WO2024201416A1 - Compositions and methods comprising plants with modified organ size and/or protein composition - Google Patents

Abstract

Plants and plant parts comprising increased GIF1 activity, and compositions and methods of producing such plants and plant parts are provided. The plants and plant parts can have a genetic mutation or transgene that increases the GIF1 activity, and can have increased seed size, increased biomass, increased yield, and/or increased protein content relative to a control plant or plant part. The mutation can be located at least partially in a GIF1 gene or homolog or regulatory region thereof. The mutation can be located at or near the G-box region in the GIF1 regulatory region, decrease binding of a GIF1 repressor (e.g., a KIX8/9- PPD1/2-MYC3/4 complex) thereto, and increase GIF1 level or activity. The plants and plant parts can further have decreased BIG SEEDS (BS) activity, e.g., a mutation in a BS gene. Also provided are plant products produced from the plants or plant parts provided herein.

Description

COMPOSITIONS AND METHODS COMPRISING PLANTS

WITH MODIFIED ORGAN SIZE AND/OR PROTEIN COMPOSITION

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/493,629, filed on March 31, 2023, the content of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which is submitted herewith in electronically readable format. The Sequence Listing file was created on March 28, 2024, is named “B88552_1600_SL.xml” and its size is 79. 1 kb. The entire contents of the Sequence Listing in the XML file are incorporated by reference herein.

FIELD OF THE INVENTION

The present disclosure relates to the field of agricultural biotechnology. More specifically, this disclosure relates to plants and plant parts having modified organ (e.g., seed) size and/or protein content, and associated methods and compositions.

BACKGROUND OF THE INVENTION

With the ever-increasing world population and the dwindling supply of arable land available for agriculture, plants with increased biomass and yield are desired. An increased plant biomass is an advantageous trait for forage crops including alfalfa, clover, birdsfoot trefoil, com, sorghum, wheat, rye, and fescue. An increase in plant yield, particularly an increase in seed yield, is advantageous for human and animal consumption and for industrial use. Crops such as soybean, com, rice, wheat, and canola account for over half the total human caloric intake, whether through direct consumption of the seeds or through consumption of products derived from animals raised on processed seeds. Seeds are also a source of sugars, oils and many kinds of metabolites used in industrial processes.

High protein content is another desirable trait for plants and seeds. For instance, soy protein is valued for its high nutritional quality for humans and livestock, and for its functional properties, such as gel and foam formation. Plants and seeds are processed through multiple steps (e.g., drying, cracking, dehulling, flaking, cooking, roasting, extruding, expelling, extracting by solvent, desolventing, toasting, precipitating) to produce different protein compositions for use in various purposes, for example plant protein meal, protein concentrates, protein extracts, and protein isolates. Plants with higher concentration or content of protein are desirable for the manufacture of various products including seed compositions, protein compositions, food and beverage products, or industrial materials. It is challenging, however, to obtain plants with both high yield and high protein content, because in breeding populations a strong inverse correlation exists between yield and protein content, or between seed size and protein content. Accordingly, providing plants and seeds that possess both high yield / increased organ (e.g., seed) size and high protein content could offer important commercial advantages.

SUMMARY OF THE INVENTION

Plants and plant parts comprising increased GIF1 activity are provided. Compositions and methods for producing such plants and plant parts, and products (e.g., seed compositions, protein compositions) produced from such plants and plant parts are also provided. The plants or plant parts of the present disclosure can have a transgene or a genetic mutation that increases the GIF1 activity, e.g., one or more mutations in at least one native GIF1 gene or homolog or in its regulatory region (e.g., promoter, 5’UTR, G- box region), increased expression levels of the GIF1 gene, increased levels or activity of the GIF 1 protein, increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control plant or plant part. The plants and plant parts can further have decreased BIG SEEDS (BS) activity, e.g., a mutation in a BS gene or homolog thereof or regulatory region thereof that reduces BIG SEEDS activity.

In one aspect, the present disclosure provides a plant or plant part comprising increased nuclear transcription factor GRF-interacting factor 1 (GIF1) activity compared to a control plant or plant part, wherein said plant or plant part comprises a genetic mutation that increases the GIF1 activity.

In some embodiments, the plant or plant part comprises increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control plant or plant part. In some embodiments, the mutation comprises one or more insertions, substitutions, or deletions in at least one GIF1 gene or homolog thereof or regulatory region thereof in the plant or plant part, wherein an expression level of said at least one GIF1 gene or homolog thereof is increased compared to an expression level a corresponding GIF1 gene or homolog thereof without said mutation, and/or level or activity of a GIF1 protein encoded by said at least one GIF1 gene or homolog thereof is increased compared to level or activity of a GIF1 protein encoded by a corresponding GIF1 gene or homolog thereof without said mutation.

In some embodiments, the mutation is located at least partially in a promoter region or 5 ’ untranslated region (5’UTR) of said at least one GIF1 gene or homolog thereof. In some embodiments, the mutation is located at least partially in a G-box region in said regulatory region of said at least one GIF1 gene or homolog thereof. In some embodiments, the mutation decreases binding of a GIF1 repressor complex to the regulatory region of said at least one GIF1 gene or homolog thereof, thereby increasing level or activity of a GIF1 protein encoded by said at least one GIF1 gene or homolog thereof.

In some embodiments, said at least one GIF1 gene or homolog thereof, before the mutation is located: (i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein said nucleic acid sequence encodes a polypeptide that retains GIF1 activity; (ii) comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-12; (iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein said polypeptide retains GIF1 activity; (iv) encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 5-8; (v) includes said regulatory region thereof, and comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein said nucleic acid sequence encodes a polypeptide that retains GIF1 activity; and/or (vi) includes said regulatory region thereof, and comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4.

In some embodiments, the mutation is located at least partially in a promoter region or 5’ untranslated region (5’UTR) of a Glycine max GIF1 gene. In some embodiments, the Glycine max GIF1 gene is selected from the group consisting of Glyma.03G249000, Glyma.l9G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l4G122500, and Glyma.OlGl 13500.

In some embodiments, the mutation is located at least partially in a G-box region for the Glycine max GIF1 gene. In some embodiments, the mutation is located at least partially in a nucleic acid sequence of any one of SEQ ID NOs: 17-19 in the G-box region of the Glycine max GIF1 gene. In some embodiments, the mutation comprises a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides located at least partially in the G-box region for the Glycine max GIF1 gene. In some embodiments, the plant or plant part comprises Glyma.03G249000 with a mutated G-box region comprising a nucleic acid sequence of any one of SEQ ID NOs: 20-24, and/or Glyma.19G246600 with a mutated G-box region comprising a nucleic acid sequence of SEQ ID NO: 25.

In some embodiments, the plant or plant part further comprises a genetic mutation that decreases BIG SEEDS (BS) activity. In some embodiments, the mutation that decreases BIG SEEDS activity comprises one or more insertions, substitutions, or deletions in at least one BS gene or homolog thereof or regulatory region thereof, wherein an expression level of said at least one BS gene or homolog thereof is increased compared to an expression level a corresponding BS gene or homolog thereof without said mutation, and/or level or activity of a BIG SEEDS protein encoded by said at least one BS gene or homolog thereof is increased compared to level or activity of a BIG SEEDS protein encoded by a corresponding BS gene or homolog thereof without said mutation.

In some embodiments, said at least one BS gene or homolog thereof, before the mutation is located: (i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 26, 27, or 49, wherein said nucleic acid sequence encodes a polypeptide that retains BIG SEEDS activity; (ii) comprises the nucleic acid sequence of SEQ ID NO: 26, 27, or 49; (iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 28, 29, or 41, wherein said polypeptide retains BIG SEEDS activity; and/or (iv) encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, 29, or 41.

In some embodiments, said mutation is located at least partially in a nucleic acid region of exon 1 or exon 2 of a Glycine max BS1 gene and/or exon 2 of a Glycine max BS2 gene. In some embodiments, the plant or plant part comprises (i) a mutated Glycine max BS1 gene comprising a deletion of nucleotides 98 through 101 of SEQ ID NO: 26, (ii) a mutated Glycine max BS1 gene comprising a deletion of nucleotides 389 through 396 of SEQ ID NO: 26, and/or (iii) a mutated Glycine max BS2 gene comprising a deletion of nucleotides 409 through 415 of SEQ ID NO: 27.

In some embodiments, said plant or plant part is a legume. In some embodiments, said plant or plant part is selected from the group consisting of soybean (Glycine max), beans (Phaseolus spp.), common bean (Phaseolus vulgaris), fava bean (Vida faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut Arachis hypogaea), lentils (Lens culinaris, Lens esculenta), lupins (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.).

In some embodiments, said plant or plant part is selected from the group consisting of com (Zea mays), Brassica species, Brassica napus, Brassica rapa, Brassica juncea, rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet, pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp ), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp ), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp ), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

In some embodiments, said plant or plant part is a seed.

In one aspect, the present disclosure provides a population of plants or plant parts comprising the plant or plant part provided herein, wherein the population comprises increased GIF1 activity, increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control population. In some embodiments, said plant or plant part is a seed, and said population is a population of seeds.

In one aspect, the present disclosure provides a method for increasing seed size, biomass, protein content, and/or yield in a plant or plant part, said method comprising increasing level or activity of GRF- interacting factor 1 (GIF1) in said plant or plant part.

In some embodiments, the method comprises introducing a genetic mutation that increases GIF1 activity into said plant or plant part, further comprising introducing the genetic mutation that increases GIF 1 activity into a plant cell, and regenerating said plant or plant part from said plant cell. In some embodiments, the mutation comprises one or more insertions, substitutions, or deletions in at least one GIF1 gene or homolog thereof or regulatory region thereof in said plant or plant part, wherein an expression level of said at least one GIF1 gene or homolog thereof is increased by said mutation, and/or level or activity of a GIF1 protein encoded by said at least one GIF1 gene or homolog thereof is increased by said mutation.

In some embodiments, the method comprises introducing the mutation to locate at least partially in a promoter region or 5’ untranslated region (5’UTR) in the regulatory region of said at least one native GIF1 gene or homolog thereof. In some embodiments, the method comprises introducing the mutation to locate at least partially in a G-box region in the regulatory region of said at least one GIF1 gene or homolog thereof. In some embodiments, the mutation decreases binding of a GIF1 repressor complex to the regulatory region of said at least one GIF1 gene or homolog thereof, thereby increasing level or activity of a GIF 1 protein encoded by said at least one GIF1 gene or homolog thereof.

In some embodiments, said at least one GIF1 gene or homolog thereof, before the mutation is introduced: (i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein said nucleic acid sequence encodes a polypeptide that retains GIF1 activity; (ii) comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-12; (iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein said polypeptide retains GIF1 activity; (iv) encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 5-8; (v) includes said regulatory region thereof, and comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein said nucleic acid sequence encodes a polypeptide that retains GIF1 activity; and/or (vi) includes said regulatory region thereof, and comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4.

In some embodiments, the mutation is introduced at least partially into a promoter region or 5 ’ untranslated region (5’UTR) of a Glycine max GIF1 gene. In some embodiments, the Glycine max GIF1 gene is selected from the group consisting of Glyma.03G249000, Glyma.19G246600, Glyma.10G 164100, Glyma.20G226500, Glyma.18G121100, Glyma.14G122500, and Glyma.OlGl 13500. In some embodiments, the mutation is introduced at least partially into a G-box region for the Glycine max GIF1 gene.

In some embodiments, the mutation is introduced at least partially into a nucleic acid sequence of any one of SEQ ID NOs: 17-19 in the G-box region of the Glycine max GIF1 gene. In some embodiments, the mutation comprises a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides introduced at least partially into the G-box region for the Glycine max GIF1 gene.

In some embodiments, the method comprises (i) introducing a mutation into a G-box region of Glyma.03G249000, thereby producing a mutated G-box region comprising a nucleic acid sequence of any one of SEQ ID NOs: 20-24, and/or (ii) introducing a mutation into a G-box region of Glyma.19G246600, thereby producing a mutated G-box region comprising a nucleic acid sequence of SEQ ID NO: 25.

In some embodiments, the method further comprises introducing a genetic mutation that decreases BIG SEEDS (BS) activity into the plant or plant part. In some embodiments, the mutation that decreases the BIG SEEDS activity comprises one or more insertions, substitutions, or deletions in at least one BS gene or homolog thereof or regulatory region thereof, wherein an expression level of said at least one BS gene or homolog thereof is increased compared to an expression level a corresponding BS gene or homolog thereof without said mutation, and/or level or activity of a BIG SEEDS protein encoded by said at least one BS gene or homolog thereof is increased compared to level or activity of a BIG SEEDS protein encoded by a corresponding BS gene or homolog thereof without said mutation.

In some embodiments, said at least one BS gene or homolog thereof, before the mutation is introduced: (i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 26, 27, or 49, wherein said nucleic acid sequence encodes a polypeptide that retains BIG SEEDS activity; (ii) comprises the nucleic acid sequence of SEQ ID NO: 26, 27, or 49; (iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 28, 29, or 41, wherein said polypeptide retains BIG SEEDS activity; and/or (iv) encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, 29, or 41.

In some embodiments, said mutation that decreases the BIG SEEDS activity is located at least partially in a nucleic acid region of exon 1 or exon 2 of a Glycine max BS1 gene and/or exon 2 of a Glycine max BS2 gene. In some embodiments, the method comprises: (i) introducing a mutation into a Glycine max BS1 gene, thereby producing a mutated Glycine max BS1 gene comprising a deletion of nucleotides 98 through 101 of SEQ ID NO: 26;

(ii) introducing a mutation into Glycine max BS1 gene, thereby producing a mutated Glycine max BS1 gene comprising a deletion of nucleotides 389 through 396 of SEQ ID NO: 26; and/or (iii) introducing a mutation into Glycine max BS2 gene, thereby producing a mutated Glycine max BS2 gene comprising a deletion of nucleotides 409 through 415 of SEQ ID NO: 27.

In some embodiments, the method comprises introducing editing reagents or a nucleic acid construct encoding said editing reagents into said plant, plant part, or plant cell.

In some embodiments, said editing reagents comprise at least one nuclease, wherein the nuclease cleaves a target site in a genome of said plant, plant part, or plant cell, and said mutation is introduced at said cleaved target site. In some embodiments, the at least one nuclease comprises a CRISPR nuclease. In some embodiments, the CRISPR nuclease is a Type II CRISPR system nuclease, a Type V CRISPR system nuclease, a Cas9 nuclease, a Casl2a (Cpfl) nuclease, a Cmsl nuclease, or ortholog of any thereof.

In some embodiments, the editing reagents comprise one or more guide RNAs (gRNAs). In some embodiments, one or more guide RNAs comprise a nucleic acid sequence complementary to a region of a GIF1 gene or homolog thereof or regulatory region thereof in the plant or plant part. In some embodiments, at least one of the one or more guide RNAs binds a promoter region or a 5’ untranslated region (5’UTR) of a Glycine max GIF1 gene in said plant or plant part. In some embodiments, at least one of the one or more guide RNAs binds G-box region of the Glycine max GIF1 gene at or adjacent to a nucleic acid sequence of any one of SEQ ID NO: 17-19. In some embodiments, the editing reagents comprise two or more gRNAs.

In some embodiments, said plant or plant part is a legume. In some embodiments, said plant or plant part is selected from the group consisting of: soybean (Glycine max), beans (Phaseolus spp.), common bean (Phaseolus vulgaris), fava bean (Vida faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentils (Lens culinaris, Lens esculenta), lupins (Lupinus spp.), white lupin Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqud), tamarind (Tamarindus indica , alfalfa (Medicago sativa), barrel medic (Medicago truncatuld), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.).

In some embodiments, said plant or plant part is selected from the group consisting of: com (Zea mays), Brassica species, Brassica napus, Brassica rapa, Brassica juncea, rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet, pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp ), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp ), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp ), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

In one aspect, the present disclosure provides a plant or plant part produced by the method provided herein, wherein said plant or plant part comprises increased GIF1 activity compared to a control plant or plant part. In some embodiments, the plant or plant part comprises increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control plant or plant part. In one aspect, the present disclosure provides said plant or plant part is a seed.

In one aspect, the present disclosure provides a population of plants or plant parts produced by the method provided herein, wherein the population comprises increased GIF 1 activity, increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control population. In some embodiments, said population is a population of seeds.

In one aspect, the present disclosure provides a seed composition produced from the plant, plant part, or population of plants or plant parts provided herein.

In one aspect, the present disclosure provides a protein and/or oil composition produced from the plant, plant part, population of plants or plant parts or seed composition provided herein.

In one aspect, the present disclosure provides a food or beverage product comprising the plant, plant part, population of plants or plant parts, seed composition, or protein and/or oil composition provided herein.

In one aspect, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence of a mutated GIF1 regulatory region comprising a GIF1 promoter and/or a 5 ’ untranslated region (5’UTR), wherein said nucleic acid sequence: (i) has at least 80% identity to a nucleic acid sequence of any one of SEQ ID NOs: 20-25; or (ii) comprises the nucleic acid sequence of any one of SEQ ID NOs: 20-25, wherein binding of a GIF1 repressor complex to the mutated GIF1 regulatory region is decreased as compared to a corresponding GIF1 regulatory region without said mutation. In one aspect, the present disclosure provides a DNA construct comprising, in operable linkage, the nucleic acid molecule comprising the nucleic acid sequence of a mutated GIF1 regulatory region provided herein, and a polynucleotide of interest.

In some embodiments, the DNA construct further comprises, in operable linkage:

(i) a nucleic acid sequence comprising a mutated Glycine max BS1 gene comprising a deletion of nucleotides 98 through 101 of SEQ ID NO: 26, a mutated Glycine max BS1 gene comprising a deletion of nucleotides 389 through 396 of SEQ ID NO: 26, or a mutated Glycine max BS2 gene comprising a deletion of nucleotides 409 through 415 of SEQ ID NO: 27; and (ii) a promoter functional in a plant cell.

In one aspect, the present disclosure provides a cell comprising the nucleic acid molecule or the DNA construct provided herein. In one embodiment, the cell is a plant cell or a bacterial cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an expression profile of a GIF1 gene copy Glyma.03G249000 in various tissues of two varieties of soybean plants based on a soy expression database. FIG. IB depicts an expression profile of a GIF1 gene copy Glyma.l9G246600 in various tissues of two varieties of soybean plants based on a soy expression database. FIG. 1C depicts an expression profile of a GIF1 gene copy Glyma.lOGl 64100 in various tissues of two varieties of soybean plants based on a soy expression database. FIG. ID depicts an expression profile of a GIF1 gene copy Glyma.20G226500 in various tissues of two varieties of soybean plants based on a soy expression database. “FPKM” refers to fragments per kilobase of exon per million reads.

FIG. 2 depicts effect of substitution (SNP) mutations at or near the G-box sequence in the promoter region of a GIF1 gene copy Glyma.03G249000 on GIF1 activity in soybean protoplasts, as measured in three replicates. “NFC” refers to normalized fold change of the intensity of the reporter gene expression as a readout for the GIF1 expression.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure now will be described more fully hereinafter. The disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will satisfy applicable legal requirements.

I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. Further, the term “a plant” may include a plurality of plants. As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or.”

The term “about” or “approximately” usually means within 5%, or more preferably within 1%, of a given value or range.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

Various embodiments of this disclosure may be presented in a range format. It should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this disclosure. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1-10 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, from 1 to 9, from 2 to 4, from 2 to 6, from 2 to 8, from 2 to 10, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. The recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values =0 and =2 if the variable is inherently continuous.

A “plant” refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., leaves, stems, roots, embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, pulp, juice, kernels, ears, cobs, husks, stalks, root tips, anthers, etc.), plant tissues, seeds, plant cells, protoplasts and/or progeny of the same. A plant cell is a biological cell of a plant, taken from a plant or derived through culture of a cell taken from a plant. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention. As used herein, a “subject plant or plant cell” is one in which genetic alteration, such as a mutation, has been effected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. As used herein, the term “mutated” or “genetically modified” or “transgenic” or “transformed” or “edited” plants, plant cells, plant tissues, plant parts or seeds refers plants, plant cells, plant tissues, plant parts or seeds that have been mutated by the methods of the present disclosure to include one or more mutations (e.g., insertions, substitutions, and/or deletions) in the genomic sequence.

As used herein, a “control plant” or “control plant part” or “control cell” or “control seed” refers to a plant or plant part or plant cell or seed that has not been subject to the methods and compositions described herein. A “control” or “control plant” or “control plant part” or “control cell” or “control seed” provides a reference point for measuring changes in phenotype of the subject plant or plant cell. A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e. with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene);

(c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell;

(d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli (e.g., sucrose) that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed. In certain instances, a control plant of the present disclosure is grown under the same environmental conditions (e.g., same or similar temperature, humidity, air quality, soil quality, water quality, and/or pH conditions) as a subject plant described herein. Similarly, a control protein or control protein composition can refer to a protein or protein composition that is isolated or derived from a control plant. In specific embodiments, a control plant, plant part, or plant cell is a plant cell that does not have a mutated nucleotide sequence in a GIF1 gene or a regulatory region of a GIF1 gene.

Plant cells possess nuclear, plastid, and mitochondrial genomes. Accordingly, by “chromosome” or “chromosomal” is intended the nuclear, plastid, or mitochondrial genomic DNA. “Genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondria or plastids) of the cell. The compositions and methods disclosed herein are not limited to mutations made in the genomic DNA of the plant nucleus, but may be used to modify the sequence of the nuclear, plastid, and/or mitochondrial genome, or to modulate the expression of a gene or genes encoded by the nuclear, plastid, and/or mitochondrial genome. In certain embodiments, a mutation is created in the genomic DNA of an organelle (e.g. a plastid and/or a mitochondrion). In certain embodiments, a mutation is created in extrachromosomal nucleic acids (including RNA) of the plant, cell, or organelle of a plant. Nonlimiting examples include creating mutations in supernumerary chromosomes (e.g. B chromosomes), plasmids, and/or vector constructs used to deliver nucleic acids to a plant. It is anticipated that new nucleic acid forms will be developed and yet fall within the scope of the claimed invention when used with the teachings described herein. As used herein, the term “gene” or “coding sequence”, herein used interchangeably, refers to a functional nucleic acid unit encoding a protein, polypeptide, or peptide. As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A gene may include a regulatory region, e.g., a promoter region or a 5 ’untranslated region, that regulates transcription or translation of the encoded gene. For example, a “GIF1 gene” includes the coding region of the GIF1 gene, and may also include the regulatory region (e.g., promoter, 5’UTR, G- box region) of the GIF1 gene. Further, a “GIF1 gene” as used herein includes a homolog of a known GIF1 gene.

As used herein, the term a “nucleic acid”, used interchangeably with a “nucleotide”, refers to a molecule consisting of a nucleoside and a phosphate that serves as a component of DNA or RNA. For instance, nucleic acids include adenine, guanine, cytosine, uracil, and thymine.

As used herein, “allele” refers to an alternative nucleic acid sequence at a particular locus. The length of an allele can be as small as one nucleotide base. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome, e.g., as occurs for different chromosomes of a heterozygous individual, or between different homozygous or heterozygous individuals in a population. “Locus” as used herein refers to a chromosome region or chromosomal region where a polymorphic nucleic acid, trait determinant, gene, or marker is located.

