60% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 65% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 70% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 75% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 80% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 85% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 90% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 95% amino-acid sequence identity with any one of SEQ ID NOS: 25-30. In some embodiments, KMD3 expresses a protein that includes a sequence with at least 99% amino-acid sequence identity with any one of SEQ ID NOS: 25-30.

[0030] In some embodiments, the altered KMD family of genes includes KMD4. In some embodiments, KMD4 includes a sequence of any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 50% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 55% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 60% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 65% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 70% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 75% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 80% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 85% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 90% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 95% nucleotide sequence identity with any one of SEQ ID NOS: 31-34. In some embodiments, KMD4 includes a sequence with at least 99% nucleotide sequence identity with any one of SEQ ID NOS: 31-34.

[0031] In some embodiments, KMD4 expresses a protein that includes any one of SEQ ID NOS: 35- 39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 50% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 55% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 60% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 65% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 70% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 75% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 80% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 85% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 90% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 95% amino-acid sequence identity with any one of SEQ ID NOS: 35-39. In some embodiments, KMD4 expresses a protein that includes a sequence with at least 99% amino-acid sequence identity with any one of SEQ ID NOS: 35-39.

[0032] Altering of KMD genes

[0033] The methods of the present disclosure may be utilized to alter KMD family of genes in various manners. For instance, in some embodiments, the altering includes, without limitation, downregulating the expression of one or more KMD family of genes, mutating one or more KMD family of genes, deleting one or more KMD family of genes, introducing an insertion to one or more KMD family of genes, removing a portion of one or more KMD family of genes, changing a nucleotide sequence of one or more KMD family of genes, altering a promoter region of one or more KMD family of genes, altering a proximate sequence that alters expression of one or more KMD family of genes, or combinations thereof.

[0034] In some embodiments, the altering includes downregulating the expression of one or more KMD family of genes. In some embodiments, the downregulating results in reduced expression of one or more KMD family of genes. In some embodiments, the downrcgulating results in eliminated expression of one or more KMD family of genes.

[0035] In some embodiments, the altering includes altering a promoter region of one or more KMD family of genes. In some embodiments, the altering includes introducing one or more mutations in one or more promoter elements.

[0036] In some embodiments, the altering includes altering a proximate sequence that alters expression of one or more KMD family of genes. In some embodiments, the proximate sequence is upstream or downstream one or more KMD family of genes. In some embodiments, the altering includes introducing one or more mutations in one or more proximate sequences.

[0037] Various methods may be utilized to alter KMD family of genes. For instance, in some embodiments, the altering includes introduction of an altering agent to the plant or seed. In some embodiments, the altering agent includes, without limitation, a microRNA interfering agent, a gene editing system, or combinations thereof.

[0038] In some embodiments, the altering agent includes a gene editing system. In some embodiments, the gene editing system includes a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system). In some embodiments, the CRISPR / Cas system includes at least one Cas nuclease and at least one guide RNA. In some embodiments, the Cas nuclease includes, without limitation, class 2 of Cas nucleases, Cas 9, Cas , Casd>2, Cpf 1 , or combinations thereof. In some embodiments, the CRISPR / Cas system includes a CRISPR / Cas9 system.

[0039] Altering agents may be introduced to plants or seeds in various manners. For instance, in some embodiments, the introduction of the altering agent occurs by a method that includes, without limitation, transfection, electroporation, particle bombardment, agrofiltration, or combinations thereof.

[0040] Altering of KMD genes may occur at various growth stages of a plant or seed. For instance, in some embodiments, the altering occurs at a seedling stage of a plant. In some embodiments, the altering occurs at an adult stage of a plant.

[0041] Altered KMD genes

[0042] The plants and seeds of the present disclosure may include various types of altered KMD genes. For instance, in some embodiments, the alteration includes one or more downregulated KMD family of genes, one or more mutated KMD family of genes, one or more deleted KMD family of genes, an insertion into one or more KMD family of genes, a removed portion of one or more KMD family of genes, a changed nucleotide sequence of one or more KMD family of genes, an altered promoter region of one or more KMD family of genes, and altered proximate sequence that alters expression of one or more KMD family of genes, or combinations thereof.

[0043] In some embodiments, the alteration includes one or more downregulated KMD family of genes. In some embodiments, the downregulation is represented by reduced expression of one or more KMD family of genes. In some embodiments, the downregulation is represented by eliminated expression of one or more KMD family of genes.

[0044] In some embodiments, the alteration includes an altered promoter region of one or more KMD family of genes. In some embodiments, the altered promoter region includes one or more mutations in one or more promoter elements.