As used herein, a “mutation” is any change in a nucleic acid sequence. Nonlimiting examples comprise insertions, deletions, duplications, substitutions, inversions, and translocations of any nucleic acid sequence, regardless of how the mutation is brought about and regardless of how or whether the mutation alters the functions or interactions of the nucleic acid. For example and without limitation, a mutation may produce altered enzymatic activity of a ribozyme, altered base pairing between nucleic acids (e.g. RNA interference interactions, DNA-RNA binding, etc.), altered mRNA folding stability, and/or how a nucleic acid interacts with polypeptides (e.g. DNA-transcription factor interactions, RNA-ribosome interactions, gRNA-endonuclease reactions, etc.). A mutation might result in the production of proteins with altered amino acid sequences (e.g. missense mutations, nonsense mutations, frameshift mutations, etc.) and/or the production of proteins with the same amino acid sequence (e.g. silent mutations). Certain synonymous mutations may create no observed change in the plant while others that encode for an identical protein sequence nevertheless result in an altered plant phenotype (e.g. due to codon usage bias, altered secondary protein structures, etc.). Mutations may occur within coding regions (e.g., open reading frames) or outside of coding regions (e.g., within promoters, terminators, untranslated elements, or enhancers), and may affect, for example and without limitation, gene expression levels, gene expression profiles, protein sequences, and/or sequences encoding RNA elements such as tRNAs, ribozymes, ribosome components, and microRNAs.

Accordingly, “plant with mutation” or “plant part with mutation” or “plant cell with mutation” or “plant genome with mutation” refers to a plant or plant part or plant cell or plant genome that contains a mutation (e.g., an insertion, a substitution, or a deletion) described in the present disclosure, such as a mutation in the nucleic acid sequence of a GIF1 gene or a regulatory region of a GIF1 gene. For example, as used herein, a plant, plant part or plant cell with mutation may refer to a plant, plant part or plant cell in which, or in an ancestor of which, at least one GIF1 gene or a regulatory region of the GIF1 gene has been deliberately mutated such that the plant, plant part or plant cell expresses a mutated (e.g., truncated) GIF1 protein or have an increased expression level of the GIF1 gene or GIF1 protein. The mutated GIF1 protein can have altered function, e.g., increased function or loss-of-function, compared to a wild-type, or control, GIF1 protein comprising no mutation.

“Genome editing” or “gene editing” as used herein refers to a type of genetic engineering by which one or more mutations (e.g., insertions, substitutions, deletions, modifications) are introduced at a specific location of the genome.

As used herein, the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or double -stranded polynucleotides. A recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector.

An expression construct can permit transcription of a particular nucleic acid sequence in a host cell (e.g., a bacterial cell or a plant cell). An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a promoter of and a nucleic acid molecule is a functional link that allows for expression of the nucleic acid molecule. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be co-transformed into the plant. Alternatively, the additional gene(s) can be provided on multiple expression cassettes or DNA constructs. The expression cassette may additionally contain selectable marker genes. Other elements that may be present in an expression cassette include those that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators), as well as those that confer certain binding affinity or antigenicity to the recombinant protein produced from the expression cassette.

As used herein, “function” of a gene, a peptide, a protein, or a molecule refers to activity of a gene, a peptide, a protein, or a molecule.

“Introduced” in the context of inserting a nucleic acid molecule (e.g., a recombinant DNA construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a plant cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., nuclear chromosome, plasmid, plastid chromosome or mitochondrial chromosome), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

As used herein with respect to a parameter, the term “increased” or “increasing” or “increase” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, or more) positive change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “increased”, “increase”, and the like encompass both a partial increase and a significant increase compared to a control.

As used herein with respect to a parameter, the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” or “loss” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) negative change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “decreased”, “reduced”, and the like encompass both a partial reduction and a complete reduction compared to a control.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

As used herein, the term “polypeptide” refers to a linear organic polymer containing a large number of amino-acid residues bonded together by peptide bonds in a chain, forming part of (or the whole of) a protein molecule. The amino acid sequence of the polypeptide refers to the linear consecutive arrangement of the amino acids comprising the polypeptide, or a portion thereof.

As used herein the terms “polynucleotide”, “polynucleotide sequence,” “nucleic acid sequence,” and “nucleic acid fragment” are used interchangeably and refer to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence (e.g., an mRNA sequence), a complementary nucleic acid sequence (cDNA), a genomic nucleic acid sequence, a synthetic nucleic acid sequence, and/or a composite nucleic acid sequences (e.g., a combination of the above). The polynucleotides provided herein encompass all forms of sequences including, but not limited to, single-stranded forms, double -stranded forms, hairpins, stem-and-loop structures, and the like.

The term “isolated” refers to at least partially separated from the natural environment e.g., from a plant cell. As used herein, the term “expression” or “expressing” refers to the transcription and/or translation of a particular nucleic acid sequence driven by a promoter.

As used herein, the terms “exogenous” or “heterologous” in reference to a nucleic acid sequence or amino acid sequence are intended to mean a sequence that is purely synthetic, that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. Thus, a heterologous nucleic acid sequence may not be naturally expressed within the plant (e.g., a nucleic acid sequence from a different species) or may have altered expression when compared to the corresponding wild type plant. An exogenous polynucleotide may be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the plant.

As used herein, by “endogenous” in reference to a gene or nucleic acid sequence or protein is intended a gene or nucleic acid sequence or protein that is naturally comprised within or expressed by a cell. Endogenous genes can include genes that naturally occur in the cell of a plant, but that have been modified in the genome of the cell without insertion or replacement of a heterologous gene that is from another plant species or another location within the genome of the modified cell.

As used herein, “fertilization” and/or “crossing” broadly includes bringing the genomes of gametes together to form zygotes but also broadly may include pollination, syngamy, fecundation and other processes related to sexual reproduction. Typically, a cross and/or fertilization occurs after pollen is transferred from one flower to another, but those of ordinary skill in the art will understand that plant breeders can leverage their understanding of fertilization and the overlapping steps of crossing, pollination, syngamy, and fecundation to circumvent certain steps of the plant life cycle and yet achieve equivalent outcomes, for example, a plant or cell of a soybean cultivar described herein. In certain embodiments, a user of this innovation can generate a plant of the claimed invention by removing a genome from its host gamete cell before syngamy and inserting it into the nucleus of another cell. While this variation avoids the unnecessary steps of pollination and syngamy and produces a cell that may not satisfy certain definitions of a zygote, the process falls within the definition of fertilization and/or crossing as used herein when performed in conjunction with these teachings. In certain embodiments, the gametes are not different cell types (i.e. egg vs. sperm), but rather the same type and techniques are used to effect the combination of their genomes into a regenerable cell. Other embodiments of fertilization and/or crossing include circumstances where the gametes originate from the same parent plant, i.e. a “self’ or “self-fertilization”. While selfing a plant does not require the transfer pollen from one plant to another, those of skill in the art will recognize that it nevertheless serves as an example of a cross, just as it serves as a type of fertilization. Thus, methods and compositions taught herein are not limited to certain techniques or steps that must be performed to create a plant or an offspring plant of the claimed invention, but rather include broadly any method that is substantially the same and/or results in compositions of the claimed invention. “Homolog” or “homologous sequence” may refer to both orthologous and paralogous sequences. Paralogous sequence relates to gene-duplications within the genome of a species. Orthologous sequence relates to homologous genes in different organisms due to ancestral relationship. Thus, orthologs are evolutionary counterparts derived from a single ancestral gene in the last common ancestor of given two species and therefore have great likelihood of having the same function. One option to identify homologs (e.g., orthologs) in monocot plant species is by performing a reciprocal BLAST search. This may be done by a first blast involving blasting the sequence-of-interest against any sequence database, such as the publicly available NCBI database which may be found at: ncbi.nlm.nih.gov. If orthologs in rice were sought, the sequence-of-interest would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI. The blast results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence-of-interest is derived. The results of the first and second blasts are then compared. An ortholog is identified when the sequence resulting in the highest score (best hit) in the first blast identifies in the second blast the query sequence (the original sequence-of-interest) as the best hit. Using the same rational a paralog (homolog to a gene in the same organism) is found. In case of large sequence families, the ClustalW program may be used [ebi.ac.uk/Tools/clustalw2/index.html], followed by a neighbor-joining tree (wikipedia.org/wiki/Neighbor-joining) which helps visualizing the clustering.

In some embodiments, the term “homolog” as used herein, refers to functional homologs of genes. A functional homolog is a gene encoding a polypeptide that has sequence similarity to a polypeptide encoded by a reference gene, and the polypeptide encoded by the homolog carries out one or more of the biochemical or physiological fimction(s) of the polypeptide encoded by the reference gene. In general, it is preferred that functional homologs and/or polypeptides encoded by functional homologs share at least some degree of sequence identity with the reference gene or polypeptide encoded by the reference gene.

Homology (e.g., percent homology, sequence identity+sequence similarity) can be determined using any homology comparison software computing a pairwise sequence alignment.

As used herein, “sequence identity,” “identity,” “percent identity,” “percentage similarity,” “sequence similarity” and the like refer to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol.266: 131-141; Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402); Zhang et al. (2000), J. Comput. Biol. 7( 1 -2) :203- 14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word size=3; gap opening penalty=-l 1; gap extension penalty=-l; and scoring matrix=BLOSUM62. As used herein, percent similarity of two nucleic acid sequences is the score based upon the following parameters for the BLASTn algorithm: word size=l 1; gap opening penalty=-5; gap extension penalty=-2; match reward=l; and mismatch penalty=-3. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff J G. (Proc Natl Acad Set 89: 10915-9 (1992)). Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

According to some embodiments, the identity is a global identity, i.e., an identity over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof.

According to some embodiments, the term “homology” or “homologous” refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences; or the identity of an amino acid sequence to one or more nucleic acid sequence. According to some embodiments, the homology is a global homology, e.g., a homology over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof. The degree of homology or identity between two or more sequences can be determined using various known sequence comparison tools which are described in WO2014/102774.

As used herein, the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term “population” refers to a set comprising any number, including one, of individuals, objects, or data from which samples are taken for evaluation, e.g., desirable traits, mutations, or quantitative trait locus (QTL) effects. The terms can relate to a population of plants to which a mutation has been introduced, or a breeding population of plants from which members are selected and crossed to produce progeny in a breeding program. A population of plants can include the progeny of a single breeding cross or a plurality of breeding crosses and can be either actual plants or plant derived material, or in silico representations of plants. The member of a population need not be identical to the population members selected for use in subsequent cycles of analyses, nor does it need to be identical to those population members ultimately selected to obtain a final progeny of plants. Often, a plant population is derived from a single biparental cross but can also derive from two or more crosses between the same or different parents. Although a population of plants can comprise any number of individuals, those of skill in the art will recognize that plant breeders commonly use population sizes ranging from one or two hundred individuals to several thousand, and that the highest performing 5-20% of a population is what is commonly selected to be used in subsequent crosses in order to improve the performance of subsequent generations of the population in a plant breeding program.

As used herein, the term “crop performance” is used synonymously with “plant performance” and refers to of how well a plant grows under a set of environmental conditions and cultivation practices. Crop performance can be measured by any metric a user associates with a crop’s productivity (e.g., yield), appearance and/or robustness (e.g., color, morphology, height, biomass, maturation rate, etc.), product quality (e.g., fiber lint percent, fiber quality, seed protein content, seed carbohydrate content, etc.), cost of goods sold (e.g., the cost of creating a seed, plant, or plant product in a commercial, research, or industrial setting) and/or a plant’s tolerance to disease (e.g., a response associated with deliberate or spontaneous infection by a pathogen) and/or environmental stress (e.g., drought, flooding, low nitrogen or other soil nutrients, wind, hail, temperature, day length, etc.). Crop performance can also be measured by determining a crop’s commercial value and/or by determining the likelihood that a particular inbred, hybrid, or variety will become a commercial product, and/or by determining the likelihood that the offspring of an inbred, hybrid, or variety will become a commercial product. Crop performance can be a quantity (e.g., the volume or weight of seed or other plant product measured in liters or grams) or some other metric assigned to some aspect of a plant that can be represented on a scale (e.g., assigning a 1-10 value to a plant based on its disease tolerance).

A “microbe” will be understood to be a microorganism, i.e. a microscopic organism, which can be single celled or multicellular. Microorganisms are very diverse and include all the bacteria, archaea, protozoa, fungi, and algae, especially cells of plant pathogens and/or plant symbionts. Certain animals are also considered microbes, e.g. rotifers. In various embodiments, a microbe can be any of several different microscopic stages of a plant or animal. Microbes also include viruses, viroids, and prions, especially those which are pathogens or symbionts to crop plants. A “pathogen” as used herein refers to a microbe that causes disease or harmful effects on plant health.

A “fungus” includes any cell or tissue derived from a fungus, for example whole fungus, fungus components, organs, spores, hyphae, mycelium, and/or progeny of the same. A fungus cell is a biological cell of a fungus, taken from a fungus or derived through culture of a cell taken from a fungus.

A “pest” is any organism that can affect the performance of a plant in an undesirable way. Common pests include microbes, animals (e.g. insects and other herbivores), and/or plants (e.g. weeds). Thus, a pesticide is any substance that reduces the survivability and/or reproduction of a pest, e.g. fungicides, bactericides, insecticides, herbicides, and other toxins.

“Tolerance” or “improved tolerance” in a plant to disease conditions (e.g. growing in the presence of a pest) will be understood to mean an indication that the plant is less affected by the presence of pests and/or disease conditions with respect to yield, survivability and/or other relevant agronomic measures, compared to a less tolerant, more "susceptible" plant. Tolerance is a relative term, indicating that a "tolerant" plant survives and/or performs better in the presence of pests and/or disease conditions compared to other (less tolerant) plants (e.g., a different soybean cultivar) grown in similar circumstances. As used in the art, “tolerance” is sometimes used interchangeably with “resistance”, although resistance is sometimes used to indicate that a plant appears maximally tolerant to, or unaffected by, the presence of disease conditions. Plant breeders of ordinary skill in the art will appreciate that plant tolerance levels vary widely, often representing a spectrum of more-tolerant or less-tolerant phenotypes, and are thus trained to determine the relative tolerance of different plants, plant lines or plant families and recognize the phenotypic gradations of tolerance.

“Yield” as used herein is defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production, leaf senescence and more. Root development, nutrient uptake, stress tolerance, photosynthetic carbon assimilation rates, and early vigor may also be important factors in determining yield. Optimizing the abovementioned factors may therefore contribute to increasing crop yield. Yield can be measured and expressed by any means known in the art. In specific embodiments, yield is measured by seed weight or volume in a given harvest area.

A plant, or its environment, can be contacted with a wide variety of “agriculture treatment agents.” As used herein, an “agriculture treatment agent”, or “treatment agent”, or “agent” can refer to any exogenously provided compound that can be brought into contact with a plant tissue (e.g. a seed) or its environment that affects a plant’s growth, development and/or performance, including agents that affect other organisms in the plant’s environment when those effects subsequently alter a plant’s performance, growth, and/or development (e.g. an insecticide that kills plant pathogens in the plant’s environment, thereby improving the ability of the plant to tolerate the insect's presence). Agriculture treatment agents also include a broad range of chemicals and/or biological substances that are applied to seeds, in which case they are commonly referred to as seed treatments and/or seed dressings. Seed treatments are commonly applied as either a dry formulation or a wet slurry or liquid formulation prior to planting and, as used herein, generally include any agriculture treatment agent including growth regulators, micronutrients, nitrogen-fixing microbes, and/or inoculants. Agriculture treatment agents include pesticides (e.g. fungicides, insecticides, bactericides, etc.) hormones (abscisic acids, auxins, cytokinins, gibberellins, etc.) herbicides (e.g. glyphosate, atrazine, 2,4-D, dicamba, etc.), nutrients (e.g. a plant fertilizer), and/or a broad range of biological agents, for example a seed treatment inoculant comprising a microbe that improves crop performance, e.g. by promoting germination and/or root development. In certain embodiments, the agriculture treatment agent acts extrace llularly within the plant tissue, such as interacting with receptors on the outer cell surface. In some embodiments, the agriculture treatment agent enters cells within the plant tissue. In certain embodiments, the agriculture treatment agent remains on the surface of the plant and/or the soil near the plant. In certain embodiments, the agriculture treatment agent is contained within a liquid. Such liquids include, but are not limited to, solutions, suspensions, emulsions, and colloidal dispersions. In some embodiments, liquids described herein will be of an aqueous nature. However, in various embodiments, such aqueous liquids that comprise water can also comprise water insoluble components, can comprise an insoluble component that is made soluble in water by addition of a surfactant, or can comprise any combination of soluble components and surfactants. In certain embodiments, the application of the agriculture treatment agent is controlled by encapsulating the agent within a coating, or capsule (e.g. microencapsulation). In certain embodiments, the agriculture treatment agent comprises a nanoparticle and/or the application of the agriculture treatment agent comprises the use of nanotechnology.

In certain embodiments, plants disclosed herein can be modified to exhibit at least one desired trait, and/or combinations thereof. The disclosed innovations are not limited to any set of traits that can be considered desirable, but nonlimiting examples include high protein content, male sterility, herbicide tolerance, pest tolerance, disease tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified seed oil, modified seed protein, modified lodging resistance, modified shattering, modified iron-deficiency chlorosis, modified water use efficiency, and/or combinations thereof. Desired traits can also include traits that are deleterious to plant performance, for example, when a researcher desires that a plant exhibits such a trait in order to study its effects on plant performance.

In certain embodiments, a user can combine the teachings herein with high-density molecular marker profiles spanning substantially the entire soybean genome to estimate the value of selecting certain candidates in a breeding program in a process commonly known as genomic selection.

The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

II. Overview of the Invention

Increased seed size, increased biomass, increased yield, and/or increased protein content in plants, plant parts, and plant products is an advantageous trait in the growing markets of food and beverages (e.g., plant-based food), feed, and industrial use. Cell proliferation and cell growth coordinately determine the organ size during the plant organogenesis. GRF interacting factor 1 (GIF1), a transcriptional co-activator, interacts with growth regulating factors (GRFs) and other molecules to control various developmental processes by regulating cell proliferation and growth in plants. The gifl single mutants can produce significantly smaller leaves, flowers, or plants with reduced cell number, while overexpression of GIF1 results in large leaf, flower, or plant size with an increase of cell number, as compared to wild-type plants. In addition, GIF1 interacts with GRFs, SWI/SNF chromatin remodeling complexes, and hormone biosynthesis- related proteins and regulate transcription of the target genes (e.g., GRF5, GRF3, COL5, ARR4, RAMOSA2, CLV3/ENDOSPERM SURROUNDING REGION 4a) for transcriptional regulation. PRAPODS (PPD1/2), an inhibitor of organ growth and development, interact with KINASE-INDUCIBLE DOMAIN INTERACTING 8/9 (KIX8/9) and TOPLESS (TPL) to form a repressor protein complex, which controls the leaf development by influencing the expression of cell division-related genes. The stability of KIX-PPD complex is regulated by an F-box protein STERILE APETALA (SAP) that acts as a part of the SKPl/Cullin/F-box E3 ubiquitin ligase complex. SAP positively regulates organ growth by targeting the KIX-PPD complex for 26S proteasome dependent degradation. Further, TPL, KIX8/9, PPD1/2, and/or transcription factors MYC3/4 can interact with one another to form a repressor complex (e.g., TPL-KIX- PPD-MYC complex). A repressor complex (e.g., KIX-PPD-MYC complex, TPL-KIX-PPD-MYC complex) associates with the G-box sequence (CACGTG, SEQ ID NO: 17) of a GIF1 promoter and represses expression of the GIF1 gene. Inhibition of the GIF1 gene negatively regulates (decreases) seed size in a plant. The transcriptional repression of GIF1 is relieved by decreased binding or loss or binding of a GIF1 repressor complex (e.g., KIX-PPD-MYC complex, TPL-KIX-PPD-MYC complex) to the G-box in the GIF1 gene promoter, e.g., by SAP modulating the KIX-PPD module for 26S proteasome degradation. Without the KIX-PPD complex, the binding of the repressor complex with the GIF1 promoter region is decreased, the GIF1 expression is increased, resulting in an increased seed size.

Modifying the native sequence of a GIF1 gene or its regulatory region (e.g., promoter, 5’UTR, G- box region) to enhance level or activity of GIF1 protein can be one approach to generate advantageous traits, such as increased seed size, increased biomass, increased yield, and/or increased protein content and increased disease tolerance, a GIF1 promoter contains a G-box region. A “G-box region” refers to a region in the regulatory region (e.g., promoter) of a gene comprising the G-box sequence, i.e., CACGTG (SEQ ID NO: 17). A G-box region is not limited to the G-box sequence, but can include the nucleotide regions adjacent to the G-box sequence, and can be about 10-200 nucleotides in length, e.g., about 10-20, 20-40, 40- 80, 80-120, 120-160, or 120-160 nucleotide long. The G-box region can be an area of transcriptional regulator (e.g., transcriptional repressor, e.g., KIX-PPD-MYC complex, TPL-KIX-PPD-MYC complex) binding. Mutations to the G-box region (i.e., the binding site of the repressor complex) of a GIF1 gene can result in conformational change and decrease the affinity of transcriptional regulator (e.g., transcriptional repressor) binding, thereby de-repressing GIF1 and increasing GIF1 level or activity.

Disclosed herein are plants or plant parts comprising a genetic mutation that increases the GIF1 activity compared to a control plant or plant part, as well as methods for making the plants or plant parts with increased GIF1 activity. Such plants or plant parts can have one or more insertions, substitutions, or deletions in at least one native (e.g., wild-type) GIF1 gene or homolog thereof or in its regulatory region. The plants or plant parts may further have decreased BIG SEEDS (BS) activity, e.g., a mutation in a BS gene or homolog thereof or regulatory region thereof that decreases the BIG SEEDS activity. The plants or plant parts can have an increased expression level of the GIF1 gene or homolog thereof, increased level or activity of the GIF1 protein encoded by the GIF1 gene or homolog thereof, altered (e.g., increased) expression or activity of GIF1 downstream target molecules that regulate organ size or protein content (e.g., GRF5, GRF3, COL5, ARR4, RA2, CLE4a), increased seed size, increased biomass, increased yield, and/or increased protein content, compared to a plant or plant part without the mutation.

Also disclosed herein are compositions and methods for producing plants, plant parts, or a population of plants or plant parts having increased seed size, increased biomass, increased yield, and/or increased protein content by introducing a genetic mutation that increases GIF1 activity. The methods disclosed herein can include introducing one or more insertions, substitutions, or deletions in at least one GIF1 gene or homolog thereof or in its regulatory region in the genome of a plant, plant part, or plant cell, such that an expression level of the GIF1 gene or homolog thereof is increased, level or activity of a GIF1 protein encoded by the GIF1 gene or homolog thereof is increased, and/or seed size, biomass, yield, or protein content is increased in the plant, plant part, or plant cell compared to a plant, plant part, or plant cell without the mutation. Said mutation can be introduced in the promoter region or 5’UTR of one or more of the GIF1 gene. The methods of the present disclosure can include introducing editing reagents (e.g., nuclease, guide RNA) into the plants or plant parts to introduce a mutation in at least one native GIF1 gene or homolog thereof or in its regulatory region. Introducing two or more guide RNAs into a plant or plant part can increase sequence diversity of mutations generated in the plant genome. The methods can further include introducing a mutation in at least one BS gene or homolog thereof or regulatory region thereof in the plant, plant part, or plant cell, such that the BIG SEEDS activity is decreased.

Also disclosed herein are a population of plants or plant parts (e.g., seeds) having increased GIF1 activity, an increased seed size, increased biomass, increased yield, and/or increased protein content, compared to a control population, and plant products (e.g., seed compositions, protein compositions, or food and beverage products) produced from the plants, plant parts, or population of plants or plant parts of the present disclosure. The plants, plant parts, a population of plants or plant parts (e.g., seeds), or plant products provided herein, including those produced using the methods disclosed herein, can have increased GIF 1 activity and/or increased seed size, increased biomass, increased yield, and/or increased protein content.

Further provided herein are nucleic acid molecules comprising a mutated GIF1 gene or its regulatory region (e.g., mutated GIF1 promoter), a DNA construct comprising such nucleic acid molecule operably linked to a promoter, and cells comprising the nucleic acid molecule or the DNA construct of the present disclosure.

III. Plants with Increased seed size, increased biomass, increased yield, and/or increased protein content

Plants and plant parts are provided herein having altered (e.g., increased) GIF1 level or activity as compared to a control plant or plant part. The plants or plant parts described herein having altered GIF1 level or activity can comprise a genetic mutation or transgene that alters (e.g., increases) GIF1 level or activity, altered (e.g., increased) expression levels of at least one GIF1 gene encoding GIF1 protein, altered (e.g., increased) GIF1 protein levels or activity, altered (e.g., increased) protein content, and/or altered (e.g., increased) pathogen tolerance compared to a control plant or plant part.

Also provided herein is a population of plants and plant parts comprising the plants and plant parts described herein having altered (e.g., increased) GIF1 level or activity. In such population of plants or plant parts, having altered GIF 1 level or activity relative to a control population, not all individual plants or plant parts need to have altered (e.g., increased) GIF1 level or activity, genetic mutation that cause altered (e.g., increased) GIF1 level or activity, or phenotypes caused by the altered (e.g., increased) GIF1 activity (e.g., increased seed size, increased biomass, increased yield, and/or increased protein content). In specific embodiments at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more plants within a given plant population have a mutation that alters (e.g., increases) the GIF1 level or activity.

The teachings herein are not limited to certain plant species, and it is envisioned that they can be modified to be useful for monocots, dicots, and/or substantially any crop and/or valuable plant type, including plants that can reproduce by self-fertilization and/or cross fertilization, hybrids, inbreds, varieties, and/or cultivars thereof. A plant or plant part of the present disclosure can be a legume, i.e., a plant belonging to the family Fabaceae (or Leguminosae), or a part (e.g., fruit or seed) of such a plant. When used as a dry grain, the seed of a legume is also called a pulse. Examples of legume include, without limitation, soybean (Glycine max), beans (Phaseolus spp.), common bean (Phaseolus vulgaris), fava bean (Vida faba), mung bean (Vigna radiata), pea (Pisum sativum and other members of the Fabaceae like Cjanus and Vigna species), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentils (Lens culinaris, Lens esculenta), lupins (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.). For example, a plant or plant part of the present disclosure can be Glycine max or Pisum sativum. Additionally, a plant or plant part of the present disclosure can be a crop plant or part of a crop plant, including legumes. Examples of crop plants include, but are not limited to, com (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), camelina (Camelina sativa), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), quinoa (Chenopodium quinoa), chicory (Cichorium intybus), lettuce (Lactuca sativa), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana spp., e.g., Nicotiana tabacum, Nicotiana sylvestris), potato (Solanum tuberosum), tomato (Solanum lycopersicum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), grapes (Vitis vinifera, Vitis riparia), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oil palm (Elaeis guineensis), poplar (Populus spp.), pea (Pisum sativum), eucalyptus (Eucalyptus spp.), oats (Avena sativa), barley (Hordeum vulgare), vegetables, ornamentals, and conifers. Additionally, a plant or plant part of the present disclosure can be an oilseed plant (e.g., canola (Brassica napus), cotton (Gossypium sp.), camelina (Camelina sativa) and sunflower (Helianthus sp.)), or other species including wheat (Triticum sp., such as Triticum aestivum L. ssp. aestivum (common or bread wheat), other subspecies of Triticum aestivum, Triticum turgidum L. ssp. durum (durum wheat, also known as macaroni or hard wheat), Triticum monococcum L. ssp. monococcum (cultivated einkom or small spelt), Triticum timopheevi ssp. timopheevi, Triticum turgigum L. ssp. dicoccon (cultivated emmer), and other subspecies of Triticum turgidum (Feldman)), barley (Hordeum vulgare), maize (Zea mays), oats (Avena sativa), or hemp (Cannabis sativa). Additionally, a plant or plant part of the present disclosure can be a forage plant or part of a forage plant. Examples of forage plants include legumes and crop plants described herein as well as grass forages including Agrostis spp., Lolium spp., Festuca spp., Poa spp., and Bromus spp.

A. Plants with altered level or activity of GIF1 protein

Provided herein are plants or plant parts (e.g., seeds) comprising altered (e.g., increased) GIF1 activity compared to a control plant or plant part. In specific embodiments, plants or plant parts provided herein comprise increased GIF 1 activity compared to a control plant or plant part, but at least partially retain the GIF1 activity. As used herein, “GIF1 activity” refers to the ability of a GIF1 protein (i) to associate with GRF or other co-activators and regulate downstream target genes and/or (ii) to regulate cell proliferation, cell growth, and/or organ size in plants or plant parts. In particular aspects, plants and plant parts (e.g., seeds, leaves) disclosed herein have a genetic mutation that alters (e.g., increases) the GIF1 activity. Also provided herein is a population of plants or plant parts (e.g., seeds) comprising altered (e.g., increased) GIF1 activity compared to a control population provided herein.

The genetic mutation that alters (e.g., increases) the GIF1 activity in the plants and plant parts provided herein can comprise one or more insertions, substitutions, or deletions in at least one native GIF1 gene or homolog thereof, or in a regulatory region of at least one native GIF1 gene or homolog thereof. The genetic mutation that alters (e.g., increases) the GIF1 activity can be located in at least one GIF1 gene or homolog thereof; in a regulatory region (e.g., promoter, 5’UTR, G-box region) of the GIF1 gene or homolog thereof; a coding region, a non-coding region, or a regulatory region of any other gene; or at any other site in the genome of the plant or plant part. A GIF 1 “gene”, as used herein, refers to any polynucleotide that encodes a polypeptide having GIF1 protein activity, including SEQ ID NOs: 1-4 and 9-12 in a GmGIFl gene. A GIF1 gene, as used herein, can refer to a polynucleotide including a regulatory region (e.g., promoter, 5’UTR, G-box region) of the GIF1 gene, such as SEQ ID NOs: 1-4 in a GmGIFl gene. A GIF1 gene can also include a homolog, ortholog, or variant, that retains GIF1 activity, of a known GIF1 gene. A “native” gene, as used herein, refers to any gene having a wild-type nucleic acid sequence, e.g., a nucleic acid sequence that can be found in the genome of a plant existing in nature, and need not naturally occur within the plant, plant part, or plant cell comprising such native gene. For example, a transgenic GIF1 gene located at a genomic site or in a plant in a non-naturally occurring matter is a “native” GIF1 gene if its nucleic acid sequence can be found in a plant existing in nature. A “regulatory region” of a gene, as used herein, refers to the region of a genome that controls expression of the gene, such as SEQ ID NOs: 13-16 in a GmGIFl gene. A regulatory region of a gene can include a genomic site where a RNA polymerase, a transcription factor, or other transcription modulators bind or where a regulatory structure or complex is formed to control mRNA synthesis of the gene, such as promoter regions, binding sites for transcription modulator proteins, 5 ’ untranslated region, and other genomic regions that contribute to regulation of transcription of the gene.

A control plant or plant part can be a plant or plant part to which a mutation or transgene (e.g., an exogenous copy of a GIF1 gene) provided herein has not been introduced, e.g., by methods of the present disclosure. Thus, a control plant or plant part (e.g., seeds, leaves) may express a native (e.g., wild-type) GIF1 gene endogenously. A control plant of the present disclosure may be grown under the same environmental conditions (e.g., same or similar temperature, humidity, air quality, soil quality, water quality, and/or pH conditions) as a plant with the mutation described herein. A plant, plant part (e.g., seeds, leaves), or a population of plants or plant parts of the present disclosure may have altered (e.g., increased) expression levels of at least one GIF1 gene or homolog thereof, altered (e.g., increased) GIF1 protein level or activity, altered (e.g., increased) protein content, and/or altered (e.g., increased) pathogen tolerance as compared to a control plant, plant part, or population, when the plant, plant part, or population of plants or plant parts of the present disclosure is grown under the same environmental conditions as the control plant or plant part.

1. Plants with one or more mutations in regulatory region of GIF 1 gene

The plants or plant parts described herein can comprise a mutation (e.g., one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions) that increases the GIF1 activity at least partially in a regulatory region of at least one (e.g., one, more than one but not all, or all) GIF1 gene. The regulatory region having the mutation can comprise a promoter region, 5’ untranslated region (5’UTR), a binding site (e.g., an enhancer sequence) for a transcription modulator protein (e.g., transcription factor), or other genomic regions that contribute to regulation of transcription or translation of at least one (e.g., one, more than one but not all, or all) GIF1 gene. As used herein, where an insertion, a substitution, or a deletion is “at least partially” in a certain nucleotide region, the whole part of the insertion, substitution, or deletion can be within the certain nucleotide region, or alternatively, can span across the certain nucleotide region and a region outside the nucleotide region. For instance, where an insertion, a substitution, or a deletion is at least partially in a regulatory region, the whole part of the insertion, the substitution, or the deletion can be within the regulatory region, or can span across the regulatory region and a region upstream or downstream of the regulatory region (e.g., exons, introns). In some embodiments, the mutation is at least partially in a promoter region of at least one (e.g., one, more than one but not all, or all) GIF1 gene. As used herein, a “promoter” refers to an upstream regulatory region of DNA prior to the ATG of a native gene, having a transcription initiation activity (e.g., function) for said gene and other downstream genes. “Transcription initiation” as used herein refers to a phase or a process during which the first nucleotides in the RNA chain are synthesized. It is a multistep process that starts with formation of a complex between a RNA polymerase holoenzyme and a DNA template at the promoter, and ends with dissociation of the core polymerase from the promoter after the synthesis of approximately first nine nucleotides. A promoter sequence can include a 5’ untranslated region (5’UTR), including intronic sequences, in addition to a core promoter that contains a TATA box capable of directing RNA polymerase II (pol II) to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence of interest. A promoter may additionally comprise other recognition sequences positioned upstream of the TATA box, and well as within the 5’UTR intron, which influence the transcription initiation rate. The one or more insertions, substitutions, and/or deletions in the promoter region of the GIF1 gene can alter the transcription initiation activity of the promoter. For example, the modified promoter can increase transcription of the operably linked nucleic acid molecule (e.g., the GIF1 gene), initiate transcription in a developmentally-regulated or temporally-regulated manner, initiate transcription in a cell-specific, cell-preferred, tissue-specific, or tissue-preferred manner, or initiate transcription in an inducible manner. A deletion, a substitution, or an insertion, e.g., introduction of a heterologous promoter sequence, a cis-acting factor, a motif or a partial sequence from any promoter, including those described elsewhere in the present disclosure, can be introduced into the promoter region of the GIF1 gene to confer an altered (e.g., increased) transcription initiation function according to the present disclosure. The mutation of a promoter region can comprise correction of the promoter sequence by: (i) detection of one or more polymorphism or mutation that enhances the activity of the promoter sequence; and (ii) correction of the promoter sequences by deletion, modification, and/or correction of the polymorphism or mutation. In some embodiments, the mutation is in the upstream region of a promoter region of at least one (e.g., one, more than one but not all, or all) GIF1 gene.

In some embodiments, a mutation is at least partially located in 5’UTR of one or more (e.g., one, more than one but not all, or all) GIF1 gene. As used herein, a “5’UTR”, used interchangeably with a 5’ untranslated region, a leader sequence, or a transcript leader, refers the region of a genomic DNA or mRNA from the transcription initiation site to the translation initiation codon (e.g., between the promoter and the translation initiation codon). The 5’UTR regulates translation of a main coding sequence of the mRNA by various mechanisms including forming complex secondary structure (e.g., pre-initiation complex regulation, closed-loop regulation) or being translated into a polypeptide that regulates translation of the main coding sequence (reinitiation of translation, cis- and trans-regulation).

In some embodiments, the plant or plant part provided herein comprises a mutation that is at least partially located in the regulatory region (e.g., promoter region or 5’UTR) of at least one (e.g., one, more than one but not all, or all) GIF1 gene. In specific embodiments, the mutation is located at least partially in a G-box region in the regulatory region of at least one GIF gene. The G-box region is part of the regulatory region that contains the G-box sequence, i.e., CACGTG (SEQ ID NO: 17), where a transcription factor (e.g., a repressor complex, e.g., KIX-PPD-MYC repressor complex) binds and negatively regulates GIF1. Mutation in the G-box region at or near the G-box sequence of the GIF1 gene can alter (e.g., decrease) binding of a transcription factor (e.g., GIF1 transcriptional repressor complex) and alter (e.g., increase) level or activity of a GIF1 protein encoded by the GIF1 gene. In specific embodiments, the plant or plant part has a mutation that decreases binding of a GIF1 repressor complex to the regulatory region of at least one GIF1 gene or homolog thereof, and increases level or activity of a GIF 1 protein encoded by said at least one GIF1 gene or homolog. Such mutation can be located at least partially into the G-box region in the regulatory region of at least one GIF gene.

In some embodiments, the mutation is located in the regulatory region of a GIF1 gene, and (i) the regulatory region, before the mutation is located, comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 13-16, wherein the regulatory region retains transcription initiation activity; (ii) the regulatory region, before the mutation is locate, comprises a nucleic acid sequence of any one of SEQ ID NOs: 13-16; (iii) the GIF1 gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; (iv) the GIF1 gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-12; (v) the GIF1 gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein the polypeptide retains GIF1 activity; (vi) the GIF1 gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8; (vii) the GIF1 gene including the regulatory region thereof, before the mutation is located, comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; and/or (viii) the GIF1 gene including said regulatory region thereof, before the mutation is located, comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4.

In some embodiments, the mutation is located at least partially in a promoter region or 5’UTR of a Glycine max GIF1 gene. The Glycine max GIF1 gene can be Glyma.03G249000, Glyma.l9G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l4G122500, or Glyma.OlGll 3500, or homolog thereof. In some embodiments, the mutation is located at least partially in a promoter region of a GmGIFl gene (e.g., SEQ ID NOs: 13-16). The mutation can be located at least partially in a G-box region of a GmGIFl gene, e.g., in a nucleic acid sequence of one or more of SEQ ID NOs: 17-19 in the Glycine max GIF1 gene. In some embodiments, the plant or plant part of the present disclosure comprises a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides located at least partially in the promoter, G- box region, and/or 5’UTR of a Glycine max GIF1 gene. In some embodiments, the plant or plant part provided herein comprises (i) Glyma.03G249000 with a mutated G-box region comprising a nucleic acid sequence of any one of SEQ ID NOs: 20-24; and/or (ii) Glyma.l9G246600 with a mutated G-box region comprising a nucleic acid sequence of SEQ ID NO: 25.

In some embodiments, a mutation is located in the gene encoding (or regulating expression of) one or more transcription factors that regulates expression of a GIF1 gene. A “transcription factor” as used herein refers to a protein (other than an RNA polymerase) that regulates transcription of a target gene. A transcription factor has DNA-binding domains to bind to specific genomic sequences such as an enhancer sequence or a promoter sequence. In some instances, a transcription factor binds to a promoter sequence near the transcription initiation site and regulate formation of the transcription initiation complex. A transcription factor can also bind to regulatory sequences, such as enhancer sequences, and modulate transcription of the target gene. The mutation in the gene encoding (or regulating expression of) a transcription factor can modulate expression or function of the transcription factor and increase expression levels of the GIF1 gene, e.g., by increasing transcription initiation activity of the GIF1 gene promoter. In some embodiments, the mutation modifies or inserts transcription factor binding sites or enhancer elements that regulates GIF1 gene expression into the regulatory region of the GIF1 gene.

In some embodiments, the mutation inserts a part or whole of one or more positive regulatory elements of the GIF1 gene into the genome of a plant cell or plant part. A “positive regulatory element” of a gene, as used herein, refers to a nucleic acid molecule that enhances expression or activity of the GIF1 gene, e.g., by enhancing transcription activity of the promoter. The positive regulatory sequence of the gene can be in a cis location or in a trans location. Positive regulatory elements of the one or more GIF1 genes can also include upstream open reading frames (uORFs). In some instances, a positive regulatory element can be inserted in a region upstream of the GIF1 gene in order to inhibit the expression and/or function of the gene.

The insertion, substitution, or deletion that is at least partially in the promoter, 5 ’ UTR, the gene encoding (or regulating expression of) one or more transcription factors that regulates expression of GIF1, or other regulatory region of a GIF1 gene can comprise insertion, substitution, or deletion of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more) nucleotides. The substitute can be a cisgenic substitute, a transgenic substitute, or both.

2. Plants with one or more mutations in at least one GIF1 gene, or its homolog, ortholo ., or variant

In some aspects, the plants and plant parts of the present disclosure comprise increased GIF1 activity and a genetic mutation that increases the GIF1 activity. The genetic mutation can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions in at least one native GIF1 gene (e.g., coding region, non-coding region, exons, introns, and/or regulatory region thereof) or homolog thereof. A plant or plant part described herein can comprise 1-2, 1-3, 1-4, 1-5, 2-5, 3-5, 4-5 (e.g., 1, 2, 3, 4, or 5) copies of GIF1 gene, e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.l8G121100, Glyma.14G 122500, and Glyma.01 G113500, each encoding a GIF 1 protein. In particular, a plant or plant part described herein can comprise at least 2 genes encoding a GIF1 protein, such as 2, 3, 4, or 5 genes that have less than 100% (e.g., less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85%) sequence identity to one another. The plant or plant part described herein can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions: in one GIF1 gene or homolog; in a regulatory region of one GIF1 gene or homolog; in more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all GIF1 genes or homologs; in regulatory regions of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all GIF1 genes or homologs; in all GIF1 genes or homologs; and/or in regulatory regions of all GIF1 genes or homologs in the plant or plant part. In one embodiment, the coding region of the GIF1 gene does not comprise a mutation (e.g., only the regulatory region of the GIF1 gene comprise a mutation). In another embodiment, the coding region of the GIF1 gene has a mutation (e.g., insertion, deletion, substitution, inversion, or truncation at N- or C-terminus) to increase protein content.

Each mutation can be heterozygous or homozygous. That is, the plants or plant parts described herein can comprise a certain mutation (e.g., comprising one or more insertions, substitutions, and/or deletions) in one allele or two (both) alleles of a GIF1 gene/homolog or its regulatory region. All mutations in the plant or plant part can be homozygous; all mutations in the plant or plant part can be heterozygous; or mutations can comprise some heterozygous mutations in certain locations of the genome and some homozygous mutations in certain locations of the genome in the plant or plant part.

In some embodiments, the mutation is located in a GIF1 gene or its regulatory region, and the GIF1 gene, before the mutation is located, (i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; (ii) comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-12; (iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein the polypeptide retains GIF1 activity; (iv) encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8; (v) includes the regulatory region thereof, and comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; and/or (vi) includes the regulatory region thereof, and comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4. In specific embodiments, the mutation that increases the GIF1 activity is located in one or two alleles of one or more (e.g., one, more than one but not all, or all) Glycine max GIF1 genes (e.g., Glyma.03G249000, Glyma.19G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.18G121100, Glyma.l4G122500, and Glyma.OlGl 13500) and/or a regulatory region thereof.

The mutation that increases the GIF1 activity in the plant or plant part disclosed herein can comprise an out-of-frame mutation of at least one (e.g., one, more than one but not all, or all) GIF1 gene or homolog thereof. Alternatively, the mutation in the plant or plant part can comprise an in-frame mutation, a nonsense mutation, or a missense mutation of at least one (e.g., one, more than one but not all, or all) GIF1 gene or homolog thereof.

A plant or plant part of the present disclosure can have a genetic mutation that increases the GIF1 activity in a gene that is a homolog, ortholog, or variant of a GIF1 gene disclosed herein and expresses a functional GIF1 protein, or in a regulatory region of such homolog, ortholog, or variant of a GIF1 gene. By “orthologs” is intended genes derived from a common ancestral gene and found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleic acid sequences and/or their encoded protein sequences share at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, plants or plant parts comprising polynucleotides that have GIF1 activity and share at least 75% sequence identity to the sequences disclosed herein are encompassed by the present disclosure and can have a genetic mutation that increases the GIF1 activity. For example, orthologs of GIF1 genes disclosed herein include, but are not limited to vigan.Gyeongwon.gnm3.annl. Vang03gl0430, vigra. VC1973A.gnm6.annl. Vradi0083s00920, Vigun06gl 14600, Phvul.006G103700, arahy. Tifrunner.gnm l.annl.L225S3,Lalb_Chrl8g0044001,Medtr7gll5410,Pisat.03G006130, pissa.Cameor.gnml.annl.Psat0s2429g0080, Ca_01247, AT5G28640, arahy.Tifrunner.gnml.annl. 71 F8WW, arahy. Tifrunner.gnml. annl.HKl FSC, pissa. Came or. gnml. annl.Psat0s3277g0080, Medtrlg080590, Pisat.06G061820,pissa.Cameor.gnml.annl.Psat0s368g0160, Ca_03768, Phvul.007Gl 89200, vigra. VC1973A.gnm6.annl. Vradi08gl3410, vigan.Gyeongwon.gnm3.annl. Vang06gl0880, Vigun07gl 56800, Lalb_Chr21gO311081 , Lalb_Chr06g0168171, wA Lalb_Chr06g0171791.

Variant sequences (e.g., homologs, orthologs) can be isolated by PCR. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Variant sequences (e.g., homologs, orthologs) may also be identified by analysis of existing databases of sequenced genomes. In this manner, variant sequences encoding GIF1 can be identified and used in the methods of the present disclosure. The variant sequences will retain the GIF1 activity.

In certain instances, mutations in any GIF1 gene in a plant, plant part, population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) can be identified by a diagnostic method described herein. Such diagnostic methods may comprise use of primers (a forward primer and a reverse primer) or an oligonucleotide probe for detecting a mutation in a GIF1 gene. For example, a pair of primers or an oligonucleotide probe can be designed to detect a mutation at or near the G-box region of a GIF1 gene, e.g., in a region comprising any one of SEQ ID NOs: 17-19, or to detect a mutation comprising any one of SEQ ID NOs: 20-25, in the G-box region of a GmGIFl gene. In certain instances, a kit comprising a set of primers and/or an oligonucleotide probe can be used for detecting mutation of GIF1 genes in a plants, plant part, or plant product (e.g., seed composition, plant protein/oil composition).

In some embodiments, the mutations, e.g., one or more insertions, substitutions, or deletions are integrated into the plant genome and the plant or the plant part is stably transformed. In other embodiments, the one or more mutations are not integrated into the plant genome and wherein the plant or the plant part is transiently transformed.