[0045] Enhanced resistance to abiotic stress

[0046] The methods of the present disclosure may form plants and seeds with various forms of resistance to abiotic stress. Additionally, the plants and seeds of the present disclosure may have various forms of resistance to abiotic stress.

[0047] For instance, in some embodiments, the enhanced resistance to abiotic stress in the plant or seed includes enhanced resistance to drought stress. In some embodiments, the enhanced resistance to abiotic stress in the plant or seed includes enhanced resistance to salt stress. In some embodiments, the enhanced resistance to salt stress in the plant or seed includes enhanced resistance to high salt concentrations above 8 dS / m for soil, or above 5120 mg / L salt for irrigation and river water. In some embodiments, the enhanced resistance to salt stress in the plant or seed includes enhanced resistance to low salt concentrations above 2 dS / m electrical conductivity for soil, or above 1280 mg / L salt for irrigation and river water.

[0048] In some embodiments, the enhanced resistance to abiotic stress in the plant or seed includes enhanced resistance to temperature stress. In some embodiments, the enhanced resistance to temperature stress in the plant or seed includes enhanced resistance to high temperatures. In some embodiments, the high temperatures include temperatures above 90 Fahrenheit. In some embodiments, the high temperatures include temperatures above 100 Fahrenheit. In some embodiments, the enhanced resistance to temperature stress in the plant or seed includes enhanced resistance to low temperatures. In some embodiments, the low temperatures include temperatures below 32 Fahrenheit. In some embodiments, the low temperatures include temperatures below 0 Fahrenheit.

[0049] Enhanced resistance to abiotic stress in a plant or seed may be identified in various manners. For instance, in some embodiments, enhanced resistance to abiotic stress in a plant or seed is identified by delayed leaf senescence, improved seed set, improved seed fill, improved seed weight, improved seed yield, improved photosynthetic capacity, or combinations thereof.

[0050] Plants and seeds

[0051] The methods of the present disclosure may be utilized to enhance resistance to abiotic stress in various plants and seeds by altering various KMD family of genes. Additionally, the plants and seeds of the present disclosure can include numerous species.

[0052] For instance, in some embodiments, the plant or seed includes, without limitation monocots2rice, corn, maize, wheat, barley, lawn grass, turf grass, biscuit grass, the Poaceae grass family, eudicots, Arabidopsis, soybean, tobacco, tomato, lettuce, common beans, potato, cotton, grapes, varieties thereof, or combinations thereof. In some embodiments, the plant or seed includes lawn or turf grass. In some embodiments, the plant or seed includes rice. In some embodiments, the plant or seed includes a seed. In some embodiments, the plant or seed includes a plant.

[0053] Methods of growing plants and seeds in a field

[0054] Further embodiments of the present disclosure pertain to methods of growing a plant or a seed in a field by applying a plant or a seed of the present disclosure to the field. The plants and seeds of the present disclosure may be applied to various fields. For instance, in some embodiments, the field experiences or is vulnerable to drought. In some embodiments, the field experiences or is vulnerable to high salt concentrations. In some embodiments, the field experiences or is vulnerable to low salt concentrations. In some embodiments, the field experiences or is vulnerable to high temperatures (e.g., temperatures above 90 Fahrenheit). In some embodiments, the field experiences or is vulnerable to low temperatures (e.g., temperatures below 32 Fahrenheit).

[0055] Applications and Advantages

[0056] The methods, plants and seeds of the present disclosure provide numerous advantages. For instance, the methods of the present disclosure represent a non-GMO method by which to confer abiotic resistance on plants and seeds. Moreover, the plants and seeds of the present disclosure have more effective and long-lasting abiotic resistance than plants and seeds that are modified through chemical treatments. As such, the methods, plants and seeds of the present disclosure can have numerous applications in growing crops while mitigating abiotic stresses.

[0057] Additional embodiments

[0058] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0059] Example 1 . Method and composition for conferring abiotic stress resistance on plants by modulating activity of an F-box protein family

[0060] In this Example, Applicants describe an approach by which to produce resistance to drought and related abiotic stresses in plants. This approach is based on reducing the activity of the altered KMD family of F-box proteins. As described in this Example, Applicants found that a reduction in expression levels of the altered KMD family of F-box proteins in rice confers drought resistance, based on delayed leaf senescence and improved seed set under drought conditions.