Also provided herein is a population of plants or plant parts (e.g., seeds) comprising the plants and plant parts having a genetic mutation that increases the GIF1 activity described herein.

One or more insertions, substitutions, or deletions located in at least one GIF1 gene or homolog or in a regulatory region of the GIF1 gene or homolog in the plant or plant part provided herein can increase the expression levels of the GIF1 gene or homolog, increase level or activity of the GIF1 protein encoded by the GIF1 gene or homolog, and/or increase organ size (e.g., seed size), biomass, yield, and/or protein content relative to a control plant or plant part without the mutation when grown under the same environmental conditions, as further described in the present disclosure.

3. Plants with increased GIF1 activity

The plants, plant parts (e.g., seeds, leaves), or plant products (e.g., seed composition, plant protein composition) of the present disclosure can comprise increased activity of GIF 1 protein compared to a control plant, plant part, or plant product. Also provided herein is a population of plants or plant parts (e.g., seeds) comprising the plants and plant parts of the present disclosure, which has increased GIF1 activity compared to a control (e.g., wild-type) population of plants or plant parts.

In particular, the GIF 1 activity in the plant, plant part, population of plants or plant parts, or plant product of the present disclosure can be increased by about 10-100%, 20-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300- 900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400- 500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more as compared to a control plant, plant part, population, or plant product.

Without wishing to be bound by theory, GIF1 is a transcriptional co-activator that interacts with growth regulating factors (GRFs) and other molecules to regulate cell proliferation and growth and control various developmental processes in plants. Expression of GIF1 is regulated in part by binding of a GIF1 repressor complex (e.g., KIX-PPD-MYC complex) to the G-box region of the GIF1 promoter. GIF1 activity can be measured by assessing level or activity of downstream target genes (e.g., GRF5, GRF3, COL5, ARR4, RA2, CLE4a) by measuring mRNA levels (e.g., quantitative RT-PCR, northern blot, serial analysis of gene expression (SAGE)), by measuring protein levels (e.g., western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from the plant or plant part using an antibody directed to the protein), or by standard functional assays or enzymatic assays for measuring activity of these downstream target proteins. GIF1 activity can also be assessed by measuring organ size (e.g., seed size, leaf size), cell counts within an organ, or total protein content. Protein content in a plant sample can be measured by, for example, protein extraction and quantitation (e.g., BCA protein assay, Lowry protein assay, Bradford protein assay), spectroscopy, near-infrared reflectance (NIR) (e.g., analyzing 700 - 2500 nm), and nuclear magnetic resonance spectrometry (NMR).

4. Plants with increased expression level o GIF 1 gene or GIF1 protein

The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure can have increased expression level of the GIF1 gene(s) or homolog, resulting in increased GIF 1 activity, as compared to the expression level of the GIF1 gene(s) or homolog in a control (e.g., wild-type) plant, plant part, a population of plants or plant parts, or plant product, e.g., a plant, plant part, a population of plants or plant parts, or plant product. Also provided herein is a population of plants or plant parts (e.g., seeds) comprising the plants and plant parts of the present disclosure, which has increased expression level of GIF1 gene(s) or GIF1 protein compared to a control (e.g., wild-type) population of plants or plant parts.

In some embodiments, the expression levels of the endogenous GIF1 gene(s) or homolog are increased by, e.g., genetic mutation or other mechanisms to up-regulate the expression of the endogenous GIF1 gene(s). The plant, plant part, population of plants or plant parts, or plant product comprising one or more insertions, substitutions, or deletions in at least one endogenous GIF1 gene or homolog or in a regulatory region thereof can have increased total expression levels of the GIF1 gene(s) or homolog as compared to a control (e.g., wild-type) plant, plant part, a population of plants or plant parts, or plant product. For example, the plant, plant part, population of plants or plant parts, or plant product can comprise a mutation in the regulatory region (e.g., promoter, 5’UTR, G-box region) of at least one endogenous GIF1 gene, e.g., at or near transcriptional repressor binding sites, e.g., a G-box region in the GIF1 promoter, that increases expression of the GIF1 gene. Alternatively or additionally, the expression levels of the GIF1 gene(s) or homolog are increased by introduction of one or more exogenous copies of a GIF1 gene into the plant or plant part. One or more exogenous copies of the GIF1 gene can be native, i.e., without mutation. Alternatively, one or more exogenous copies of the GIF1 gene can have a mutation that increases GIF1 level or activity.

In particular, the expression levels of GIF1 gene(s) or homolog in the plant, plant part, a population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) of the present disclosure can be increased by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70- 100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300- 1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more as compared to a control plant, plant part, a population of plants or plant parts, or plant product. In specific embodiments, the copy of GIF1 gene or homolog that contributes to an increased expression (e.g., up-regulation, overexpression) of the GIF1 gene or homolog is an endogenous or exogenous copy of a Glycine max GIF1 gene (e.g., Glyma.03G249000, Glyma.19G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.18G121100, Glyma.14G 122500, and Glyma.01G113500). Expression levels of the GIF1 gene or homolog can be measured by any standard methods for measuring mRNA levels of a gene, including quantitative RT-PCR, northern blot, and serial analysis of gene expression (SAGE).

The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure can have increased expression of the GIF 1 protein, as compared to the expression level of the GIF1 protein in a control (e.g., wild-type) plant, plant part, a population of plants or plant parts, or plant product. In particular, the expression levels of a full length GIF1 protein in the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure can be increased as compared to a control plant, plant part, a population of plants or plant parts, or plant product. A “full-length” GIF 1 protein, as used herein, refers to a GIF 1 protein comprising the complete amino acid sequence of a wild-type GIF1 protein, e.g., encoded by a native GIF1 gene, and having the complete function of a wildtype GIF1 protein. Additionally or alternatively, the expression levels of a functional fragment, variant, or ortholog of GIF 1 protein can be increased in the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure as compared to a control plant, plant part, a population of plants or plant parts, or plant product. In some embodiments, the levels of GIF1 protein encoded by the endogenous GIF1 gene(s) or homolog are increased by, e.g., genetic mutation or other mechanisms to up-regulate the expression of the endogenous GIF1 protein. The plant, plant part, population of plants or plant parts, or plant product comprising one or more insertions, substitutions, or deletions in at least one endogenous GIF1 gene or homolog (e.g., in the regulatory region, coding region, and/or non-coding region) can have increased expression level of GIF1 protein as compared to a control (e.g., wild-type) plant, plant part, a population of plants or plant parts, or plant product. For example, the plant, plant part, population of plants or plant parts, or plant product can comprise a mutation in the regulatory region (e.g., promoter, 5’UTR, G-box region) of at least one endogenous GIF1 gene, e.g., at or near transcriptional repressor binding sites, e.g., the G-box region, that increases expression of the GIF1 protein. Alternatively or additionally, the levels of GIF1 protein are increased by introduction of one or more exogenous copies of a GIF1 gene into the plant or plant part. One or more exogenous (e.g., transgenic) copies of the GIF1 gene can be from the same, related, or different plant species. One or more exogenous copies of the GIF1 gene can be native, i.e., without mutation; alternatively, one or more exogenous copies of the GIF1 gene can have a mutation (e.g., in the regulatory region, coding region, and/or non-coding region) that increases GIF1 level or activity.

In the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure, expression of GIF1 protein (e.g., functional GIF1 protein), e.g., encoded by endogenous and/or exogenous copies of the GIF1 gene(s), is increased by about 10-100%, 20-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300- 900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400- 500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, as compared to expression of GIF1 protein in a control plant, plant part, a population of plants or plant parts, or plant product. In certain embodiments, the GIF1 protein is encoded by endogenous or exogenous copies of the Glycine max GIF1 gene (e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l 4G 122500, and Glyma.OlGl 13500). Expression of a GIF1 protein in a plant, plant part, a population of plants or plant parts, or plant product can be determined by one or more standard methods of determining protein levels. For example, expression of a GIF1 protein can be determined by western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from a plant, plant part, a population of plants or plant parts, or plant product using an antibody directed to the GIF1 protein.

5. Plants with enhanced function of GIF1 protein

The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure can have increased function in the GIF1 protein, resulting in increased GIF1 activity, as compared to the GIF1 protein in a control plant, plant part, or plant product. Also provided herein is a population of plants or plant parts (e.g., seeds) comprising the plants and plant parts of the present disclosure, which has increased function of the GIF1 protein compared to a control (e.g., wildtype) population of plants or plant parts. The plant, plant part, population of plants or plant parts, or plant product can have a mutation in at least one endogenous GIF1 gene or homolog thereof (e.g., in the regulatory, coding, and/or non-coding regions) that enhances function of GIF1 protein. Additionally or alternatively, the plant, plant part, population of plants or plant parts, or plant product can have an exogenous copy of a GIF1 gene encoding GIF1 protein with enhanced function. A control plant, plant part, a population of plants or plant parts, or plant product can be a plant, plant part, a population of plants or plant parts, or plant product without the mutation, without an exogenous copy of a GIF1 gene, or otherwise having wild-type GIF1 activity. The GIF1 protein with increased function can comprise a mutation compared to a wild-type GIF1 protein that causes enhanced GIF1 function. In some embodiments, the function or activity of the GIF1 protein is increased by about 10-100%, 20-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300- 900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400- 500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, as compared to function or activity of a wildtype GIF1 protein. In certain embodiments, the GIF1 protein with enhanced function is encoded by an endogenous or exogenous copy of mutated Glycine max GIF1 gene (e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.IOG 164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l4G122500, and Glyma.OlGll 3500).

Function of a GIF1 protein in a plant, plant part, a population of plants or plant parts, or plant product can be measured by assessing level or activity of downstream target genes (e.g., GRF5, GRF3, COL5, ARR4, RA2, CLE4a) by measuring mRNA levels (e.g., quantitative RT-PCR, northern blot, serial analysis of gene expression (SAGE)), by measuring protein levels (e.g., western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from the plant or plant part using an antibody directed to the protein), or by standard functional assays or enzymatic assays for measuring activity of these downstream target proteins. GIF1 activity can also be assessed by measuring organ size (e.g., seed size, leaf size), cell counts within an organ, or total protein content. Protein content in a plant sample can be measured by, for example, protein extraction and quantitation (e.g., BCA protein assay, Lowry protein assay, Bradford protein assay), spectroscopy, near-infrared reflectance (NIR) (e.g., analyzing 700 - 2500 nm), and nuclear magnetic resonance spectrometry (NMR).

6. Plants with decreased BIG SEEDS activity

The plant, plant part (e.g., seeds, leaves), population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) of the present disclosure having increased GIF1 activity, e.g., comprising a mutation or an exogenous gene copy that increases GIF1 activity, can further have decreased BIG SEEDS activity as compared to a control plant or plant part. The plants or plant parts described herein having decreased BIG SEEDS level or activity can comprise a genetic mutation that decreases BIG SEEDS level or activity, decreased expression levels of at least one BS gene encoding BIG SEEDS protein, decreased BIG SEEDS protein levels or activity, increased activity of one or more target molecules regulated by BIG SEEDS and regulating organ growth or size [e.g., growth-regulating factor (GRF), GRF1, GRF5, GRF -interacting factor (GIF), GIF1, GIF2, cyclin D3;3 (CYCD3;3), histone 4 (H4)], increased organ (e.g., seed) size, increased biomass or yield (e.g., seed yield), and/or increased amino acid or protein content compared to a control plant or plant part. As used herein, “BIG SEEDS (BS) activity” refers to the ability of BIG SEEDS (i) to regulate organ growth or size and/or (ii) to regulate protein or amino acid content, by for instance regulating activity of its downstream target molecules [e.g., growth-regulating factor (GRF), GRF1, GRF5, GRF -interacting factor (GIF), GIF1, GIF2, cyclin D3;3 (CYCD3;3), histone 4 (H4)] that regulate organ growth or size, in plant or plant part.

In particular aspects, plants and plant parts (e.g., seeds, leaves), or a population of plants or plant parts disclosed herein (e.g., having increased GIF1 activity) further have a genetic mutation that decreases the BIG SEEDS activity. Also provided herein is a population of plants or plant parts (e.g., seeds) comprising altered BIG SEEDS activity compared to a control population provided herein.

The genetic mutation that decreases the BIG SEEDS activity in the plants and plant parts provided herein can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, or deletions in at least one native BS gene or homolog thereof, or in a regulatory region of at least one native BS gene or homolog thereof. The genetic mutation that decreases the BIG SEEDS activity can be located in at least one native BS gene or homolog thereof; in a regulatory region of the native BS gene or homolog thereof; a coding region, a non-coding region, or a regulatory region of any other gene; or at any other site in the genome of the plant or plant part.

A plant or plant part described herein can comprise 1-2, 1-3, 1-4, 1-5, 2-5, 3-5, 4-5 (e.g., 1, 2, 3, 4, or 5) copies of BS gene, e.g., BS1 and BS2 genes, each encoding a BIG SEEDS protein. In particular, a plant or plant part described herein can comprise at least 2 genes encoding a BIG SEEDS protein, such as 2, 3, 4, or 5 genes that have less than 100% (e.g., less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85%) sequence identity to one another. The plant or plant part described herein can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions: in one BS gene or homolog; in a regulatory region of one BS gene or homolog; in more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all BS genes or homologs; in regulatory regions of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all BS genes or homologs; in all BS genes or homologs; and/or in regulatory regions of all BS genes or homologs in the plant or plant part. Each mutation can be heterozygous or homozygous. That is, the plants or plant parts described herein can comprise a certain mutation (e.g., comprising one or more insertions, substitutions, and/or deletions) in one allele or two (both) alleles of aBS gene/homolog or its regulatory region. All mutations in the plant or plant part can be homozygous; all mutations in the plant or plant part can be heterozygous; or mutations can comprise some heterozygous mutations in certain locations of the genome and some homozygous mutations in certain locations of the genome in the plant or plant part. In some embodiments, the mutation that decreases the BIG SEEDS activity can be located in one or two alleles of a BS gene or homolog thereof comprising a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 26, 27, or 49 and encoding a polypeptide that retains BIG SEEDS activity, for example the nucleic acid sequence of SEQ ID NO: 26, 27, or 49; and/or a regulatory region of the BS gene or homolog thereof comprising such nucleic acid sequence. Additionally, the mutation can be located in one or two alleles of a BS gene or homolog thereof encoding a polypeptide comprising an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 28, 29, or 41 and retaining BIG SEEDS activity, for example a polypeptide comprising the amino acid sequence of SEQ ID NO: 28, 29, or 41; and/or a regulatory region of the BS gene or homolog thereof encoding such polypeptide. In specific embodiments, the mutation that decreases the BIG SEEDS activity is located in one or two alleles of one or more (e.g., one, more than one but not all, or all) Glycine max BS genes, such as a Glycine max BS1 gene, a Glycine max BS2 gene, or one or more Pisum sativum BS genes, and/or a regulatory region thereof.

In the plant or plant part provided herein comprising a mutation that decreases the BIG SEEDS activity, at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertion, substitution, or deletion can be located at least partially in a nucleic acid region of exon 1 or 2 of a Glycine max BS1 gene (set forth as SEQ ID NOs: 30 and 32, respectively) or exon 1 or 2 of a Glycine max BS2 gene (set forth as SEQ ID NOs: 31 and 33, respectively). In some embodiments, the plant or plant part of the present disclosure comprises a deletion of about 4-8 nucleotides at least partially in the nucleic acid region of exon 1 or 2 of a Glycine max BS1 gene or exon 2 of a Glycine max BS2 gene. For example, the plant or plant part of the present disclosure can comprise one or two alleles of (i) a mutated Glycine max BS1 gene comprising a deletion of nucleotides 98 through 101 of SEQ ID NO: 26, (ii) a mutated Glycine max BS1 gene comprising a deletion of nucleotides 389 through 396 of SEQ ID NO: 26, and/or (iii) a mutated Glycine max BS2 gene comprising a deletion of nucleotides 409 through 415 of SEQ ID NO: 27.

The mutation that decreases the BIG SEEDS activity in the plant or plant part disclosed herein can comprise an out-of-frame mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) BS gene or homolog thereof. Alternatively, the mutation in the plant or plant part can comprise an inframe mutation, a nonsense mutation, or a missense mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) BS gene or homolog thereof.

A plant or plant part of the present disclosure can have a genetic mutation that decreases the BIG SEEDS activity in a gene that is a homolog, ortholog, or variant of a BS gene disclosed herein and expresses a functional BIG SEEDS protein, or in a regulatory region of such homolog, ortholog, or variant of a BS gene. Functions of orthologs are often highly conserved among species. Thus, plants or plant parts comprising polynucleotides that have BIG SEEDS activity and share at least 75% sequence identity to the sequences disclosed herein are encompassed by the present disclosure and can have a genetic mutation that decreases the BIG SEEDS activity. For example, orthologs of BS genes disclosed herein include, but are not limited to yellow pea BS1 (Pisum sativum, the nucleic acid sequence and amino acid sequence set forth as SEQ ID NO: 40 and 41, respectively), barrel medic BS1 (Medicago truncatula, NCBI ID: KM668032.1), Alfalfa BS1 (Medicago sativa, NCBI ID: KM668033.1), common bean BS1 (Phaseolus vulgaris, NCBI ID: KM668018.1), and Peruvian cotton BS1, BS2, BS3 (Gossypium raimondii, NCBI IDs: KM668013.1, KM668014.1, KM668015.1).

In certain instances, mutations in any BS gene in a plant, plant part, population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) can be identified by a diagnostic method described herein. Such diagnostic methods may comprise use of primers for detecting mutation in a BS gene. For example, a forward primer set forth as SEQ ID NO: 36 and a reverse primer set forth as SEQ ID NO: 37 can be used for detection of mutation in Glycine max BS1 or BS2 gene near the binding site of the GmBSl/GmBS2 guide RNA1, for example a mutation generated by introducing the GmBSl/GmBS2 guide RNA1 into the plant or plant part. A forward primer set forth as SEQ ID NO: 38 and a reverse primer set forth as SEQ ID NO: 39 can be used for detection of mutation in Glycine max BS1 or BS2 gene near the binding site of the GmBSl/GmBS2 guide RNA 4, for example a mutation generated by introducing the GmBSl/GmBS2 guide RNA 4 into the plant or plant part. In certain instances, a kit comprising a set of primers can be used for detecting mutation of BS genes in plants, plant parts, or plant product (e.g., seed composition, plant protein composition). For example, a kit comprising the forward primer SEQ ID NO: 36 and the reverse primer SEQ ID NO: 37, and a kit comprising the forward primer SEQ ID NO: 38 and the reverse primer SEQ ID NO: 39 can be used for detection of mutation in BS1 or BS2 gene in plants, plant parts, or plant products (e.g., seed composition, plant protein compositions) near the binding site of the GmBSl/GmBS2 guide RNA1 and guide RNA4, respectively.

In some embodiments, the mutations, e.g., one or more insertions, substitutions, or deletions are integrated into the plant genome and the plant or the plant part is stably transformed. In other embodiments, the one or more mutations are not integrated into the plant genome and wherein the plant or the plant part is transiently transformed.

The BIG SEEDS activity in the plant, plant part, population of plants or plant parts, or plant product of the present disclosure can be reduced by about 10-99%, 20-99%, 30-99%, 40-99%, 50-99%, 60-99%, 70- 99%, 80-99%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, or 100% (e.g., by about 10-20%, 20- 30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-99%, 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to a control plant, plant part, population, or plant product.

Without wishing to be bound by theory, BIG SEEDS is a transcription regulator and a group II member of the TIFY family of proteins. BIG SEEDS interact with Novel Interactor of JAZ (NINJA), an adaptor protein that interacts with the transcription corepressors TOPLESS (TPL) and TOPLESS- RELATED PROTEINs (TPRs), to suppress downstream gene expression. BIG SEEDS activity can be measured by measuring expression levels of one or more downstream target genes, e.g., growth-regulating factor 1 and 5 (GRF1 and GRF5), GRF-interacting factor 1 and 2 (GIF1 and GIF2), cyclin D3;3 (CYCD3;3), and HISTONE4 (H4) by quantitative RT-PCR, northern blot, serial analysis of gene expression (SAGE), or any other methods for measuring mRNA levels. BIG SEEDS activity can also be measured by measuring levels of proteins encoded by one or more downstream target genes, e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4, by for instance western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from the plant or plant part using an antibody directed to the protein. BIG SEEDS activity can also be measured by measuring activity of downstream target proteins, e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4, by standard functional assays or enzymatic assays for measuring activity of these proteins. In certain embodiments, decrease in BIG SEEDS activity can be assessed by increase in expression levels (e.g., mRNA or protein levels) or activity of the BIG SEEDS downstream target molecules (e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4). For example, expression levels (e.g., mRNA or protein levels) or activity of the BIG SEEDS downstream target molecules (e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4) can be increased by about 10-100%, 20-100%, 30-100%, 40- 100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100- 1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200- 900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300- 400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%,

20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, as compared to a control plant or plant part.

The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure, e.g., comprising one or more insertions, substitutions, or deletions in at least one native BS gene or homolog or in a regulatory region of such BS gene or homolog can have reduced expression level of the BS gene(s), the BIG SEEDS protein, or homolog thereof as compared to the expression level in a control plant, plant part, a population of plants or plant parts, or plant product, e.g., a plant, plant part, a population of plants or plant parts, or plant product without such mutation. The expression levels of BS gene(s) or homolog or a BIG SEEDS protein in the plant, plant part, a population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) of the present disclosure can be reduced by about 10-99%, 20-99%, 30-99%, 40-99%, 50-99%, 60-99%, 70-99%, 80-99%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to a control plant, plant part, a population of plants or plant parts, or plant product. In specific embodiments, the BS gene or homolog is a BS1 gene and/or a BS2 gene, e.g., a Glycine max BS1 gene and/or a Glycine max BS2 gene. Expression levels of the BS gene or homolog can be measured by any standard methods for measuring mRNA levels of a gene, including quantitative RT-PCR, northern blot, and serial analysis of gene expression (SAGE). Expression levels of the BIG SEEDS protein in a plant, plant part, a population of plants or plant parts, or plant product can also be measured by any standard methods for measuring protein levels, including western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from a plant, plant part, a population of plants or plant parts, or plant product using an antibody directed to the BIG SEEDS protein encoded by the BS gene.

The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure, e.g., comprising one or more insertions, substitutions, or deletions in at least one native BS gene or homolog or in a regulatory region of such BS gene or homolog can have loss- of-function or reduced function in the BIG SEEDS protein, e.g., loss of BIG SEEDS activity or reduced BIG SEEDS activity, as compared to the BIG SEEDS protein in a control plant, plant part, or plant product. The function or activity of the BIG SEEDS protein encoded by the BS gene or homolog having a mutation (e.g., one or more insertions, substitutions, or deletions) in the gene or its regulatory region can be reduced by about 10-99%, 20-99%, 30-99%, 40-99%, 50-99%, 60-99%, 70-99%, 80-99%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to function or activity of a control BIG SEEDS protein encoded by a control BS gene or homolog without such mutation. In certain embodiments, the BIG SEEDS protein is encoded by the BS1 gene and/or the BS2 gene, e.g., Glycine max BS1 gene and/or Glycine max BS2 gene.

7. Plants with increased seed size, increased biomass, increased yield, and/or increased protein content

The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure, e.g., comprising a mutation or an exogenous gene copy that increases GIF1 activity, can have increased organ size (e.g., seed size), increased biomass, increased yield, and/or increased protein content as compared to a control (e.g., wild-type) plant, plant part, or plant product. Also provided herein is a population of plants or plant parts (e.g., seeds) comprising the plants and plant parts of the present disclosure, which has increased organ size (e.g., increased average organ size), increased biomass, increased yield, and/or increased protein content as compared to a control population. “Organ” as used herein refers to a functional and structural unit of a plant including but not limited to seeds, leaves, roots, stems, flowers, fruits. In specific embodiments, the plant, plant part, population of plants or plant parts, or plant product provided herein, having increased GIF1 activity, further has decreased BIG SEEDS activity (e.g., a genetic mutation that decreases BIG SEEDS activity) provided herein. Decrease in BIG SEEDS activity can upregulate a downstream target gene GIF1 and increase GIF1 activity, as disclosed herein. Further, increased GIF1 activity and decreased BIG SEEDS activity (e.g., a genetic mutation that increases GIF1 activity and a genetic mutation that decreases BIG SEEDS activity) can have additive or synergistic effect on organ size, biomass, yield, and/or protein content. For example, plants, plant parts, a population of plants or plant parts, or plant products having modifications (e.g., mutations) that increase GIF1 activity as well as modifications (e.g., mutations) that decrease BIG SEEDS activity can have greater organ size (e.g., seed size), greater biomass, greater yield, and/or greater protein content relative to control plants, plant parts, population of plants or plant parts, or plant products having a modification (e.g., mutation) that increases GIF1 activity but not a modification (e.g., mutation) that decreases BIG SEEDS activity, or having a modification (e.g., mutation) that decreases BIG SEEDS activity but not a modification (e.g., mutation) that increases GIF1 activity other than the effect of the decreased BIG SEEDS activity. The organ size, biomass, yield, and/or protein content can be increased by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-50%, 30-50%, 40-50%, 50-50%, 60-50%, 70-50%, 100-500%, 200-500%, 300-500%, 400-500%, 500-500%, 600-500%, 700-500%, 800-500%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 500% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, or more than 500%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, or more in plants, plant parts, a population of plants or plant parts, or plant products having modifications that increase GIF1 activity as well as modifications that decrease BIG SEEDS activity relative to control plants, plant parts, population of plants or plant parts, or plant products having a modification that increases GIF1 activity but not a modification that decreases BIG SEEDS activity, or having a modification that decreases BIG SEEDS activity but not a modification that increases GIF1 activity other than the effect of the decreased BIG SEEDS activity.

A control plant, plant part, a population of plants or plant parts, or plant product can comprise a plant or plant part to without a mutation or an exogenous gene copy provided herein. Thus, a control plant, plant part, a population of plants or plant parts, or plant product has a wild-type GIF1 activity (and a wildtype BIG SEEDS activity), and may express a native (e.g., wild-type) GIF1 gene (and BS gene) endogenously or transgenically. A plant, plant part, a population of plants or plant parts, or plant product of the present disclosure can have increased seed size, increased biomass, increased yield, and/or increased protein content as compared to a control plant, plant part, a population of plants or plant parts, or plant product, when the plant or plant part of the present disclosure is grown under the same environmental conditions (e.g., same or similar temperature, humidity, air quality, soil quality, water quality, and/or pH conditions) as the control plant or plant part.

In some embodiments, organ size (e.g., seed size, leaf size), plant biomass, or yield (e.g., seed yield) of the plant or plant part of the present disclosure is increased by about 10-100%, 20-100%, 30-100%, 40- 100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-

1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200- 900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300- 400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more as compared to a control plant or plant part. In specific embodiments, seed size, leaf size, and/or seed yield is increased in the plants or plant parts provided herein relative to a control plant or plant part by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300- 900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400- 500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,

200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. Organ size can be measured by measuring parameters (e.g., seed diameter, stem length, leaf width and length) or calculating organ size based on measured parameters according to the standard methods. For instance, leaf area (LA) can be estimated by using the formula: LA = 2.0185 x L x W, where L is length and W is width (Richter et al. 2014 Bragantici 73(4):416-425), with an R² of 0.9747. Yield and biomass can be measured and expressed by standard methods, for example weight or volume of seeds, fruits, leaves, or whole plants harvested from a given harvest area.

In some embodiments, total protein content of the plant or plant part of the present disclosure can be increased by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20- 90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500- 1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70- 80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more in the plants or plant parts of the present disclosure as compared to a control plant or plant part. In some embodiments, total protein content, as expressed by % dry weight, in the plant, plant part, or a population of plant or plant parts provided herein is greater than that in control plant, plant part, or population, and the difference (by subtraction) is about 0.25-10%, 0.5-10%, 0.75-10%, 1.0-10%, 1.5-10%, 2-10%, 2.5-10%, 3-10%, 3.5-10%, 4-10%, 4.5-10%, 5-10%, 6-10%, 7-10%, 8-10%, 9-10%, or more than 10% (e.g., by about 0.25-0.5%, 0.5-0.75%, 0.75-1.0%, 1.0-1.5%, 1.5-2.0%, 2.0-2.5%, 2.5-3.0%, 3.0-3.5%, 3.5-4.0%, 4.0-4.5%, 4.5-5.0%, 5-6%, 6-7%, 7-8%, or 8-9%, 9-10%, or more than 10%), by about 0.25%, 0.5%, 0.75%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, or more, or at least 0.25%, 0.5%, 0.75%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, or more by dry weight.

In specific embodiments, provided herein are seeds or a population of seeds having seed protein content greater than control seeds or a control population of seeds (e.g., control seeds or population having native GIF1 (and native BS), reference seeds or population, commodity seeds or population). The seeds can be legume seeds, e.g., pea seeds or soybean seeds. Typical pea cultivars average approximately 20-30% protein in the seed in dry weight (Meng & Cloutier, 2014 Microencapsulation in the Food Industry: A Practical Implementation Guide § 20.5). In contrast, the pea seeds or a population of pea seeds provided herein can have seed protein content of at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more by dry weight. Seed protein content of typical soybean cultivars ranges approximately 36-46% in dry weight (Rizzo & Baroni 2018 Nutrients 10( 1):43 ; Grieshop & Fahey 2001 J Agric Food Chem 49(5):2669-73; Garcia et al. 1997 Crit Rev Food Set Nutr 37(4) :361-91). In contrast, the soybean seeds or a population of soybean seeds provided herein can have seed protein content of at least 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% or more by dry weight.

Protein content in a plant sample can be measured by standard methods, for example by protein extraction and quantitation (e.g., BCA protein assay, Lowry protein assay, Bradford protein assay), spectroscopy, near-infrared reflectance (NIR) (e.g., analyzing 700 - 2500 nm), and nuclear magnetic resonance spectrometry (NMR).

In specific embodiments, the plant, plant part, or a population of plants or plant parts of the present disclosure have the trait of increased organ (e.g., seed) size, biomass, and/or yield (e.g., seed yield) as well as the trait of increased protein content as compared to a control plant, plant part, population of plants or plant parts, or plant product.

In specific embodiments, provided herein are seeds and a population of seeds with increased GIF1 activity (and decreased BS activity) provided herein, having an increased seed size, increased biomass, increased yield, and/or increased protein content as compared to control seeds or a population of seeds. B. Plant parts and plant products

The present disclosure provides plant parts and plant products obtained from the plant of the present disclosure. A “plant part”, as used herein, refers to any part of a plant, including seeds (e.g., a representative sample of seeds), plant cells, embryos, pollen, ovules, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, juice, pulp, nectar, stems, branches, and bark. A “plant product”, as used herein, refers to any product or composition produced from the plant, including any oil products, sugar products, fiber products, protein products (such as protein concentrate, protein isolate, flake, or other protein product), seed hulls, meal, or flour, for a food, feed, aqua, or industrial product, plant extract (e.g., sweetener, antioxidants, alkaloids, etc.), plant concentrate (e.g., whole plant concentrate or plant part concentrate), plant powder (e.g., formulated powder, such as formulated plant part powder (e.g., seed flour)), plant biomass (e.g., dried biomass, such as crushed and/or powdered biomass), grains, plant protein composition, plant oil composition, and food and beverage products containing plant compositions (e.g., plant parts, plant extract, plant concentrate, plant powder, plant protein, plant oil, and plant biomass) described herein. Plant parts and plant products provided herein can be intended for human or animal consumption.

As used herein, a “protein product” or “protein composition” refers to any protein composition or product isolated, extracted, and/or produced from plants or plant parts (e.g., seed) and includes isolates, concentrates, and flours, e.g., soy protein composition, soy protein concentrate (SPC), soy protein isolate (SPI), soy flour, flake, white flake, texturized vegetable protein (TVP), or textured soy protein (TSP)). A plant protein composition can be a concentrated protein solution (e.g., soybean protein concentrate solution) in which the protein is in a higher concentration than the protein in the plant from which the protein composition is derived. The protein composition can comprise multiple proteins as a result of the extraction or isolation process. In specific embodiments, the protein composition can further comprise stabilizers, excipients, drying agents, desiccating agents, anti-caking agents, or any other ingredient to make the protein fit for the intended purpose. The protein composition can be a solid, liquid, gel, or aerosol and can be formulated as a powder. The protein composition can be extracted in a powder form from a plant and can be processed and produced in different ways, such as: (i) as an isolate - through the process of wet fractionation, which has the highest protein concentration; (ii) as a concentrate - through the process of dry fractionation, which are lower in protein concentration; and/or (Hi) in textured form - when it is used in food products as a substitute for other products, such as meat substitution (e.g. a “meat” patty). Protein isolate can be derived from defatted soy flour with a high solubility in water, as measured by the nitrogen solubility index (NSI). The aqueous extraction is carried out at a pH below 9. The extract is clarified to remove the insoluble material and the supernatant liquid is acidified to a pH range of 4-5. The precipitated protein-curd is collected and separated from the whey by centrifuge. The curd can be neutralized with alkali to form the sodium proteinate salt before drying. Protein concentrate can be produced by immobilizing the soy globulin proteins while allowing the soluble carbohydrates, whey proteins, and salts to be leached from the defatted flakes or flour. The protein is retained by one or more of several treatments: leaching with 20-80% aqueous alcohol/solvent, leaching with aqueous acids in the isoelectric zone of minimum protein solubility, pH 4-5; leaching with chilled water (which may involve calcium or magnesium cations), and leaching with hot water of heat-treated defatted protein meal/flour (e.g., soy meal/flour). Any of the process provided herein can result in a product that is 70% protein, 20% carbohydrates (2.7 to 5% crude fiber), 6% ash and about 1% oil, but the solubility may differ. As an example, one ton (t) of defatted soybean flakes can yield about 750 kg of soybean protein concentrate. “Texturized vegetable protein” (TVP), “Textured vegetable protein”, also referred to as “textured soy protein” (TSP), soy meat, or soya chunks refers to a defatted plant (e.g., soy) flour product, a by-product of extracting plant (e.g., soybean) oil. It can be used as a meat analogue or meat extender. It is quick to cook, with a protein content comparable to certain meats. TVP can be produced from any protein-rich seed meal left over from vegetable oil production. A wide range of pulse seeds other than soybean, such as lentils, peas, and fava beans, or peanut may be used for TVP production. TVP can be made from high protein (e.g., 50%) soy isolate, flour, or concentrate, and can also be made from cottonseed, wheat, and oats. It is extruded into various shapes (chunks, flakes, nuggets, grains, and strips) and sizes, exiting the nozzle while still hot and expanding as it does so. The defatted thermoplastic proteins are heated to 150-200 °C, which denatures them into a fibrous, insoluble, porous network that can soak up as much as three times its weight in liquids. As the pressurized molten protein mixture exits the extruder, the sudden drop in pressure causes rapid expansion into a puffy solid that is then dried. As much as 50% protein when dry, TVP can be rehydrated at a 2: 1 ratio, which drops the percentage of protein to an approximation of ground meat at 16%. TVP can be used as a meat substitute. When cooked together, TVP can help retain more nutrients from the meat by absorbing juices normally lost. Also provided herein are methods of isolating, extracting, or preparing any of the protein compositions or protein products provided herein from plants or plant parts.

In specific embodiments, the plant protein compositions provided herein are obtained from a legume plant (e.g., Pisum sativum, Glycine max) that contains a mutation or a transgene that increases GIF1 activity, e.g., one or more insertions, substitutions, or deletions in at least one native GIF1 gene or homolog (e.g., in a regulatory region, a coding region, and/or a non-coding region), or an exogenous copy of a GIF1 gene; and optionally a mutation that decreases BIG SEEDS activity, e.g., one or more insertions, substitutions, or deletions in at least one native BS gene or homolog or regulatory region thereof.

Food and/or beverage products of the present disclosure can contain plant compositions, e.g., seed composition, plant protein compositions of the present disclosure. Food and/or beverage products can be meant for human or animal consumption. Food and/or beverage products of the present disclosure can include animal feed, shakes (e.g., protein shakes), health drinks, alternative meat products (e.g., meatless burger patties, meatless sausages), alternative egg products (e.g., eggless mayo), non-dairy products (e.g., non-dairy whipped toppings, non-dairy milk, non-dairy creamer, non-dairy milk shakes, non-diary ice cream), energy bars (e.g., protein energy bars), infant formula, baby foods, cereals, baked goods, edamame, tofu, and tempeh. A food and/or beverage product that contains plant compositions obtained from plants or plant parts of the present disclosure can have desired traits (e.g., increased GIF activity, decreased BS activity, increased protein content) compared to a similar or comparable food and/or beverage product that contains plant compositions obtained from a control plant or plant part.

Plant parts (e.g., seeds) and plant products (e.g., plant biomass, seed compositions, protein compositions, food and/or beverage products) as disclosed herein can be meant for consumption by agricultural animals or for use as feed in an agriculture or aquaculture system. In specific embodiments, plant parts and plant products include animal feed (e.g., roughages - forage, hay, silage; concentrates - cereal grains, soybean cake) intended for consumption by bovine, porcine, poultry, lambs, goats, or any other agricultural animal. In some embodiments, plant parts and plant products include aquaculture feed for any type of fish or aquatic animal in a farmed or wild environment including, without limitation, trout, carp, catfish, salmon, tilapia, crab, lobster, shrimp, oysters, clams, mussels, and scallops.

Seeds of the present disclosure include a representative sample of seeds, from a plant of the present disclosure. A plant or plant part of the present disclosure can be a crop plant, a forage plant, or part of a crop plant or forage plant.

As provided herein, the plant parts, population of plant parts, and plant products (e.g., seed compositions, plant protein compositions, and plant-based food/beverage products) of the present disclosure can contain a mutation that increases GIF1 activity, e.g., one or more insertions, substitutions, or deletions in at least one native GIF1 gene or homolog or in a regulatory region of such GIF1 gene or homolog, e.g., a substitution of 1-10 nucleotides or a deletion of about 4-12 nucleotides at least partially in the G-box region of a Glycine max GIF1 gene. Additionally, the plant parts, population of plant parts, and plant products of the present disclosure can have one or more exogenous copies of a native or mutated GIF1 gene. The mutation can be located at least partially in the regulatory region, coding region, or non-coding region of the exogenous copy of the GIF1 gene. The plant parts, population of plant parts, and plant products of the present disclosure can have increased GIF1 activity, increased expression level of the GIF1 gene or homolog, increased expression level of the GIF1 protein, increased function or activity of the GIF1 protein, increased expression or activity of GIF 1 downstream target molecules that regulate cell and organ growth and development (e.g., GRF5, GRF3, COL5, ARR4, RA2, CLE4a), increased seed size, increased biomass, increased yield, and/or increased protein content as compared to a control plant part, population, or plant product, e.g., comprising wild-type GIF1 level or activity.

The plant parts, population of plant parts, and plant products of the present disclosure can further contain a mutation that decreases BIG SEEDS activity, e.g., one or more insertions, substitutions, or deletions in at least one native BS gene or homolog or regulatory region thereof, e.g., a deletion of about 4-8 nucleotides at least partially in the nucleic acid region of exon 1 or 2 of a Glycine max BS1 gene or exon 1 or 2 of a Glycine max BS2 gene. The plant parts, population of plant parts, and plant products of the present disclosure can further have decreased BIG SEEDS activity, decreased expression level of the BS gene, the BIG SEEDS protein, or homolog, decreased function or activity of the BIG SEEDS protein, and/or increased activity of one or more target molecules regulated by BIG SEEDS and regulating organ growth or size (e.g., GRF, GRF1, GRF5, GIF, GIF1, GIF2, cyclin D3;3, histone 4) compared to a control plant parts, population of plant parts, and plant products. Modifications that increase GIF1 activity and modifications that decrease BIG SEEDS activity can have additive or synergistic effects on organ (e.g., seed) size, biomass, yield, or protein content in the plant parts, population of plant parts, and plant products of the present disclosure, such that they have greater organ (e.g., seed) size, greater biomass or yield (e.g., seed yield), and/or greater protein content compared to a control plant part, population of plant parts, or plant product having modifications that increase GIF1 activity but not modifications that decrease BIG SEEDS activity, or having modifications that decrease BIG SEEDS activity but not modifications that increase GIF1 activity beyond the increase in GIF1 activity caused by the decreased BIG SEEDS activity.

IV. Increasing Protein Content in Plants

Methods are provided herein for altering (e.g., increasing) protein content and/or altering (e.g., increasing) disease tolerance in a plant or plant part. In some aspects, the methods comprise increasing GIF 1 activity in the plant or plant part, by, e.g., increasing level or activity of a GIF1 protein. Level or activity of GIF 1 in a plant or plant part can be increased by any methods known in the art for increasing protein activity or increasing gene expression, including the methods provided herein. For example, level or activity of GIF1 can be increased by increasing level or activity of at least one endogenous gene encoding GIF1; or by introducing an exogenous copy of a GIF1 gene (native or mutated) into the plant or plant part.

In one aspect, the methods comprise increasing level or activity of at least one endogenous gene encoding GIF1 in said plant or plant part. In some aspects, the methods comprise introducing a genetic mutation that alters (e.g., increases) GIF1 activity into a plant or plant part. The method can further comprise introducing the genetic mutation that alters (e.g., increases) GIF1 activity into a plant cell, and regenerating a plant or plant part from the plant cell (e.g., transformed plant cell). The methods provided herein can alter (e.g., increase) GIF1 level or activity, alter (e.g., increase) expression levels of at least one GIF1 gene encoding GIF1 protein, alter (e.g., increase) GIF1 protein levels or activity, alter (e.g., increase) activity of one or more target molecules regulated by GIF1 and regulating cell or tissue growth or development and/or disease, alter (e.g., increase) protein content, and/or alter (e.g., increase) disease tolerance in the plant or plant part compared to a control plant or plant part. A control plant or plant part can be a plant or plant part to which a mutation or a transgene (e.g., an exogenous copy of a GIF1 gene) provided herein has not been introduced, e.g., by methods of the present disclosure. Thus, a control plant or plant part (e.g., seeds, leaves) may express a native (e.g., wild-type) GIF1 gene endogenously. A control plant of the present disclosure may be grown under the same environmental conditions (e.g., same or similar temperature, humidity, air quality, soil quality, water quality, and/or pH conditions) as a plant to which the mutation is introduced according to the methods provided herein.

Also provided herein are plants, plant parts (e.g., seeds, leaves), a population of plants or plant parts, or plant product (e.g., seed composition, plant protein compositions) produced according to the methods of the present disclosure. Such plants, plant parts, a population of plants or plant parts, or plant products may have the mutation or transgene that increases GIF1 activity, altered (e.g., increased) expression levels of at least one GIF1 gene or homolog thereof, altered (e.g., increased) GIF1 protein levels or activity, altered (e.g., increased) activity of one or more target molecules regulated by GIF1 and regulating cell or tissue growth or development , and/or altered (e.g., increased) protein content, as compared to a control plant, plant part, population of plants or plant parts, when the plant, plant part, or population of plants or plant parts of the present disclosure is grown under the same environmental conditions as the control plant or plant part. The plant, plant part, or population of plants or plant parts produced according to the methods provided herein can further have increased disease tolerance compared to a control plant, plant part, or population of plants or plant parts. In the population of plants or plant parts, having altered GIF1 level or activity relative to a control population, not all individual plants or plant parts need to have altered (e.g., increased) GIF1 level or activity, genetic mutation that cause altered (e.g., increased) GIF1 level or activity, or phenotypes caused by the altered (e.g., increased) GIF1 activity (e.g., increased seed size, increased biomass, increased yield, and/or increased protein content). In specific embodiments at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more plants within a given plant population have a mutation that alters the GIF1 level or activity.

A. Altering expression or function of GIF1 in plants

Provided herein are compositions and methods for altering (e.g., increasing) protein content in a plant or plant part by introducing a genetic mutation that alters (e.g., increases) GIF1 activity into a plant or plant part. The method can further comprise introducing the genetic mutation that alters (e.g., increases) GIF1 activity into a plant cell, and regenerating a plant or plant part from the plant cell (e.g., transformed plant cell). The genetic mutation that is introduced into the plant or plant part according to the methods provided herein can comprise one or more insertions, substitutions, or deletions into the genome of the plant or plant part. The genetic mutation that alters (e.g., increases) the GIF1 activity can be introduced into at least one native GIF1 gene or homolog thereof; a regulatory region (e.g., promoter, 5’UTR, G-box region) of the native GIF1 gene or homolog thereof; in a coding region, a non-coding region, or a regulatory region of any other gene; or at any other site in the genome of the plant or plant part.

1. Introducing regulatory modi fications

The methods described herein can comprise introducing a mutation that increases the GIF1 activity, e.g., one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions at least partially into a regulatory region of at least one (e.g., one, more than one but not all, or all) GIF1 gene. A “regulatory region” of a gene can include a genomic site that modulate transcription or translation of the gene, e.g., where a RNA polymerase, a transcription factor, or other transcription or translation modulators bind, or where a regulatory structure or complex is formed, and include a promoter region, 5’ UTR, a binding site for transcription modulator proteins (e.g., transcription factors), and other genomic regions that contribute to regulation of transcription or translation of the gene. A regulatory region of the gene can be located in the 5’ region from the coding region of the gene. For example, one or more insertions, substitutions, and/or deletions can be introduced at least partially into a promoter region, 5’UTR, a binding site (e.g., an enhancer sequence) for a transcription modulator protein (e.g., transcription factor), or other genomic regions that contribute to regulation of transcription or translation of at least one (e.g., one, more than one but not all, or all) GIF1 gene, to confer to the plant or plant part an increased GIF1 activity.

In some embodiments, the methods provided herein include introducing a mutation at least partially into a promoter region of at least one (e.g., one, more than one but not all, or all) GIF1 gene. The one or more insertions, substitutions, and/or deletions in the promoter region of the GIF1 gene can alter the transcription initiation activity of the promoter. For example, the modified promoter can increase transcription of the operably linked nucleic acid molecule (e.g., the GIF1 gene), initiate transcription in a developmentally-regulated or temporally-regulated manner, initiate transcription in a cell-specific, cellpreferred, tissue-specific, or tissue-preferred manner, or initiate transcription in an inducible manner. A deletion, a substitution, or an insertion, e.g., introduction of a heterologous promoter sequence, a cis-acting factor, a motif or a partial sequence from any promoter, including those described elsewhere in the present disclosure, can be introduced into the promoter region of the GIF1 gene to confer an altered (e.g., increased) transcription initiation function according to the present disclosure.