[0061] A CRISPR-Cas9 based approach of gene editing was used to reduce expression of the four- member KMD gene family of rice, thus avoiding concerns with genetically modified organisms (GMOs), which would facilitate translation of this genetic tool into commercial crops. These results indicate that use of molecular genetic approaches to reduce expression of KMD family members generally (e.g. CRISPR-Cas9, RNAi) or more specifically such as by introducing mutations in their promoter elements can serve to confer drought resistance on plants of interest. Furthermore, these results also indicate that alternative methods by which to reduce KMD activity, such as through chemical inhibitors, would also serve to confer drought resistance on plants of interest. The effects of salt stress on plants are due in part to the drought stress response, and so manipulation of KMD activity is predicted to also serve for resistance to other abiotic stresses such as salt stress.

[0062] Example 1,1, Background

[0063] Drought and salinity stress are the major abiotic stresses limiting crop productivity worldwide, with over 30% of all arable land affected by drought stress and 20% by salt stress. The Poaceae grass family, which includes grains such as rice, maize, wheat, and barley, are the most important caloric food sources worldwide and are all affected by these abiotic stresses. Rice and wheat are particularly sensitive, drought alone affecting more than 50% of the world’s rice production.

[0064] Plants respond to drought and salt stress through independent and overlapping mechanisms. For example, although salt stress results in ionic imbalances, its effects on osmotic pressure also result in dehydration and induction of drought responses, including stomatai closure, production of reactive oxygen species (ROS), and the accumulation of various osmotic agents such as proline and sugars. The level of overlap in the plant response to drought and salinity stress, and their significance for crop productivity, has inspired molecular and breeding approaches to achieve shared tolerance for both.

[0065] The molecular approaches to date have primarily relied upon increased expression of transgenes, and it would be advantageous to employ genome editing approaches to generate non- transgenic plants that possess increased tolerance to drought and salt stress. Furthermore, climate change and rising sea levels will exacerbate the effects of water scarcity and soil salinity on crop productivity, increasing the importance of studies on stress physiology and the employment of new strategies to improve drought and stress tolerance in important crop species.

[0066] The KMD family of F-box proteins (FIGS. 1A-1B) are found throughout the plant kingdom, including the eudicot Arabidopsis thaliana, the monocot Oryza sativa, and mosses such as Physcomitrella patens (Pp) and Selaginella moellendorffii (Sm). F-box proteins target their substrates for degradation by participation in SCF complexes, the F-box component of the complex serving to recruit substrates to the SCF complex for degradation. The F-box proteins typically target multiple substrates and that is the case for the KMD family of F-box proteins.

[0067] KMD substrates are involved in transcriptional regulation (type-B RRs) of the signaling pathway for the plant hormone cytokinin, resulting in changes in gene expression. Additional KMD substrates (PALs) are involved in regulating the biosynthetic pathway for phenylpropanoids, potentially affecting levels of such phenolic compounds, such as anthocyanins, flavonoids, coumarins, stilbenes, tannins, and lignin.

[0068] A role in regulating multiple pathways has resulted in alternative gene designations for this family of F-box proteins (KMD and KFB), as designated: KMD1 / KFB20; KMD2 / KFB01; KMD3 / KFB39; and KMD4 / KFB50. Additional uncharacterized targets may also contribute to the roles that the KMD family play in plant growth, development, and stress responses.

[0069] As such, it is difficult to predict the effect of down-regulating KMD activity on plant growth and development, and more specifically, the response to abiotic stresses such as drought and salt stress.

[0070] Historically, abscisic acid (ABA) has been the phytohormone most closely associated with the abiotic stress response. However, the plant hormone cytokinin may also play a role, although there are conflicting data on its role, pointing to the difficulty in predicting the outcome from manipulating cytokinin activity in plants.

[0071] Cytokinins arc adenine derivatives that regulate diverse aspects of plant growth and development, including key roles in regulating seed yield and root architecture. Applicants’ understanding of the metabolism and perception of cytokinin has made great strides in recent years, mostly from studies of the model eudicot Arabidopsis. Cytokinin levels are controlled primarily through its metabolism, and the basic pathways for its biosynthesis and degradation have been elucidated.

[0072] The biosynthesis of cytokinin involves three key enzymes: isopentenyltransferase (IPT), cytochrome P450 enzyme CYP735A, and LONELY GUY (LOG) nucleoside 5' monophosphate (FIG. 2). These act in sequence to add a prenyl group to the N6position of ADP / ATP, hydroxylate the isoprenoid side chain, and then activate the cytokinin by converting it to the free base form. The level of active cytokinin can be decreased through conjugation to sugars, es glucose, or by degradation by cytokinin oxidases (FIG. 2).