In some embodiments, the methods provided herein include introducing a mutation at least partially into 5’UTR of one or more (e.g., one, more than one but not all, or all) GIF1 gene and alter (e.g., increase) translation regulation activity.

The promoter or 5’UTR activity to regulate transcription or translation of one or more GIF1 genes can be modified by insertion of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,

77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more) nucleotides. Additionally or alternatively, the promoter or 5’UTR activity of one or more of GIF1 genes can be modified by deletion of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,

79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more) nucleotides.

The promoter sequence of one or more GIF1 genes can also be modified by replacement of the promoter sequence with one or more substitutes. In particular, the substitute can be a cisgenic substitute, a transgenic substitute, or both.

In some instances, the promoter sequence of one or more GIF1 genes is modified by correction of the promoter sequence. A promoter sequence can be corrected by deletion or modification of one or more polymorphisms or mutations that would, without correction, reduce the activity of the promoter or 5’UTR. In some instances, the promoter or 5’UTR sequence of one or more GIF1 genes is modified by insertion, deletion, and/or modification of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more) upstream nucleotide sequences. In some instances, the promoter or 5’UTR sequence of one or more GIF1 genes is modified by addition, insertion, and/or engineering of cis-acting factors that interact with and modify the promoter sequence.

In some embodiments, the methods provided herein include introducing a mutation that is at least partially located in the regulatory region (e.g., promoter region or 5’UTR) of at least one (e.g., one, more than one but not all, or all) GIF1 gene. In specific embodiments, the mutation is located at least partially in a G-box region in the regulatory region of at least one GIF gene. The G-box region is part of the regulatory region that contains the G-box sequence, i.e., CACGTG (SEQ ID NO: 17), where a transcription factor (e.g., a repressor complex, e.g., KIX-PPD-MYC repressor complex) binds and negatively regulates GIFT Mutation in the G-box region at or near the G-box sequence of the GIF1 gene can alter (e.g., decrease) binding of a transcription factor (e.g., GIF1 transcriptional repressor complex) and alter (e.g., increase) level or activity of a GIF1 protein encoded by the GIF1 gene. In specific embodiments, the methods introduces a mutation that decreases binding of a GIF 1 repressor complex to the regulatory region of at least one GIF1 gene or homolog thereof, and increases level or activity of a GIF 1 protein encoded by said at least one GIF1 gene or homolog. Such mutation can be introduced at least partially into the G-box region in the regulatory region of at least one GIF gene.

In some embodiments, the methods include introducing a mutation to locate at least partially into the regulatory region of a GIF1 gene, and (i) the regulatory region comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 13-16, wherein the regulatory region retains transcription initiation activity; (ii) the regulatory region comprises a nucleic acid sequence of any one of SEQ ID NOs: 13-16; (iii) the GIF1 gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; (iv) the GIF1 gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-12; (v) the GIF1 gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein the polypeptide retains GIF1 activity; (vi) the GIF1 gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8; (vii) the GIF1 gene including the regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; and/or (viii) the GIF1 gene including said regulatory region thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4.

In some embodiments, the mutation is introduced at least partially into a promoter region or 5’UTR of a Glycine max GIF1 gene. The Glycine max GIF1 gene can be Glyma.03G249000, Glyma.19G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.18G121100, Glyma.l4G122500, or Glyma.01G113500, or homolog thereof. In some embodiments, the mutation is located at least partially in a promoter region of a GmGIFl gene (e.g., SEQ ID NOs: 13-16). The mutation can be introduced at least partially into a G-box region of a GmGIFl gene, e.g., in a nucleic acid sequence of one or more of SEQ ID NOs: 17-19 in the Glycine max GIF1 gene. The mutation to be introduced can comprise a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides to locate at least partially in the promoter, G-box region, and/or 5’ UTR of a Glycine max GIF1 gene. In some specific embodiments, the methods comprise (i) introducing a mutation into a G-box region of Glyma.03G249000, thereby producing a mutated G-box region comprising a nucleic acid sequence of any one of SEQ ID NOs: 20-24; and/or (ii) introducing a mutation into a G-box region of Glyma.19G246600, thereby producing a mutated G-box region comprising a nucleic acid sequence of SEQ ID NO: 25.

Function and/or expression of the one or more GIF1 genes can be increased or inhibited by modulation of expression of one or more transcription factor genes. In some instances, a transcription factor binds to a promoter sequence near the transcription initiation site and regulate formation of the transcription initiation complex. A transcription factor can also bind to regulatory sequences, such as enhancer sequences, and modulate transcription of the target gene. The methods provided herein can include introducing a mutation in the gene encoding (or regulating expression of) a transcription factor to modulate expression or function of the transcription factor and increase expression levels of a downstream gene, e.g., the GIF1 gene, e.g., by increasing transcription initiation activity of the GIF1 gene promoter.

In some embodiments, the methods include introducing a mutation that modifies or inserts transcription factor binding sites or enhancer elements that regulates GIF1 gene expression into the regulatory region of the GIF1 gene. Function and/or expression of the one or more GIF1 genes can also be increased by insertion, modification, and/or engineering of transcription factor binding sites or enhancer elements. For example, insertion of new transcription factor binding sites or enhancer elements can increase function and/or expression of GIF1 genes. Alternatively, modification and/or engineering of existing transcription factor binding sites or enhancer elements can increase function and/or expression of GIF1 genes.

Function and/or expression of the one or more GIF1 genes can also be increased or inhibited by insertion of one or more positive regulatory elements of the gene. For example, to inhibit the expression and/or function of the GIF1 gene, a part or whole of one or more positive regulatory elements of the GIF1 gene can be inserted in the genome of a plant cell or plant part. The positive regulatory sequence of the gene can be in a cis location. Alternatively, the positive regulatory sequence of the gene may be in a trans location. Positive regulatory elements of the one or more GIF1 genes can also include upstream open reading frames (uORFs). In some instances, the methods provided herein include inserting a positive regulatory sequence into a region upstream of the GIF1 gene in order to increase the expression and/or function of the gene.

2. Introducing mutation to GIF 1 gene, or its homolog, ortholog, or variant

In some aspects, the methods of the present disclosure comprise introducing a genetic mutation that increases the GIF1 activity into a plant or plant part. The genetic mutation that is introduced into the plant or plant part can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions in at least one native GIF1 gene or homolog thereof (e.g., coding region, non-coding region, exons, introns, and/or regulatory region thereof) in a genome of said plant or plant part. A plant or plant part described herein can comprise 1-2, 1-3, 1-4, 1-5, 2-5, 3-5, 4-5 (e.g., 1, 2, 3, 4, or 5) copies of GIF1 gene, e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l4G122500, and Glyma.OlGl 13500, each encoding a GIF1 protein. In particular, the plant or plant part to which the mutation is introduced according to the methods can comprise at least 2 genes encoding a GIF1 protein, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes that have less than 100% (e.g., less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85%) sequence identity to one another. The methods can comprise introducing one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions: into one GIF1 gene or homolog; into a regulatory region of one GIF1 gene or homolog; into more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all GIF1 genes or homologs; into regulatory regions of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all GIF1 genes or homologs; into all GIF1 genes or homologs; and/or into regulatory regions of all GIF1 genes or homologs in the plant or plant part. In one embodiment, the methods include introducing a mutation into the area excluding the coding region of the GIF1 gene (e.g., introducing a mutation only into the regulatory region of the GIF1 gene). In another embodiment, the methods include including a mutation at least partially into the coding region of the GIF1 gene (e.g., insertion, deletion, substitution, inversion, or truncation at N- or C-terminus) to increase protein content in the plant or plant part.

Each mutation that is introduced in to the plant or plant part can be heterozygous or homozygous. That is, the method can introduce a certain mutation (e.g., comprising one or more insertions, substitutions, and/or deletions) in one allele or two (both) alleles of a GIF1 gene/homolog or its regulatory region. All mutations introduced into the plant or plant part can be homozygous; all mutations introduced into the plant or plant part can be heterozygous; or mutations can comprise some heterozygous mutations in certain locations of the genome and some homozygous mutations in certain locations of the genome in the plant or plant part.

In some embodiments, the mutation is introduced into a GIF1 gene or its regulatory region, and (i) the GIF1 gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; (ii) the GIF1 gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 9- 12; (iii) the GIF1 gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein the polypeptide retains GIF1 activity; (iv) the GIF1 gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8; (v) the GIF1 gene including the regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; and/or (vi) the GIF1 gene including said regulatory region thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4. In specific embodiments, the mutation that increases the GIF1 activity is introduced into one or two alleles of one or more (e.g., one, more than one but not all, or all) Glycine max GIF1 genes (e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l 4G 122500, and Glyma.OlGl 13500) and/or a regulatory region thereof.

The mutation introduced into the plant or plant part according to the methods of the present disclosure can comprise an out-of-frame mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) GIF1 gene or homolog thereof. Alternatively, the mutation introduced into the plant or plant part according to the methods can comprise an in-frame mutation, a nonsense mutation, or missense mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) GIF1 gene or homolog thereof.

A genetic mutation that increases the GIF1 activity can be introduced into a gene that is a homolog, ortholog, or variant of a GIF1 gene disclosed herein and expresses a GIF1 protein with GIF1 function, or in a regulatory region of such homolog, ortholog, or variant of a GIF1 gene, according to the methods provided herein. For example, the mutation (e.g., one or more insertions, substitutions, or deletions that increase the GIF 1 activity) can be introduced into orthologs of GIF1 genes including, without limitation, vigan.Gyeongwon.gnm3.annl. Vang03gl0430, vigra. VC1973A.gnm6.annl. Vradi0083s00920, Vigun06gl 14600, Phvul.006G103700, arahy. Tifrunner.gnm l.annl.L225S3,Lalb_Chrl8g0044001,Medtr7gl 15410, Pisat.03G006130,pissa.Cameor.gnml.annl.Psat0s2429g0080, Ca_01247,AT5G28640, arahy. Tifrunner.gnml.annl. 71 F8 WW, arahy. Tifrunner. gnml .annl .HK1 FSC, pissa.Cameor.gnml.annl.Psat0s3277g0080, Medtrlg080590, Pisat.06G061820, pissa.Cameor. gnml. annl. Psat0s368g0160, Ca_03768, Phvul.007Gl 89200, vigra. VC1973A.gnm6.annl. Vradi08gl3410, vigan.Gyeongwon.gnm3. annl . Vang06gl0880, Vigun07gl 56800, Lalb_Chr21gO311081 , Lalb_Chr06g0168171, wA Lalb_Chr06g0171791.

Variant sequences (e.g., homologs, orthologs) can be isolated by PCR. In this manner, variant sequences encoding GIF1 can be identified and used in the methods of the present disclosure. The variant sequences will retain the GIF1 activity.

In certain instances, mutations introduced into any GIF1 gene or its regulatory region in a plant, plant part, a population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) according to the methods provided herein can be identified by a diagnostic method described herein. Such diagnostic methods may comprise use of primers for detecting mutation in a GIF1 gene. For example, a pair of primers or an oligonucleotide probe can be designed to detect a mutation at or near the G- box region of a GIF1 gene, e.g., in a region comprising any one of SEQ ID NOs: 17-19, or to detect a mutation comprising any one of SEQ ID NOs: 20-25, in the G-box region of a GmGIFl gene. In certain instances, a kit comprising a set of primers and/or an oligonucleotide probe can be used for detecting mutation of GIF1 genes in a plants, plant part, or plant product (e.g., seed composition, plant protein/oil composition). In some embodiments, the one or more mutations are integrated into the plant genome and the plant or the plant part is stably transformed according to the methods. In other embodiments, the one or more mutations are not integrated into the plant genome and wherein the plant or the plant part is transiently transformed according to the methods.

Introducing one or mutations insertions, substitutions, or deletions into at least one GIF1 gene or homolog or in a regulatory region of such GIF1 gene or homolog in the genome of the plant or plant part can increase the expression levels of the GIF1 gene or homolog, increase level or activity of the GIF1 protein encoded by the GIF1 gene or homolog, increase GIF1 activity, increase organ size (e.g., seed size), biomass, yield, and/or protein content relative to a control plant or plant part without the mutation when grown under the same environmental conditions, as further described in the present disclosure.

3. Increasing GIF1 activity

The methods of the present disclosure can increase activity of GIF1 in plants, plant parts (e.g., seeds, leaves), a population of plants or plant parts, or plant products (e.g., seed composition, plant protein composition) compared to a control (e.g., wild-type) plant, plant part, a population of plants or plant parts, or plant product. In particular, methods provided herein can increase the GIF 1 activity in the plant, plant part, a population of plants or plant parts, or plant product by about 10-100%, 20-100%, 30-100%, 40-100%, 50- 100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%,

200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300- 900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400- 500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more as compared to a control plant, plant part, a population of plants or plant parts, or plant product. A control plant or plant part can be a plant or plant part into which a mutation or other modification has not been introduced, a reference variety of plant or plant part, or a commonly available variety of plant or plant part. One skilled in the art can select an appropriate control.

GIF1 activity can be measured by assessing level or activity of downstream target genes (e.g., GRF5, GRF3, COL5, ARR4, RA2, CLE4a) by measuring mRNA levels (e.g., quantitative RT-PCR, northern blot, serial analysis of gene expression (SAGE)), by measuring protein levels (e.g., western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from the plant or plant part using an antibody directed to the protein), or by standard functional assays or enzymatic assays for measuring activity of these downstream target proteins. GIF1 activity can also be assessed by measuring organ size (e.g., seed size, leaf size), cell counts within an organ, or total protein content. Protein content in a plant sample can be measured by, for example, protein extraction and quantitation (e.g., BCA protein assay, Lowry protein assay, Bradford protein assay), spectroscopy, nearinfrared reflectance (NIR) (e.g., analyzing 700 - 2500 nm), and nuclear magnetic resonance spectrometry (NMR).

The methods of the present disclosure can increase expression level of the GIF1 gene or homolog in the plant, plant part (e.g., seeds, leaves), a population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) as compared to the expression level of the GIF1 gene or homolog in a control (e.g., wild-type) plant, plant part, a population of plants or plant parts, or plant product.

In some embodiments, the methods provided herein increase the expression levels of the endogenous GIF1 gene(s) or homolog by, e.g., introducing a genetic mutation or additional mechanism to up-regulate the expression of the endogenous GIF1 gene(s). For example, the methods provided herein can introduce a mutation in the regulatory region (e.g., promoter, 5’UTR, G-box region) of at least one endogenous GIF1 gene, e.g., at or near transcriptional repressor binding sites, e.g., a G-box region in the GIF1 promoter, that increases expression of the GIF1 gene.

Alternatively or additionally, the methods provided herein can include introducing one or more copies of a GIF1 gene, e.g., a polynucleotide encoding a functional GIF1 protein or functional fragment thereof, into a plant or plant part. The methods can further include introducing a polynucleotide encoding a functional GIF1 protein into a plant cell, and regenerating a plant or plant part overexpressing GIF1 from the plant cell. One or more exogenous (e.g., transgenic) copies of the GIF1 gene can be from the same, related, or different plant species. One or more exogenous copies of the GIF1 gene can be native, i.e., without mutation; alternatively, one or more exogenous copies of the GIF1 gene can have a mutation (e.g., in the regulatory region, coding region, and/or non-coding region) that increases GIF1 level or activity. A polynucleotide comprising a sequence of a GIF1 gene or encoding a functional GIF1 protein or functional fragment thereof can be assembled within a DNA construct with an operably-linked promoter molecule, which can be a homologous (native) GIF1 promoter or a heterologous promoter functional in a plant cell. By “heterologous promoter” is intended a sequence that is not naturally operably linked with the nucleic acid molecule of interest. For instance, a 2x35s promoter or a promoter (native or heterologous) comprising an exogenous or synthetic motif sequence may be operably linked to the polynucleotide comprising a sequence of a GIF1 gene or encoding a functional GIF1 protein or functional fragment thereof. The GIF 1 -encoding polynucleotide sequences or the promoter sequence may each be homologous, native, heterologous, or foreign to the plant host. The methods can include transiently or stably transforming a plant, plant part, or plant cell with such DNA construct according to standard methods of plant cell transformation described in the present disclosure. Upon transformation, the plant, plant part, or plant cell can express or accumulate polynucleotides comprising a native or an altered (e.g., mutated, alternatively spliced) sequence of a GIF1 gene or a GIF1 gene transcript, or a GIF1 protein encoded by the polynucleotides, thereby increasing level of the GIF1 in the plant, plant part, or plant cell. In particular, the methods provided herein can increase the expression levels of GIF1 gene or homolog in the plant, plant part, a population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60- 100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400- 900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40- 50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500- 600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more as compared to a control plant, plant part, a population of plants or plant parts, or plant product. In specific embodiments, the copy of GIF1 gene or homolog that contributes to an increased expression (e.g., up-regulation, overexpression) of the GIF1 gene or homolog is an endogenous or exogenous copy of a Glycine max GIF1 gene (e.g., Glyma.03G249000, Glyma.19G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.18G121100, Glyma.l4G122500, and Glyma.01G113500). Expression levels of the GIF1 gene or homolog can be measured by any standard methods for measuring mRNA levels of a gene, including quantitative RT-PCR, northern blot, and serial analysis of gene expression (SAGE). Expression levels of the GIF1 gene or homolog in a plant, plant part, a population of plants or plant parts, or plant product can also be measured by any standard methods for measuring protein levels, including western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from a plant, plant part, a population of plants or plant parts, or plant product using an antibody directed to the GIF1 protein encoded by the GIF1 gene.

The methods of the present disclosure can increase expression levels of the GIF1 protein, e.g., the GIF1 in the plant, plant part (e.g., seeds, leaves), a population of plants or plant parts, and plant product (e.g., seed composition, plant protein compositions), as compared to the expression level of the GIF1 protein in a control plant, plant part, a population of plants or plant parts, or plant product. In particular, the methods provided herein can increase the expression levels of a full length GIF1 protein (e.g., a GIF1 protein having the complete amino acid sequence and function of a wild-type GIF1 protein, e.g., encoded by a native GIF1 gene) in the plant, plant part, a population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) as compared to a control plant, plant part, a population of plants or plant parts, or plant product. Additionally or alternatively, the methods provided herein can increase the expression levels of a functional fragment, variant, or ortholog of GIF 1 protein in the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure as compared to a control plant, plant part, a population of plants or plant parts, or plant product. In some embodiments, the methods increase the levels of GIF1 protein encoded by the endogenous GIF1 gene(s) or homolog by, e.g., introducing genetic mutation into at least one endogenous GIF1 gene or homolog (e.g., in the regulatory region, coding region, and/or non-coding region) or other mechanisms to up-regulate the expression of the endogenous GIF1 protein. For example, the method can include introducing a mutation in the regulatory region (e.g., promoter, 5’UTR, G- box region) of at least one endogenous GIF1 gene, e.g., at or near transcriptional repressor binding sites, e.g., the G-box region, that increases expression of the GIF1 protein. Alternatively or additionally, the methods can include increasing the levels of GIF 1 protein by introducing one or more exogenous copies of a GIF1 gene into the plant or plant part. One or more exogenous (e.g., transgenic) copies of the GIF1 gene can be from the same, related, or different plant species. One or more exogenous copies of the GIF1 gene can be native, i.e., without mutation; alternatively, one or more exogenous copies of the GIF1 gene can have a mutation (e.g., in the regulatory region, coding region, and/or non-coding region) that increases GIF1 level or activity.

In particular, the methods provided herein can increase expression of GIF1 protein (e.g., functional GIF1 protein), e.g., encoded by endogenous and/or exogenous copies of the GIF1 gene(s), by about 10- 100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, as compared to expression of GIF1 protein in a control plant, plant part, a population of plants or plant parts, or plant product. In certain embodiments, the methods increase level of the GIF 1 protein encoded by endogenous or exogenous copies of the Glycine max GIF1 gene (e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.IOG 164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l4G122500, and Glyma.01G113500). Expression of a GIF1 protein in a plant, plant part, a population of plants or plant parts, or plant product can be determined by one or more standard methods of determining protein levels. For example, expression of a GIF1 protein can be determined by western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from a plant, plant part, a population of plants or plant parts, or plant product using an antibody directed to the GIF1 protein.

5. Enhancing GIF1 protein function

The methods of the present disclosure can enhance function in the GIF1 in the plant, plant part (e.g., seeds, leaves), population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) as compared to a control plant, plant part, a population of plants or plant parts, or plant product. The methods provided herein can introduce a mutation into at least one endogenous GIF1 gene or homolog thereof (e.g., in the regulatory, coding, and/or non-coding regions) to enhance function of GIF1 protein in the plant, plant part, population of plants or plant parts, or plant product. Additionally or alternatively, the methods can introduce an exogenous copy of a GIF1 gene encoding GIF1 protein (e.g., with enhanced function) into the plant, plant part, or population of plants or plant parts, such that the GIF 1 protein function is enhanced in the plant, plant part, plant population, or plant product. A control plant, plant part, a population of plants or plant parts, or plant product can be a plant, plant part, a population of plants or plant parts, or plant product without the mutation, without an exogenous copy of a GIF1 gene, or otherwise having wild-type GIF1 activity. The GIF1 protein with increased function can comprise a mutation compared to a wild-type GIF1 protein that causes enhanced GIF1 function. In some embodiments, the methods increase the GIF1 protein function (e.g., by introducing a mutation into the GIF1 gene or homolog or its regulatory region, or by introducing an exogenous copy of a GIF1 gene encoding a GIF1 protein with enhanced function) by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80- 100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400- 1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, as compared to function of a wild-type GIF1 protein. In certain embodiments, the GIF 1 protein with enhanced function is encoded by an endogenous or exogenous copy of mutated Glycine max GIF1 gene (e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.IOG 164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l4G122500, and Glyma.OlGl 13500).

Function of a GIF1 protein in a plant, plant part, a population of plants or plant parts, or plant product can be measured by assessing level or activity of downstream target genes (e.g., GRF5, GRF3, COL5, ARR4, RA2, CLE4a) by measuring mRNA levels (e.g., quantitative RT-PCR, northern blot, serial analysis of gene expression (SAGE)), by measuring protein levels (e.g., western blot analysis, ELISA, or dot blot analysis of a protein sample obtained from the plant or plant part using an antibody directed to the protein), or by standard functional assays or enzymatic assays for measuring activity of these downstream target proteins. GIF1 activity can also be assessed by measuring organ size (e.g., seed size, leaf size), cell counts within an organ, or total protein content. Protein content in a plant sample can be measured by, for example, protein extraction and quantitation (e.g., BCA protein assay, Lowry protein assay, Bradford protein assay), spectroscopy, near-infrared reflectance (NIR) (e.g., analyzing 700 - 2500 nm), and nuclear magnetic resonance spectrometry (NMR).

6. Decreasing BIG SEEDS activity The methods of the present disclosure, e.g., to increase the GIF1 activity in a plant or plant part, optionally include decreasing the BIG SEEDS activity in the plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition). Levels or activity of BIG SEEDS in a plant or plant part can be reduced by any methods known in the art for reducing protein activity or reducing gene expression, including the methods provided herein.