[0073] The cytokinin response in a tissue is also dependent on its sensitivity to the hormone, as determined by expression of the elements that compose its signal transduction pathway (FIG. 2). The initial pathway for cytokinin signal transduction is a multi-step phosphorelay that incorporates cytokinin receptors (HKs), histidine-containing phosphotransfer proteins (AHPs), and type-B response regulators (RRs). These relay the cytokinin signal from the membrane to the nucleus, where the type-B RRs function as transcription factors to regulate gene expression. The type-A RRs are among the targets whose expression is induced by the type-B RRs, and these function as negative feedback regulators for the cytokinin response.

[0074] The KMD family of F-box proteins target the type-B RRs for degradation, functioning as an additional negative regulator of the cytokinin response (FIGS. 1A-1B and 2). In rice, as in most plants, these signaling elements are encoded by multi-gene families. For example, there are 13 type- B RRs and 13 type-A RRs in the rice genome. The KMD family is composed of four members in both Arabidopsis and rice (FIGS. 1A-1B and 2).

[0075] There is conflicting data on the role that cytokinin plays in regulating resistance to drought and abiotic stresses in plants. The most extensive studies have been performed in the model plant Arabidopsis, where genetic studies generally support a role for cytokinin as a negative regulator for resistance to drought and abiotic stress. Specifically, cytokinin-insensitive Arabidopsis lines derived from loss-of-function mutations of the cytokinin receptors and type-B RRs generally display an increased tolerance to drought and salt stress. Likewise, cytokinin-deficient mutants of Arabidopsis exhibit enhanced tolerance to the abiotic stresses, whereas lines with increased cytokinin levels exhibit decreased tolerance.

[0076] These data from Arabidopsis suggest that an increase in cytokinin activity would confer increased sensitivity to drought stress. However, additional studies suggest that increased levels of endogenous cytokinin can potentially confer abiotic stress resistance. A scncsccncc / strcss-induciblc promoter driving expression of IPT (promotes cytokinin biosynthesis), resulted in increased drought resistance in eudicots, such as tobacco and cotton, as well as in monocots, such as rice, maize, and creeping bentgrass. The positive effects of increased cytokinin content on the response to drought stress could be ascribed to maintenance of photosynthesis, higher water content, increased antioxidant activity, and / or increases in various metabolites such as proline and sucrose, resulting in increased drought tolerance and an increase in grain yield.

[0077] Other studies provide further conflicting data on the potential role of cytokinin in regulating the abiotic stress response. For example, a reduction in AHP levels by RNAi, which reduced cytokinin sensitivity, resulted in decreased resistance to salt stress in rice seedlings, whereas the type-A RR loss- of-function mutant rr9,10, exhibited increased salt tolerance in seedlings. In addition, knockdown of a CKX expressed in the rice inflorescence improved reproductive growth under salt stress conditions. In contrast, one recent study in which active cytokinin levels were reduced by overexpressing a cytokinin modifying glucosyltransferase resulted in improved drought and salt tolerance in rice seedlings.

[0078] In addition, expression of a CKX gene to reduce cytokinin levels in the roots of barley and tobacco resulted in enhanced drought resistance. Furthermore, drought stress reduced cytokinin levels in rice seedling shoots, potentially due to antagonism between ABA and cytokinin activity. Overall, based on the prior data, it has not been possible to predict the effects of changing cytokinin activity on abiotic stress responses in plants.

[0079] Not only do the KMDs target several type-B RRs for degradation to reduce cytokinin activity (FIG. 2), they also target phenylalanine ammonia-lyases (PALs) for degradation, PALs catalyzing the initial step for phenylpropanoid biosynthesis. By regulating PAL protein levels for the first step in phenylpropanoid biosynthesis, the KMDs can potentially affect levels of such phenolic compounds as anthocyanins, flavonoids, coumarins, stilbenes, and tannins, as well as lignin, which serves as structural component for cell walls. Conflicting data are available on the role of PAL genes in regulating drought resistance.

[0080] In Arabidopsis, the loss-of-function mutant pall pal2 is drought resistant, suggesting that down-regulation of PAL activity can confer drought resistance. However, paradoxically, some drought resistance was reported to be conferred on Arabidopsis by overexpressing a PAL from the alpine plant Fritillaria unibracteata. In the monocot Brachypodium, PAL loss-of-function mutants, although conferring substantial changes on plant growth and development, along with pathogen sensitivity, had no discernable effect on drought resistance. Overall, based on the prior data, it has not been possible to predict the effects of changing PAL activity on abiotic stress responses in plants.

[0081] The KMD substrates are involved in transcriptional regulation (type-B RRs), resulting in changes in gene expression, as well as enzymatic catalysis (PALs), resulting in changes in levels of secondary metabolites. Which of these substrates are degraded in response to KMD activity, and the extent to which they are degraded, is likely to be dependent on the tissue type, stage of development, environmental inputs, and factors such as substrate phosphorylation.

[0082] Furthermore, because the KMD family of F-box proteins does not participate directly in these pathways, but rather regulates the protein levels of pathway components, activity of the pathway signaling elements is modulated rather than constitutively inhibited or activated, resulting in more subtle effects on activity of the pathways. Additional uncharacterized targets may also contribute to the roles that the KMD family play in regulating abiotic stress responses. These factors further complicate an ability to predict the effect of down-regulating KMD activity on plant growth and development, and more specifically, the response to abiotic stresses such as drought and salt stress.

[0083] Example 1 ,2. Generation of loss-of-function mutations in rice genes encoding members of the KMD F-box protein family

[0084] Phylogenetic analysis indicates that the KMD family of F-box proteins is conserved in land plants, including eudicots, monocots, and moss, supporting a general role in the regulating protein turnover in plants (FIGS. 1A-1B). Initial studies on the KMDs were in the eudicot Arabidopsis, and supported a role in the regulation of multiple pathways.

[0085] To determine the roles that KMDs play in a monocot, Applicants chose rice as an experimental species. Rice is an ideal monocot in which to perform such analyses because of its small genome size, the availability of a reference genome, and its case of transformation. Because of conservation of gene sequence among cereals, information obtained from the study of rice has significance for other agronomically important species such as maize, wheat, barley, rye, and sorghum. A conserved role for the KMD family in cereals and grasses is supported by phylogenetic interdigitation of the rice KMD family members with KMD family members from the monocots maize, wheat, barley, and biscuit grass (FIG. IB). Furthermore, the phylogenetic relationship of the monocot KMDs with those of the eudicot Arabidopsis support common roles in monocots and eudicots. Conserved functionality of the rice KMDs with those of Arabidopsis is also supported by the finding that overexpression of a rice KMD in Arabidopsis phenocopies is found upon overexpression of the Arabidopsis KMDs in Arabidopsis.

[0086] To functionally characterize the rice KMDs, Applicants generated a CRISPR-Cas9 construct that targets the four rice KMD genes (FIGS. 1A-1B), introduced this into the Japonica rice cultivar Kitaake, and generated multiple loss-of-function mutations in the gene family, including kmdl, 2, 3, 4 quadruple mutants. Insertion-deletion (indel) mutations in KMD1, KMD2, KMD3, and KMD4 were identified by sequencing.

[0087] Applicants focused on lines with frameshift mutations because these are predicted to be null mutations, brought the mutations to homozygosity, and eliminated the CRISPR-Cas9 vector by segregation to stabilize the mutations. Applicants identified and characterized higher-order kmd mutants from two independent transformation events. In event E5 (triple mutant kind 2,3,4 and quadruple mutant kmdl, 2, 3, 4) and event E6 (quadruple mutant kmdl, 2, 3, 4 all four kmd genes arc predicted to be null.

[0088] Applicants’ initial intent with generating kmd mutant lines in rice was to follow up on their studies of the KMD gene family in Arabidopsis, and to compare the role of the KMD family in cytokinin signaling between monocots and eudicots. However, following an inadvertent break in the rice plant watering schedule, Applicants observed that leaves of KMD event #5 mutant lines kmd2,3,4 and kmdl, 2, 3, 4 exhibited less senescence and drying out than the wild-type control plants. This suggested that the kmd mutants might be more resistant to drought stress, and Applicants therefore set up controlled experiments to test this hypothesis.

[0089] As described herein, experiments making use of different drought stress regimes and at different stages of rice growth, confirm this hypothesis and demonstrate that the kmd mutant plants arc resistant to drought stress. Mutations in the rice KMD family confer drought resistance based on physiological analyses.

[0090] Applicants performed experiments to determine the effects of the kmd mutants on the drought stress response in rice, focusing on reproductive growth during pre-anthesis and post-anthesis stages, developmental time points at which drought can significantly impact grain yield. For these experiments, Applicants grew rice plants under flooded conditions (volumetric water content, VWC >90%), with drought stress imposed by the removal of water, one of the morphological responses to drought stress for rice being leaf curling, which Applicants observed in the wild-type when the soil volumetric water content (VWC) dropped below 20%.