In some aspects, the methods comprise introducing a genetic mutation that decreases BIG SEEDS (BS) activity into a plant or plant part. The method can further comprise introducing the genetic mutation that decreases BIG SEEDS activity into a plant cell, and regenerating a plant or plant part from the plant cell (e.g., transformed plant cell). The methods provided herein can decrease BIG SEEDS (BS) level or activity, decrease expression levels of at least one BS gene encoding BIG SEEDS protein, decrease BIG SEEDS protein levels or activity, increase activity of one or more target molecules regulated by BIG SEEDS and regulating organ growth or size [e.g., growth-regulating factor (GRF), GRF1, GRF5, GRF -interacting factor (GIF), GIF1, GIF2, cyclin D3;3 (CYCD3;3), histone 4 (H4)], increase organ (e.g., seed) size, biomass, or yield (e.g., seed yield), and/or increase amino acid or protein content in the plant or plant part compared to a control plant or plant part. A control plant or plant part can be a plant or plant part to which a mutation provided herein has not been introduced, e.g., by methods of the present disclosure. Thus, a control plant or plant part (e.g., seeds, leaves) may express a native (e.g., wild-type) BS gene endogenously or transgenically. A control plant of the present disclosure may be grown under the same environmental conditions (e.g., same or similar temperature, humidity, air quality, soil quality, water quality, and/or pH conditions) as a plant to which the mutation is introduced according to the methods provided herein.

The genetic mutation that decreases the BIG SEEDS activity, which is introduced into a plant or plant part according to the methods provided herein, can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, or deletions in at least one native BS gene or homolog thereof, or in a regulatory region of at least one native BS gene or homolog thereof. The genetic mutation that decreases the BIG SEEDS activity can be introduced into at least one native BS gene or homolog thereof; in a regulatory region of the native BS gene or homolog thereof; a coding region, a non-coding region, or a regulatory region of any other gene; or at any other site in the genome of the plant or plant part.

The mutation can be introduced into a plant or plant part having 1-2, 1-3, 1-4, 1-5, 2-5, 3-5, 4-5 (e.g., 1, 2, 3, 4, or 5) copies of BS gene, e.g., BS1 and BS2 genes, each encoding a BIG SEEDS protein. The BS gene copies can have less than 100% (e.g., less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85%) sequence identity to one another. The method provided herein can include introducing one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions: in one BS gene or homolog; in a regulatory region of one BS gene or homolog; in more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all BS genes or homologs; in regulatory regions of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all BS genes or homologs; in all BS genes or homologs; and/or in regulatory regions of all BS genes or homologs in the plant or plant part. Each mutation can be heterozygous or homozygous. That is, a certain mutation (e.g., comprising one or more insertions, substitutions, and/or deletions) can be introduced into one allele or two (both) alleles of a BS gene/homolog or its regulatory region. All mutations to be introduced can be homozygous; all mutations in the plant or plant part can be heterozygous; or mutations can comprise some heterozygous mutations in certain locations of the genome and some homozygous mutations in certain locations of the genome in the plant or plant part.

In some embodiments, the mutation that decreases the BIG SEEDS activity can be located in one or two alleles of a BS gene or homolog thereof comprising a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 26, 27, or 49 and encoding a polypeptide that retains BIG SEEDS activity, for example the nucleic acid sequence of SEQ ID NO: 26, 27, or 49; and/or a regulatory region of the BS gene or homolog thereof comprising such nucleic acid sequence. Additionally, the mutation can be located in one or two alleles of a BS gene or homolog thereof encoding a polypeptide comprising an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 28, 29, or 41 and retaining BIG SEEDS activity, for example a polypeptide comprising the amino acid sequence of SEQ ID NO: 28, 29, or 41; and/or a regulatory region of the BS gene or homolog thereof encoding such polypeptide. In specific embodiments, the mutation that decreases the BIG SEEDS activity is located in one or two alleles of one or more (e.g., one, more than one but not all, or all) Glycine max BS genes, such as a Glycine max BS1 gene, a Glycine max BS2 gene, or one or more Pisum sativum BS genes, and/or a regulatory region thereof.

The methods can include introducing a mutation that decreases the BIG SEEDS activity, such that at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertion, substitution, or deletion is introduced at least partially into a nucleic acid region of exon 1 or 2 of a Glycine max BS1 gene (set forth as SEQ ID NOs: 30 and 32, respectively) or exon 1 or 2 of a Glycine max BS2 gene (set forth as SEQ ID NOs: 31 and 33, respectively). In some embodiments, a deletion of about 4-8 nucleotides is introduced to locate at least partially in the nucleic acid region of exon 1 or 2 of a Glycine max BS1 gene or exon 2 of a Glycine max BS2 gene. For example, the methods can introduce (i) a deletion of nucleotides 98 through 101 into one or both alleles of the Glycine max BS1 gene having the nucleic acid sequence of SEQ ID NO: 26, (ii) a deletion of nucleotides 389 through 396 into one or more alleles of the Glycine max BS1 gene having the nucleic acid sequence of SEQ ID NO: 26, and/or (iii) a deletion of nucleotides 409 through 415 into one or both alleles of Glycine max BS2 gene having the nucleic acid sequence of SEQ ID NO: 27.

The mutation to be introduced according to the methods provided herein can be an out-of-frame mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) BS gene or homolog thereof. Alternatively, the mutation can be an in-frame mutation, a nonsense mutation, or a missense mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) BS gene or homolog thereof.

The methods provided herein can introduce a genetic mutation that decreases the BIG SEEDS activity into a gene that is a homolog, ortholog, or variant of a BS gene disclosed herein and expresses a functional BIG SEEDS protein, or in a regulatory region of such homolog, ortholog, or variant of a BS gene, e.g., polynucleotides that have BIG SEEDS activity and share at least 75% sequence identity to the sequences disclosed herein. For example, the methods can include introducing a mutation into an ortholog of BS gene such as yellow pea BS1 (Pisum sativum, the nucleic acid sequence and amino acid sequence set forth as SEQ ID NO: 40 and 41, respectively), barrel medic BS1 (Medicago truncatula, NCBI ID: KM668032.1), Alfalfa BS1 (Medicago sativa, NCBI ID: KM668033.1), common bean BS1 (Phaseolus vulgaris, NCBI ID: KM668018.1), and Peruvian cotton BS1, BS2, BS3 (Gossypium raimondii, NCBI IDs: KM668013.1, KM668014.1, KM668015.1).

In certain instances, mutations introduced according to the methods into any BS gene in a plant, plant part, population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) can be identified by a diagnostic method described herein. Such diagnostic methods may comprise use of primers for detecting mutation in a BS gene. For example, a forward primer set forth as SEQ ID NO: 36 and a reverse primer set forth as SEQ ID NO: 37 can be used for detection of mutation in Glycine max BS1 or BS2 gene near the binding site of the GmBSl/GmBS2 guide RNA1, for example a mutation generated by introducing the GmBSl/GmBS2 guide RNA 1 into the plant or plant part. A forward primer set forth as SEQ ID NO: 38 and a reverse primer set forth as SEQ ID NO: 39 can be used for detection of mutation in Glycine max BS1 or BS2 gene near the binding site of the GmBSl/GmBS2 guide RNA 4, for example a mutation generated by introducing the GmBSl/GmBS2 guide RNA 4 into the plant or plant part. In certain instances, a kit comprising a set of primers can be used for detecting mutation of BS genes in plants, plant parts, or plant product (e.g., seed composition, plant protein composition). For example, a kit comprising the forward primer SEQ ID NO: 36 and the reverse primer SEQ ID NO: 37, and a kit comprising the forward primer SEQ ID NO: 38 and the reverse primer SEQ ID NO: 39 can be used for detection of mutation in BS1 or BS2 gene in plants, plant parts, or plant products (e.g., seed composition, plant protein compositions) near the binding site of the GmBSl/GmBS2 guide RNA1 and guide RNA4, respectively.

In some embodiments, the methods integrates the mutations into the plant genome and stably transforms the plant or the plant part. In other embodiments, the methods do not integrate the mutations into the plant genome and transiently transforms the plant or the plant part.

The methods provided herein can reduce BIG SEEDS activity in the plant, plant part, population of plants or plant parts, or plant product by about 10-99%, 20-99%, 30-99%, 40-99%, 50-99%, 60-99%, 70- 99%, 80-99%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, or 100% (e.g., by about 10-20%, 20- 30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-99%, 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to a control plant, plant part, population, or plant product. BIG SEEDS activity can be measured by measuring expression levels of one or more downstream target genes, e.g., growth-regulating factor 1 and 5 (GRF1 and GRF5), GRF-interacting factor 1 and 2 (GIF1 and GIF2), cyclin D3;3 (CYCD3;3), and HISTONE4 (H4) by quantitative RT-PCR, northern blot, serial analysis of gene expression (SAGE), or any other methods for measuring mRNA levels. BIG SEEDS activity can also be measured by measuring levels of proteins encoded by one or more downstream target genes, e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4, by for instance western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from the plant or plant part using an antibody directed to the protein. BIG SEEDS activity can also be measured by measuring activity of downstream target proteins, e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4, by standard functional assays or enzymatic assays for measuring activity of these proteins. In certain embodiments, decrease in BIG SEEDS activity can be assessed by increase in expression levels (e.g., mRNA or protein levels) or activity of the BIG SEEDS downstream target molecules (e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4). For example, expression levels (e.g., mRNA or protein levels) or activity of the BIG SEEDS downstream target molecules (e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4) can be increased by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200- 900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300- 400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%,

300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, as compared to a control plant or plant part.

The methods can reduce expression level of the BS gene(s), the BIG SEEDS protein, or homolog thereof in a plant, plant part, population of plants or plant parts, or plant product as compared to the expression level in a control plant, plant part, population of plants or plant parts, or plant product, e.g., without a mutation is introduced. The expression levels of BS gene(s) or homolog or a BIG SEEDS protein in the plant, plant part, a population of plants or plant parts, or plant product (e.g., seed composition, plant protein composition) of the present disclosure can be reduced by about 10-99%, 20-99%, 30-99%, 40-99%, 50-99%, 60-99%, 70-99%, 80-99%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,

75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to a control plant, plant part, a population of plants or plant parts, or plant product. In specific embodiments, the BS gene or homolog is a BS1 gene and/or a BS2 gene, e.g., a Glycine max BS1 gene and/or a Glycine max BS2 gene. Expression levels of the BS gene or homolog can be measured by any standard methods for measuring mRNA levels of a gene, including quantitative RT-PCR, northern blot, and serial analysis of gene expression (SAGE). Expression levels of the BIG SEEDS protein in a plant, plant part, a population of plants or plant parts, or plant product can also be measured by any standard methods for measuring protein levels, including western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from a plant, plant part, a population of plants or plant parts, or plant product using an antibody directed to the BIG SEEDS protein encoded by the BS gene.

The methods provided herein can reduce function in the BIG SEEDS protein in a plant, plant part, population of plants or plant parts, or plant product, as compared to the BIG SEEDS protein in a control plant, plant part, population, or plant product. The function or activity of the BIG SEEDS protein encoded by the BS gene or homolog having a mutation (e.g., one or more insertions, substitutions, or deletions) in the gene or its regulatory region can be reduced by about 10-99%, 20-99%, 30-99%, 40-99%, 50-99%, 60-99%, 70-99%, 80-99%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to function or activity of a control BIG SEEDS protein encoded by a control BS gene or homolog without such mutation. In certain embodiments, the BIG SEEDS protein is encoded by the BS1 gene and/or the BS2 gene, e.g., Glycine max BS1 gene and/or Glycine max BS2 gene.

In specific embodiments, the methods provided herein for increasing GIF1 activity in a plant or plant part further includes decreasing BIG SEEDS activity (e.g., by introducing a genetic mutation that decreases BIG SEEDS activity) in the plant or plant part. Decrease in BIG SEEDS activity can upregulate a downstream target gene GIF1 and increase GIF1 activity, as disclosed herein. Further, methods for increasing GIF1 activity and decreasing BIG SEEDS activity (e.g., a genetic mutation that increases GIF1 activity and a genetic mutation that decreases BIG SEEDS activity) can have additive or synergistic effect on organ size, biomass, yield, and/or protein content, as further described herein.

B. Introducing mutations into the genome of plant cells

Introducing one or more mutations into the plant genome, e.g., into at least one GIF1 gene (e.g., Glycine max GIF1, e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.l8G121100, Glyma.l4G122500, and Glyma.OlGl 13500) or its regulatory region (and optionally into at least one BS gene (e.g., Glycine max BS1 or BS2 gene) or its regulatory region), and modulating the level or activity of GIF 1 in a plant or plant part may be achieved in any method of creating a change in a nucleic acid in a plant. For example, one or more mutations can be introduced into the plant genome, e.g., into at least one GIF1 gene (e.g., Glycine max GIF1) or its regulatory region (and optionally into at least one BS gene) through the use of precise genome -editing technologies to modulate the expression of the endogenous or transgenic sequence. In this manner, a nucleic acid sequence can be inserted, substituted, or deleted proximal to or within a native plant sequence corresponding to at least one GIF1 gene or homolog thereof or regulatory region thereof through the use of methods available in the art. Such methods include, but are not limited to, use of a nuclease designed against the plant target genomic sequence of interest (D’Halluin et al 2013 Plant Biotechnol J 11: 933-941), such as the Type II CRISPR system, the Type V CRISPR system, the CRISPR-Cas9 system, the CRISPR-Casl2a (Cpfl) system, the transcription activator-like effector nuclease (TALEN) system, the zinc finger nuclease (ZFN) system, and other technologies for precise editing of genomes [Feng et al. 2013 Cell Research 23: 1229-1232, Podevin et al. 2013 Trends Biotechnology 31: 375- 383, Wei et al. 2013 J Gen Genomics 40:281-289, Zhang et al (2013) WO 2013/026740, Zetsche et al. 2015 Cell 163:759-771]; Natronohacterium gregoryi Argonauie-mc<Ba.\.c DNA insertion (Gao et al. 2016 Nat Biotechnol doi: 10.1038/nbt.3547); Cre-lox site-specific recombination (Dale et al. 1995 Plant JT.649-659 Lyznik, et al. 2007 Transgenic Plant J 1: 1-9; FLP-FRT recombination (Li et al. 2009 Plant Physiol 151: 1087-1095); Bxbl-mediated integration (Yau et al. 2011 Plant J 701: 147-166); zinc -finger mediated integration (Wright et al. 2005 Plant 744:693-705); Cai et al. 2009 Plant Mol Biol 69:699-709); and homologous recombination (Lieberman-Lazarovich and Levy 2011 Methods Mol Biol 701: 51-65; Puchta 2002 Plant Mol Biol 48: 173-182). Reagents and compositions that can be used for introducing one or more mutations into plants or plant parts according to the methods of the present disclosure are herein described.

1. Editing reagent

Inserting, substituting, or deleting one or more nucleotides at a precise location of interest in at least one GIF1 gene and/or a regulatory region of the GIF1 gene (and optionally at least one BS gene and/or a regulatory region of the BS gene) in a plant or plant part may be achieved by introducing into the plant or plant part a system (e.g., a gene editing system), reagents (e.g., editing reagents), or a construct for introducing mutations at the target site of interest in a genome of a plant cell. A “gene editing system”, “editing system”, “gene editing reagent”, and “editing reagent” as used herein, refer to a set of one or more molecules or a construct comprising or encoding the one or more molecules for introducing one or more mutations in the genome. An exemplary gene editing system or editing reagents comprise a nuclease and/or a guide RNA. Also disclosed herein is a construct (e.g., a DNA construct, a recombinant DNA construct) for introducing one or more mutations in plants or plant parts. A construct can comprise an editing system or polynucleotides encoding editing reagents (e.g., nuclease, guide RNA, base editor) each operably linked to a promoter.

As used herein, the terms “nuclease” and “endonuclease” are used interchangeably to refer to naturally-occurring or engineered enzymes, which cleave a phosphodiester bond within a polynucleotide chain. The cleavage could be a single strand cleavage or a double strand cleavage. In certain embodiments, the nuclease lacks cleavage activity and is referred to as nuclease dead. Nucleases that can be used in precise genome-editing technologies to modulate the expression of the native sequence (e.g., at least one GIF1 gene and/or a regulatory region of the GIF1 gene) include, but are not limited to, meganucleases designed against the plant genomic sequence of interest (D’Halluin et al (2013) Plant Biotechnol J 11: 933-941); Cas9 endonuclease; Casl2a (Cpfl) endonuclease; ortholog of Cas 12a endonuclease; Cmsl endonuclease; transcription activator-like effector nucleases (TALENs); zinc finger nucleases (ZFNs); and a deactivated CRISPR nuclease (e.g., a deactivated Cas9, Casl2a, or Cmsl endonuclease) fused to a transcriptional regulatory element (Piatek et al. (2015) Plant Biotechnol J 13:578-589). In some embodiments, the editing system or the editing reagents comprise a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), and/or a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease. In some embodiments, the editing reagents comprise a CRISPR nuclease. In some embodiments, the CRISPR nuclease is a Cas 12a nuclease, herein used interchangeably with a Cpfl nuclease, e.g., a McCpfl nuclease. In some embodiments, the CRISPR nuclease is a Cas 12a nuclease ortholog, e.g., Lb5Casl2a, CMaCasl2a, BsCasl2a, BoCasl2a, MlCasl2a, Mb2Casl2a, TsCasl2a, and MAD7 endonucleases.

A nuclease system can introduce insertion, substitution, or deletion of genetic elements at a predefined genomic locus by causing a double-strand break at said predefined genomic locus and, optionally, providing an appropriate DNA template for insertion. This strategy is well-understood and has been demonstrated previously to insert a transgene at a predefined location in the cotton genome (D’Halluin et al. 2013 Plant Biotechnol. 11: 933-941). For example, a Casl2a (Cpfl) endonuclease coupled with a guide RNA (gRNA) designed against the genomic sequence of interest (i.e., at least one GIF1 gene and/or a regulatory region of the GIF1 gene) can be used (i.e., a CRISPR-Casl2a system). Alternatively, a Cas9 endonuclease coupled with a gRNA designed against the genomic sequence of interest (a CRISPR-Cas9 system), or a Cmsl endonuclease coupled with a gRNA designed against the genomic sequence of interest (a CRISPR-Cmsl) can be used. Other nuclease systems for use with the methods of the present invention include the CRISPR systems (e.g., Type I, Type II, Type III, Type IV, and/or Type V CRISPR systems (Makarova et al 2020 Nat Rev Microbiol 18:67-83)) with their corresponding gRNA(s), the TALEN system, the ZFN system, the meganuclease system, and the like. Alternatively, a deactivated CRISPR nuclease (e.g., a deactivated Cas9, Cas 12a, or Cmsl endonuclease) fused to a transcriptional regulatory element can be targeted to the regulatory region (e.g., upstream regulatory region) of at least one GIF1 gene, thereby modulating the transcription of the GIF1 gene (Piatek et al. 2015 Plant Biotechnol J 13:578-589). Sitespecific introduction of mutations of plant cells by biolistic introduction of a ribonucleoprotein comprising a nuclease and suitable guide RNA has been demonstrated (Svitashev et al. 2016 Nat Commun doi: 10.1038/ncomms 13274), and is herein incorporated by reference. For example, a CRISPR system comprises a CRISPR nuclease (e.g., CRISPR-associated (Cas) endonuclease or variant or ortholog thereof, such as Cas 12a or Cas 12a ortholog) and a guide RNA. A CRISPR nuclease associates with a guide RNA that directs nucleic acid cleavage by the associated endonuclease by hybridizing to a recognition site in a polynucleotide. The guide RNA directs the nuclease to the target site and the endonuclease cleaves DNA at the target site. The guide RNA comprises a direct repeat and a guide sequence, which is complementary to the target recognition site. In certain embodiments, the CRISPR system further comprises atracrRNA (trans- activating CRISPR RNA) that is complementary (fully or partially) to the direct repeat sequence present on the guide RNA. The CRISPR-Casl2a system may comprise at least one guide RNA (gRNA) operatively arranged with the ortholog endonuclease for genomic editing of a target DNA binding the gRNA. The system may comprise a CRISPR-Casl2a expression system encoding the Casl2a ortholog nucleases and crRNAs (CRISPR RNAs) for forming gRNAs that are coactive with the Casl2a nucleases. A “TALEN” nuclease is an endonuclease comprising a DNA-binding domain comprising a plurality of TAL domain repeats fused to a nuclease domain or an active portion thereof from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, and yeast HO endonuclease. A “zinc finger nuclease” or “ZFN” refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, and yeast HO endonuclease.

The editing system, editing reagents, or construct described herein can comprise one or more guide RNAs (gRNAs), or gRNA cassette, to drive mutations at the locus of at least one GIF1 gene or the regulatory region of the GIF1 gene, and optionally at least one BS gene or the regulatory region of the BS gene. “Guide RNA” as used herein refers to an RNA molecule that function as guides for RNA- or DNA- targeting enzymes, e.g., nucleases. In some instances, a gRNA can comprise a targeting region (i.e., spacer) that is complementary to a targeted sequence as well as another region that allows the gRNA to form a complex with a nuclease (e.g., a CRISPR nuclease) of interest.

For example, the editing system, the editing reagent, or the construct of the present disclosure may contain a gRNA cassette, comprising one or more gRNAs or encoding one or more gRNAs, to drive one or more insertions, substitutions, or deletions (e.g., a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides) in the promoter or 5’UTR of one or both alleles of a GIF1 gene, e.g., a Glycine max GIF1 gene (e.g., Glyma.03G249000, Glyma.l9G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.l8G121100, Glyma.14G122500, and Glyma.OlGl 13500), and optionally, in one or both alleles of a BS gene e.g., a Glycine max BS gene. The one or more gRNAs can be designed to specifically target a regulatory region (e.g., promoter, 5’UTR, G-box region) of a GIF1 gene (and optionally a BS gene), or exons or introns of a GIF1 gene (and optionally a BS gene). In some embodiments, one or more gRNAs are specific to a G-box region in a GIF1 gene promoter. Optionally, one or more gRNAs are specific to exon 1 or exon 2 of a BS gene.

For example, the gRNA can be specific to a nucleic acid sequence having at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-4 and 9-16, and optionally, any one of SEQ ID NOs: 26, 27, and 30-33. The gRNA can be specific to the nucleic acid sequence of any one of SEQ ID NOs: 1-4 and 9-16 (and optionally any one of SEQ ID NOs: 26, 27, and 30-33) and/or can drive a deletion at least partially in the 5’ regulatory region (e.g., promoter, 5’UTR, G-box region), exons, and/or introns of the Glycine max GIF1 gene and optionally the BS gene, or active homolog thereof. In particular instances, the gRNA can facilitate binding of an RNA guided nuclease that cleaves a region of at least one GIF1 gene, a regulatory region of the GIF1 gene, the G-box region of the GIF1 promoter, and optionally a region of at least one BS gene or regulatory region thereof, and cause non-homologous end joining or homology-directed repair to introduce a mutation at the cleavage site.

The methods provided herein can comprise introducing into the plant, plant part, or plant cell two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs specific to a nucleic acid sequence having at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-4 and 9-16 (and optionally any one of SEQ ID NOs: 26, 27, and 30-33). The two or more gRNA can be specific to the nucleic acid sequence of any one of SEQ ID NOs: 1-4 and 9-16 (and optionally any one of SEQ ID NOs: 26, 27, and 30-33) and/or can drive one or more deletions at least partially in the 5’ regulatory region (e.g., promoter, 5’UTR, G-box region), exons, and/or introns of the Glycine max GIF1 gene, and optionally the Glycine max BS gene, or active homolog thereof in the plant, plant part, or plant cell. In some instances, introducing two or more gRNAs along with other editing reagents (e.g., nuclease) into the plant, plant part, or plant cell increases sequence diversity of mutations (e.g., insertions, substitutions, deletions) generated at or near the target site, as compared to introducing one gRNA.