[0091] In addition, Applicants examined the effects of two different drought stress regimes: (1) one round of extended drought stress; and (2) two rounds of short-term drought stress. The effects of drought on photosynthetic capacity were determined by measuring maximum quantum yield (Fv / Fm) for leaf photosynthesis. The effects of drought on grain yield were determined by measuring the seed set of the grain -producing rice panicles.

[0092] Applicants’ results indicate that kmd mutants are resistant to drought stress based on the analysis of photosynthetic capacity, leaf senescence, and seed yield. Furthermore, the results indicate that different levels of drought resistance can be obtained by using different mutant combinations of the kmd family.

[0093] FIGS. 3A-3B show the effects of five days of drought stress applied pre-anthesis, followed by rcwatcring, for wildtypc controls and the event 5 kmd mutants (kmd2,3,4-E5 and kmdl,2,3,4-E5). Drought stress resulted in extensive leaf senescence for the wild-type, but damage from drought stress was mitigated in the kmd mutants, where the leaves remained green (FIG. 3A). In addition, only one seed set for the nine panicles examined of the wildtype controls (decrease in seed set of >99%), compared to 51% and 35% decreases in seed set for the kmd2,3,4-E5 and kmdl,2,3,4-E5 lines, respectively (FIG. 3B). These results demonstrate that the kmd mutants are resistant to drought stress under these growth conditions based on both leaf senescence and seed yield.

[0094] FIGS. 4A-4C show the effects of two rounds of drought stress applied post-anthesis, followed by rewatering, for wildtype controls and the event 5 kind mutants (kmd2,3,4-E5 and kmdl,2,3,4-E5). Each round of drought stress was applied for three days, with plants watered for one day before the second round of drought stress. The first round of drought stress resulted in a sharp drop in photosynthetic capacity in the wild type, with only a minor decrease during the first round of drought stress for the kmd mutant lines (FIG. 4A). Significantly, the kind mutants exhibited no drop in photosynthetic capacity during the second round of drought stress, indicative that ‘priming’ occurred in response to the first round of drought stress, further protecting the lines when subsequently exposed to another round of drought (FIG. 4B).

[0095] During the first round of drought stress, the wild-type control exhibited leaf curling after two days of drought stress, but the kmd lines did not exhibit leaf curling until three days of drought stress. Furthermore, the wildtype showed more excessive curling and bending of leaves, while the mutant leaves curled but remained upright. The kmd mutant lines also recovered and uncurled following the initial drought stress treatment (FIG. 3B). Drought stress resulted in an 88% decrease in seed set for the wild type, compared to 9% and 28% decreases for the kmd2,3,4 and kmdl,2,3,4 lines, respectively (FIG. 4C). These results demonstrate that the kmd mutants are resistant to drought stress under these growth conditions based on leaf senescence, photosynthetic capacity, and seed yield.

[0096] FIGS. 5A-5D show the effects of two rounds of drought stress, making use of the event 5 kmd mutants (kmd2,3,4-E5 and kindl,2,3,4-E5) as well as the independently derived event 6 quadruple mutant kmdl / 2 / 3 / 4-E6. For this experiment, the soil volumetric water content (VWC) was -33% after one day, -16% after two days, and -7% after three days.

[0097] As seen in FIGS. 5A-5B, the quadruple kmdl / 2 / 3 / 4 mutants from event 5 and event 6 both maintain similar levels of photosynthetic capacity when subjected to drought stress under both preanthesis and post-anthesis conditions. Furthermore, as seen in FIGS. 5C-5D, the quadruple kmdl / 2 / 3 / 4 mutants from event 5 and event 6 both prevent leaf senescence to a similar extent under drought stress both pre-anthesis and post- anthesis conditions. These results demonstrate that two independently derived kmdl / 2 / 3 / 4 mutant lines exhibit drought resistance.

[0098] Example 1,3. Summary

[0099] The KMD family of F-box proteins arc involved in negative regulation of the cytokinin response and have also been implicated in negatively regulating phenylpropanoid biosynthesis. As described in this Example, Applicants have used a Crispr / Cas9-bascd approach to reduce the expression of KMD gene family members in rice. Reduced expression of the KMD family results in drought resistance and increased grain yield in rice under drought stress conditions.

[0100] This modification of KMD gene expression thus provides a new approach by which to protect plants from drought stress. Without being bound by theory, the effect of KMD mutants may arise due to the KMDs targeting certain proteins to regulate their abundance. Additionally, because there is overlap in the mechanisms by which plants respond to abiotic stress, the same approach may also provide resistance under other abiotic stress conditions. These include salinity stress, which overlaps with the drought stress response, as well as high and low temperature, and heavy metal stress.

[0101] Without further elaboration, it is believed that one skilled in the ail can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

CLAIMS1. A method of enhancing abiotic stress resistance in a plant or seed, said method comprising: altering one or more KMD family of genes of the plant or seed, wherein the one or more KMD family of genes is selected from the group consisting of KMD1, KMD2, KMD3, KMD4, a gene encoding a protein with at least 40% amino-acid sequence identity with any one of KMD 1-4, or combinations thereof.

2. The method of claim 1, wherein the one or more KMD family of genes comprises KMD1, KMD2, KMD 3, and KMD4.

3. The method of claim 1, wherein the one or more KMD family of genes is selected from the group consisting of:KMD1 , wherein KMD1 comprises a sequence of any one of SEQ ID NOS: 1-4 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 1-4, or wherein KMD1 expresses a protein comprising any one of SEQ ID NOS: 5-10 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 5-10;KMD2, wherein KMD2 comprises a sequence of any one of SEQ ID NOS: 11-14 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 11-14, or wherein KMD2 expresses a protein comprising any one of SEQ ID NOS: 15-20 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 15-20;KMD3, wherein KMD3 comprises a sequence of any one of SEQ ID NOS: 21-24 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 21-24, or wherein KMD3 expresses a protein comprising any one of SEQ ID NOS: 25-30 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 25-30;KMD4, wherein KMD4 comprises a sequence of any one of SEQ ID NOS: 31-34 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 31-34, or wherein KMD4 expresses a protein comprising any one of SEQ ID NOS: 35-39, or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 35-39; or combinations thereof.

4. The method of claim 1, wherein the altering comprises downregulating the expression of one or more KMD family of genes, mutating one or more KMD family of genes, deleting one or more KMD family of genes, introducing an insertion to one or more KMD family of genes, removing a portion of one or more KMD family of genes, changing a nucleotide sequence of one or more KMD family of genes, altering a promoter region of one or more KMD family of genes, altering a proximate sequence that alters expression of one or more KMD family of genes, or combinations thereof.

5. The method of claim 1, wherein the altering comprises downregulating the expression of one or more KMD family of genes, wherein the downregulating results in reduced or eliminated expression of one or more KMD family of genes.

6. The method of claim 1, wherein the altering comprises introduction of an altering agent to the plant or seed, wherein the altering agent is selected from the group consisting of a microRNA interfering agent, a gene editing system, or combinations thereof.

7. The method of claim 6, wherein the altering agent comprises a gene editing system, wherein the gene editing system comprises a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system), wherein the CRISPR / Cas system comprises at least one Cas nuclease and at least one guide RNA.

8. The method of claim 1 , wherein the altering occurs at a seedling stage of a plant.

9. The method of claim 1, wherein the altering occurs at an adult stage of a plant.

10. The method of claim 1, wherein the enhanced resistance to abiotic stress in the plant or seed comprises enhanced resistance to drought stress, enhanced resistance to salt stress, enhanced resistance to temperature stress, or combinations thereof.

11. The method of claim 1, wherein the plant or seed is selected from the group consisting of monocots, rice, com, maize, wheat, barley, lawn grass, turf grass, biscuit grass, the Poaceae grass family^ cudicots. Arabidopsis, soybean, tobacco, tomato, lettuce, common beans, potato, cotton, grapes, varieties thereof, or combinations thereof.

12. The method of claim 1, wherein the plant or seed comprises lawn or turf grass.

13. The method of claim 1, wherein the plant or seed comprises rice.

14. A plant or a seed comprising one or more altered KMD family of genes, wherein the one or more altered KMD family of genes is selected from the group consisting of KMD1, KMD2, KMD3, KMD4, a gene encoding a protein with at least 40% amino-acid sequence identity with any one of KMD 1-4, or combinations thereof, and wherein the plant or seed demonstrates enhanced resistance to abiotic stress.

15. The plant or seed of claim 14, wherein the one or more KMD family of genes comprises KMD1, KMD2, KMD3, and KMD4.

16. The plant or seed of claim 14, wherein the one or more KMD family of genes is selected from the group consisting of:KMD1 , wherein KMD1 comprises a sequence of any one of SEQ ID NOS: 1-4 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 1-4, or wherein KMD1 expresses a protein comprising any one of SEQ ID NOS: 5-10 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 5-10;KMD2, wherein KMD2 comprises a sequence of any one of SEQ ID NOS: 11-14 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 11-14, or wherein KMD2 expresses a protein comprising any one of SEQ ID NOS: 15-20 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 15-20;KMD3, wherein KMD3 comprises a sequence of any one of SEQ ID NOS: 21-24 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 21-24, or wherein KMD3 expresses a protein comprising any one of SEQ ID NOS: 25-30 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 25-30;KMD4, wherein KMD4 comprises a sequence of any one of SEQ ID NOS: 31-34 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 31-34, or wherein KMD4 expresses a protein comprising any one of SEQ ID NOS: 35-39, or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 35-39; or combinations thereof.

17. The plant or seed of claim 14, wherein the alteration comprises one or more downregulated KMD family of genes, one or more mutated KMD family of genes, one or more deleted KMD family of genes, an insertion into one or more KMD family of genes, a removed portion of one or more KMD family of genes, a changed nucleotide sequence of one or more KMD family of genes, an altered promoter region of one or more KMD family of genes, and altered proximate sequence that alters expression of one or more KMD family of genes, or combinations thereof.

18. The plant or seed of claim 14, wherein the alteration comprises one or more downregulated KMD family of genes, wherein the downregulation is represented by reduced or eliminated expression of one or more KMD family of genes.

19. The plant or seed of claim 14, wherein the enhanced resistance to abiotic stress in the plant or seed comprises enhanced resistance to drought stress, enhanced resistance to salt stress, enhanced resistance to temperature stress, or combinations thereof.

20. The plant or seed of claim 14, wherein the plant or seed is selected from the group consisting of monocots^ rice, com, maize, wheat, barley, lawn grass, turf grass, biscuit grass, the Poaceae grass family^ cudicots. Arabidopsis, soybean, tobacco, tomato, lettuce, common beans, potato, cotton, grapes, varieties thereof, or combinations thereof, or combinations thereof.

21. The plant or seed of claim 14, wherein the plant or seed comprises lawn or turf grass.

22. The plant or seed of claim 14, wherein the plant or seed comprises rice.

23. A method of growing a plant or a seed in a field, said method comprising: applying a plant or a seed to the field, wherein the plant or seed comprises one or more altered KMD family of genes, wherein the one or more altered KMD family of genes is selected from the group consisting of KMD1, KMD2, KMD3, KMD4, a gene encoding a protein with at least 40% amino-acid sequence identity with any one of KMD 1-4, or combinations thereof, and wherein the plant or seed demonstrates enhanced resistance to abiotic stress.

24. The method of claim 23, wherein the one or more KMD family of genes comprises KMD1, KMD2, KMD 3, and KMD4.

25. The method of claim 23, wherein the one or more KMD family of genes is selected from the group consisting of:KMD1, wherein KMD1 comprises a sequence of any one of SEQ ID NOS: 1-4 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 1-4, or wherein KMD1 expresses a protein comprising any one of SEQ ID NOS: 5-10 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 5-10;KMD2, wherein KMD2 comprises a sequence of any one of SEQ ID NOS: 11-14 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 11-14, or wherein KMD2 expresses a protein comprising any one of SEQ ID NOS: 15-20 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 15-20;KMD3, wherein KMD3 comprises a sequence of any one of SEQ ID NOS: 21-24 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 21-24, or wherein KMD3 expresses a protein comprising any one of SEQ ID NOS: 25-30 or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 25-30;KMD4, wherein KMD4 comprises a sequence of any one of SEQ ID NOS: 31-34 or a sequence with at least 40% nucleotide sequence identity with any one of SEQ ID NOS: 31-34, or wherein KMD4 expresses a protein comprising any one of SEQ ID NOS: 35-39, or a sequence with at least 40% amino-acid sequence identity with any one of SEQ ID NOS: 35-39; or combinations thereof.

26. The method of claim 23, wherein the alteration comprises one or more downregulated KMD family of genes, one or more mutated KMD family of genes, one or more deleted KMD family of genes, an insertion into one or more KMD family of genes, a removed portion of one or more KMD family of genes, a changed nucleotide sequence of one or more KMD family of genes, an altered promoter region of one or more KMD family of genes, and altered proximate sequence that alters expression of one or more KMD family of genes, or combinations thereof.

27. The method of claim 23, wherein the alteration comprises one or more downregulated KMD family of genes, wherein the downregulation is represented by reduced or eliminated expression of one or more KMD family of genes.

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