The targeting region (i.e. spacer) of a gRNA that binds to the region of at least one GIF1 gene or a regulatory region of the GIF1 gene (and optionally to the region of at least one BS gene or a regulatory region of the BS gene) for use in the method described herein can be about 100-300 nucleotides long, with the targeting region therein about 10-40 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long). For example, the targeting region of a gRNA for use in the method described herein may be about 24 nucleotides in length. In some embodiments, the targeting region of a gRNA that targets a BS gene is encoded by a nucleic acid sequence comprising a nucleic acid sequence having at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 34 or 35. In particular instances, the targeting region of a gRNA that targets a BS gene is encoded by a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 34 or 35. The methods provided herein can comprise introducing into the plant, plant part, or plant cell one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs, at least one of which comprising a nucleic acid sequence encoded by a nucleic acid sequence that shares at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 34 or 35 or a nucleic acid sequence of SEQ ID NO: 34 or 35. The methods provided herein can comprise introducing into the plant, plant part, or plant cell two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs, wherein two of the gRNAs each comprise a nucleic acid sequence encoded by: a nucleic acid sequence that shares at least 80% sequence identity with the nucleic acid sequence of SEQ ID NOs: 34 and 35.

The gRNA or a combination of two or more gRNAs provided herein can introduce a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides at least partially in the 5’ regulatory region (e.g., promoter, 5’UTR, G-box region) or the coding region (e.g., exons, introns) of a Glycine max GIF1 gene in the plant, plant part, or plant cell. For example, the one or more gRNAs provided herein can direct a nuclease to a specific target site at a G-box region in a Glycine max GIF1 promoter and introduce into the plant, plant part, or plant cell: (i) a mutation in the G-box region of Glyma.03G249000, resulting in a nucleic acid sequence of any one of SEQ ID NOs: 20-24; and/or (ii) a mutation in the G-box region of Glyma.19G246600 , resulting in a nucleic acid sequence of SEQ ID NO: 25.

Optionally, the gRNA or a combination of two or more gRNAs provided herein can introduce a deletion of about 4-8 nucleotides at least partially in the 5’ regulatory region (e.g., promoter, 5’UTR, G-box region) or the coding region (e.g., exons, introns) of a Glycine max BS gene in the plant, plant part, or plant cell. For example, the one or more gRNAs provided herein can direct a nuclease to a specific target site at a G-box region in a Glycine max GIF1 promoter and introduce into the plant, plant part, or plant cell (i) a deletion of nucleotides 98 through 101 of SEQ ID NO: 26, (ii) a deletion of nucleotides 389 through 396 of SEQ ID NO: 26, and/or (iii) a deletion of nucleotides 409 through 415 of SEQ ID NO: 27.

In some embodiments, a gene editing efficiency of the one or more gRNAs is 0.3% or greater (e.g., 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%).

Editing system or editing reagents can also include base editing components. For example, cytosine base editing (CBE) reagents, which change a C-G base pair to a T-A base pair, comprise a single guide RNA, a nuclease (e.g., dCas9, CAS9 nickase), a cytidine deaminase (e.g., APOBEC1), and a uracil DNA glycosylase inhibitor (UGI). Adenine base editing (ABE) reagents, which change an A-T base pair to a G-C base pair comprise a deaminase, (TadA), a nuclease (e.g., dCas or Cas nickase), and a guide RNA.

The gene editing system (e.g., CRISPR-Casl2a system), editing reagents, or a construct of the present disclosure can comprise at least one CRISPR RNA (crRNA) regulatory element operably linked to at least one nucleotide sequence encoding a crRNA for producing gRNA for targeting a target sequence, and at least one regulatory element, which may be the same as or different from the crRNA regulatory element, operably linked to a nucleotide sequence encoding the endonuclease, for generation of a CRISPR editing structure (e.g., CRISPR-Casl2a editing structure) by which the gRNA targets the target sequence and the CRISPR endonuclease cleaves a target DNA to alter gene expression in the cell, and wherein the CRISPR- associated nuclease, and the gRNA, do not naturally occur together. In such system, the at least one crRNA regulatory element may comprise one or more than one RNA polymerase II (Pol II) promoter, or alternatively, a single transcript unit (STU) regulatory element, or one or more of ZmUbi, OsU6, OsU3, and U6 promoters.

The methods described herein, comprising introducing into such plant a non-naturally occurring heterologous CRISPR-Cas 12a genomic editing system of a type as variously described herein, can cause the editing reagents to introduce mutations in at least one GIF1 gene or a regulatory region of the GIF1 gene and alter the level or activity of GIF1 gene or GIF1 protein. The gene editing system (e.g., the CRISPR- Casl2a system) can target PAM sites such as TTN, TTV, TTTV, NTTV, TATV, TATG, TATA, YTTN, GTTA, and/or GTTC.

Such methods of introducing mutations into plants, plant parts, or plant cells may be carried out at moderate temperatures, e.g., below 25° C. and above temperature producing freezing or frost damage of the plant. The methods provided herein may be performed on a wide variety of plants. In particular embodiments, the methods provided herein can be carried out to introduce mutations into the Glycine max plant at one or more GIF1 genes or a regulatory region of the GIF1 gene, and optionally at one or more BS genes or a regulatory region of the BS gene.

Methods disclosed herein are not limited to certain techniques of mutagenesis. Any method of creating a change in a nucleic acid of a plant can be used in conjunction with the disclosed invention, including the use of chemical mutagens (e.g. ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), methanesulfonate, sodium azide, aminopurine, etc.), genome/gene editing techniques (e.g. CRISPR-like technologies, TALENs, zinc finger nucleases, and meganucleases), physical mutagens (e.g., ionizing radiation (e.g. ultraviolet, gamma rays)), T-DNA, transposons, temperature alterations, long-term seed storage, tissue culture conditions, targeting induced local lesions in a genome, sequence-targeted and/or random recombinases, etc. It is anticipated that new methods of creating a mutation in a nucleic acid of a plant will be developed and yet fall within the scope of the claimed invention when used with the teachings described herein. Any editing system or editing reagents for use in any genome-editing methods including those described herein can be expressed in a plant or plant part.

2. Promoter

As used herein, “promoter” refers to a regulatory region of DNA that is capable of driving expression of a sequence in a plant or plant cell. A number of promoters may be used in the practice of the disclosure, e.g., to express editing reagents in plants, plant parts, or plant cells. The promoter may have a constitutive expression profile. Constitutive promoters include the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3 :2723- -2730); ALS promoter (U.S. Patent No. 5,659,026), and the like.

Alternatively, promoters for use in the methods of the present disclosure can be tissue-preferred promoters. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513- 524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5) : 773-778; Lam (1994) Results Prohl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Leaf-preferred promoters are also known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto el a/. (1994) Plant Cell Physiol. 35(5):773-778; Gotor el a/. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Alternatively, promoters for use in the methods of the present disclosure can be developmentally- regulated promoters. Such promoters may show a peak in expression at a particular developmental stage. Such promoters have been described in the art, e.g., US Patent No. 10,407,670; Gan and Amasino (1995) Science 270: 1986-1988; Rinehart et al. (1996) Plant Physiol 112: 1331-1341; Gray-Mitsumune et al. (1999) Plant Mol Biol 39: 657-669; Beaudoin and Rothstein (1997) Plant Mol Biol 33: 835-846; Genschik et al. (1994) Gene 148: 195-202, and the like.

Alternatively, promoters for use in the methods of the present disclosure can be promoters that are induced following the application of a particular biotic and/or abiotic stress. Such promoters have been described in the art, e.g., Yi et al. (2010) Planta 232: 743-754; Yamaguchi- Shinozaki and Shinozaki (1993) Mol Gen Genet 236: 331-340; U.S. Patent No. 7,674,952; Rerksiri et al. (2013) Sci World J 2013 : Article ID 397401; Khurana et al. (2013) PLoS One 8: e54418; Tao et al. (2015) Plant Mol Biol Rep 33: 200-208, and the like.

Alternatively, promoters for use in the methods of the present disclosure can be cell-preferred promoters. Such promoters may preferentially drive the expression of a downstream gene in a particular cell type such as a mesophyll or a bundle sheath cell. Such cell-preferred promoters have been described in the art, e.g., Viret et aZ. (1994) Proc Natl Acad USA 91: 8577-8581; U.S. Patent No. 8,455,718; U.S. Patent No. 7,642,347; Sattarzadeh et al. (2010) Plant Biotechnol J 8: 112-125; Engelmann et al. (2008) Plant Physiol 146: 1773-1785; Matsuoka et al. (1994) Plant J 6: 311-319, and the like.

It is recognized that a specific, non-constitutive expression profile may provide an improved plant phenotype relative to constitutive expression of a gene or genes of interest. For instance, many plant genes are regulated by light conditions, the application of particular stresses, the circadian cycle, or the stage of a plant’s development. These expression profiles may be important for the function of the gene or gene product in planta. One strategy that may be used to provide a desired expression profile is the use of synthetic promoters containing cis -regulatory elements that drive the desired expression levels at the desired time and place in the plant. Cis-regulatory elements that can be used to alter gene expression in planta have been described in the scientific literature (Vandepoele et al. (2009) Plant Physiol 150: 535-546; Rushton et al. (2002) Plant Cell 14: 749-762). Os-regulatory elements may also be used to alter promoter expression profiles, as described in Venter (2007) Trends Plant Sci 12: 118-124.

3. Transfer DNA

Nucleic acid molecules comprising transfer DNA (T-DNA) sequences can be used in the practice of the disclosure, e.g., to express editing reagents in plants, plant parts, or plant cells. For example, a construct of the present disclosure may contain T-DNA of tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens . Alternatively, a recombinant DNA construct of the present disclosure may contain T-DNA of tumor-inducing (Ti) plasmid of Agrobacterium rhizogenes. The vir genes of the Ti plasmid may help in transfer of T-DNA of a recombinant DNA construct into nuclear DNA genome of a host plant. For example, Ti plasmid of Agrobacterium tumefaciens may help in transfer of T-DNA of a recombinant DNA construct of the present disclosure into nuclear DNA genome of a host plant, thus enabling the transfer of a gRNA of the present disclosure into nuclear DNA genome of a host plant (e.g., a pea plant).

4. Regulatory signal

Construct described herein may contain regulatory signals, including, but not limited to, transcriptional initiation sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter "Sambrook 11"; Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and the references cited therein.

5. Reporter genes /selectable marker genes

Reporter genes or selectable marker genes may be included in the expression cassettes of the present invention. Examples of suitable reporter genes known in the art can be found in, for example, Jefferson, et al., (1991) in Plant Molecular Biology Manual, ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33; DeWet, et al., (1987) Mol. Cell. Biol. 7:725-737; Goff, et al., (1990) EMBO J. 9:2517-2522; Kain, et al., (1995) Bio Techniques 19:650-655 and Chiu, et al., (1996) Current Biology 6:325-330, herein incorporated by reference in their entirety.

Selectable marker genes for selection of transformed cells or tissues can include genes that confer antibiotic resistance or resistance to herbicides. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209-213; Meijer, et al., (1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron, et al., (1985) Plant Mol. Biol. 5: 103-108 and Zhijian, et al., (1995) Plant Science 108:219-227); streptomycin (Jones, et al., (1981) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5: 131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol. 7: 171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol. 15: 127-36); bromoxynil (Stalker, et al., (1988) Science 242:419-423); glyphosate (Shaw, et al., (1986) Science 233:478- 481 and US Patent Application Serial Numbers 10/004,357 and 10/427,692); phosphinothricin (DeBlock, et al., (1987) EMBO J. 6:2513-2518), herein incorporated by reference in their entirety.

Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), spectinomycin/streptinomycin resistance (SpcR, AAD), and hygromycin phosphotransferase (HPT or HGR) as well as genes conferring resistance to herbicidal compounds. Herbicide resistance genes generally code for a modified target protein insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. For example, resistance to glyphosate has been obtained by using genes coding for mutant target enzymes, 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS). Genes and mutants for EPSPS are well known, and further described below. Resistance to glufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterial genes encoding PAT or DSM-2, a nitrilase, an AAD-1, or an AAD-12, each of which are examples of proteins that detoxify their respective herbicides.

Herbicides can inhibit the growing point or meristem, including imidazolinone or sulfonylurea, and genes for resistance/tolerance of acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) for these herbicides are well known. Glyphosate resistance genes include mutant 5-enolpyruvylshikimate-3- phosphate synthase (EPSPs) and dgt-28 genes (via the introduction of recombinant nucleic acids and/or various forms of in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosate acetyl transferase (GAT) genes, respectively). Resistance genes for other phosphono compounds include bar and pat genes from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes, and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). Exemplary genes conferring resistance to cyclohexanediones and/or aryloxyphenoxypropanoic acid (including haloxyfop, diclofop, fenoxyprop, fluazifop, quizalofop) include genes of acetyl coenzyme A carboxylase (ACCase); Accl-Sl, Accl-S2 and Accl-S3. Herbicides can also inhibit photosynthesis, including triazine (psbA and ls+ genes) or benzonitrile (nitrilase gene). Further, such selectable markers can include positive selection markers such as phosphomannose isomerase (PMI) enzyme.

Selectable marker genes can further include, but are not limited to genes encoding: 2,4-D; SpcR; neomycin phosphotransferase II; cyanamide hydratase; aspartate kinase; dihydrodipicolinate synthase; tryptophan decarboxylase; dihydrodipicolinate synthase and desensitized aspartate kinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase (NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolate reductase (DHFR); phosphinothricin acetyltransferase; 2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase; 5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase; acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32 kD photosystem II polypeptide (psbA). Selectable marker genes can further include genes encoding resistance to: chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; and phosphinothricin.

Other selectable marker genes that could be employed on the expression constructs disclosed herein include, but are not limited to, GUS (beta-glucuronidase; Jefferson, (1987) Plant Mol. Biol. Rep. 5:387), GFP (green fluorescence protein; Chalfie, et al., (1994) Science 263:802), luciferase (Riggs, et al., (1987) Nucleic Acids Res. 15(19):8115 and Luehrsen, et al., (1992) Methods Enzymol. 216:397-414), red fluorescent protein (DsRFP, RFP, etc), beta-galactosidase, and the maize genes encoding for anthocyanin production (Ludwig, et al., (1990) Science 247:449), and the like (See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, N.Y., 2001), herein incorporated by reference in their entirety. The above list of selectable marker genes is not meant to be limiting. Any reporter or selectable marker gene are encompassed by the present disclosure.

6. Terminator

A transcription terminator may also be included in the expression cassettes of the present invention. Plant terminators are known in the art and include those available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141- 149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

7. Vector

Disclosed herein are vectors containing constructs (e.g., recombinant DNA constructs encoding editing reagents) of the present disclosure. As used herein, “vector” refers to a nucleotide molecule (e.g., a plasmid, cosmid), bacterial phage, or virus for introducing a nucleotide construct, for example, a recombinant DNA construct, into a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance. In some embodiments, provided herein are expression cassettes located on a vector comprising a gRNA sequence specific for at least one GIF1 gene or a regulatory region of the GIF1 gene, and optionally a gRNA sequence specific for at least one BS gene or a regulatory region of the BS gene.

In some embodiments, a vector is a plasmid containing a recombinant DNA construct of the present disclosure. For example, the present disclosure may provide a plasmid containing a recombinant DNA construct that comprises a gRNA to drive mutations at the locus of at least one GIF1 gene or the regulatory region of the GIF1 gene, and optionally at least one BS gene or the regulatory region of the BS gene.

In some embodiments, a vector is a recombinant virus containing a recombinant DNA construct of the present disclosure. For example, the present disclosure may provide a recombinant virus containing a recombinant DNA construct that comprises a gRNA, wherein the gRNA can drive mutations at the locus of at least one GIF1 gene or the regulatory region of the GIF1 gene, and optionally at least one BS gene or the regulatory region of the BS gene. A recombinant virus described herein can be a recombinant lentivirus, a recombinant retrovirus, a recombinant cucumber mosaic virus (CMV), a recombinant tobacco mosaic virus (TMV), a recombinant cauliflower mosaic virus (CaMV), a recombinant odontoglossum ringspot virus (ORSV), a recombinant tomato mosaic virus (ToMV), a recombinant bamboo mosaic virus (BaMV), a recombinant cowpea mosaic virus (CPMV), a recombinant potato virus X (PVX), a recombinant Bean yellow dwarf virus (BeYDV), or a recombinant turnip vein-clearing virus (TVCV).

8. Cells

Also provided herein are cells comprising the reagent (e.g., editing reagent, e.g., nuclease, gRNA), the system (e.g., gene editing system), the construct (e.g., expression cassette), and/or the vector of the present disclosure for introducing mutations into at least one GIF1 gene and/or a regulatory region of the GIF1 gene, and optionally at least one BS gene or the regulatory region of the BS gene. The cell can be a plant cell, a bacterial cell, and a fungal cell. The cell can be a bacterium, e.g., an Agrobacterium tumefaciens, containing the gRNA targeting at least one GIF1 gene and/or a regulatory region of the GIF1 gene (or optionally at least one BS gene or the regulatory region of the BS gene) and driving mutations at the target site of interest. The cells of the present disclosure may be grown, or have been grown, in a cell culture.

C. Increasing organ size, biomass, or yield and/or increasing protein content in plants

The methods of the present disclosure, by introducing a mutation or an exogenous gene copy that increases GIF1 activity (and optionally a mutation that decreases BIG SEEDS activity) into plants, plant parts, or plant cells and/or regenerating plants from transformed cells, can increase organ (e.g., seed, leaf) size, biomass, or yield, and/or can increase protein content in the plants, plant parts (e.g., seeds, leaves), population of plants or plant parts, or plant products (e.g., seed composition, plant protein composition) as compared to a control (e.g., wild-type) plant, plant part, population of plants or plant parts, or plant product.

In specific embodiments, the methods provided herein increases GIF1 activity, and also decreased BIG SEEDS activity. Decrease in BIG SEEDS activity can upregulate a downstream target gene GIF1 and increase GIF1 activity, as disclosed herein. Further, increased GIF1 activity and decreased BIG SEEDS activity (e.g., a genetic mutation that increases GIF1 activity and a genetic mutation that decreases BIG SEEDS activity) can have additive or synergistic effect on organ size, biomass, yield, and/or protein content. For example, methods of introducing modifications (e.g., mutations) that increase GIF1 activity as well as modifications (e.g., mutations) that decrease BIG SEEDS activity can have greater organ size (e.g., seed size), greater biomass, greater yield, and/or greater protein content relative to control plants, plant parts, population of plants or plant parts, or plant products having a modification (e.g., mutation) that increases GIF1 activity but not a modification (e.g., mutation) that decreases BIG SEEDS activity, or having a modification (e.g., mutation) that decreases BIG SEEDS activity but not a modification (e.g., mutation) that increases GIF1 activity other than the effect of the decreased BIG SEEDS activity. The organ size, biomass, yield, and/or protein content can be increased by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-50%, 30-50%, 40-50%, 50-50%, 60-50%, 70-50%, 100-500%, 200-500%, 300-500%, 400-500%, 500-500%, 600-500%, 700-500%, 800-500%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 500% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, or more than 500%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, or more in plants, plant parts, a population of plants or plant parts, or plant products that have been introduced modifications that increase GIF1 activity as well as modifications that decrease BIG SEEDS activity relative to control plants, plant parts, population of plants or plant parts, or plant products that have been introduced a modification that increases GIF1 activity but not a modification that decreases BIG SEEDS activity, or a modification that decreases BIG SEEDS activity but not a modification that increases GIF1 activity other than the effect of the decreased BIG SEEDS activity. A control plant, plant part, a population of plants or plant parts, or plant product can comprise a plant or plant part to which a mutation or an exogenous gene copy provided herein has not been introduced, e.g., by methods of the present disclosure. Thus, a control plant, plant part, a population of plants or plant parts, or plant product has a wild-type GIF1 activity (and a wild-type BIG SEEDS activity), and may express an endogenous (e.g., wild-type) GIF1 gene (and an endogenous (e.g., wild-type) BS gene). A plant, plant part, a population of plants or plant parts, or plant product of the present disclosure can have increased seed size, increased biomass, increased yield, and/or increased protein content as compared to a control plant, plant part, a population of plants or plant parts, or plant product, when the plant or plant part of the present disclosure is grown under the same environmental conditions (e.g., same or similar temperature, humidity, air quality, soil quality, water quality, and/or pH conditions) as the control plant or plant part.

In some embodiments, the methods provided herein can increase organ size (e.g., seed size, leaf size), plant biomass, or yield (e.g., seed yield) of the plant or plant part, or population of plants or plant parts by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30- 90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800-1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200-300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900- 1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more as compared to a control plant, plant part, or population. Organ size can be measured by measuring parameters (e.g., seed diameter, stem length, leaf width and length) or calculating organ size based on measured parameters according to the standard methods. For instance, leaf area (LA) can be estimated by using the formula: LA = 2.0185 x L x W, where L is length and W is width (Richter et al. 2014 Bragantia 73(4):416- 425), with an R² of 0.9747. Yield or biomass can be measured and expressed by standard methods, for example weight or volume of seeds, fruits, leaves, or whole plants harvested from a given harvest area.

In some embodiments, the methods can increase total protein content by about 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 20-90%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 100-1000%, 200-1000%, 300-1000%, 400-1000%, 500-1000%, 600-1000%, 700-1000%, 800- 1000%, 200-900%, 300-900%, 400-900%, 500-900%, 600-900%, 700-900%, or more than 1000% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-200%, 200- 300%, 300-400%, 400-500%, 500-600%, 600-700%, 700-800%, 800-900%, 900-1000%, or more than 1000%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more in the plants, plant parts, or population of plants or plant parts of the present disclosure as compared to a control plant or plant part. In some embodiments, the methods can increase total protein content, as expressed by % dry weight, in the plant, plant part, or a population of plant or plant parts, and the increase is about 0.25-10%, 0.5-10%, 0.75-10%, 1.0-10%, 1.5-10%, 2-10%, 2.5-10%, 3-10%, 3.5-10%, 4-10%, 4.5-10%, 5-10%, 6-10%, 7-10%, 8-10%, 9-10%, or more than 10% (e.g., by about 0.25-0.5%, 0.5-0.75%, 0.75-1.0%, 1.0-1.5%, 1.5-2.0%, 2.0-2.5%, 2.5-3.0%, 3.0-3.5%, 3.5-4.0%, 4.0-4.5%, 4.5-5.0%, 5-6%, 6-7%, 7-8%, or 8-9%, 9-10%, or more than 10%), by about 0.25%, 0.5%, 0.75%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, or more, or at least 0.25%, 0.5%, 0.75%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, or more when compared to (by subtraction) that in a control plant, plant part, or population.

In specific embodiments, the methods increase protein content in seeds or a population of seeds compared to control seeds or a control population of seeds (e.g., control seeds or population having native GIF1 and BS, reference seeds or population, commodity seeds or population). The seeds can be legume seeds, e.g., pea seeds or soybean seeds. The methods can increase the protein content of pea seeds or a population of pea seeds to at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more by dry weight, wherein typical pea cultivars average approximately 20-30% protein in the seed in dry weight (Meng & Cloutier, 2014 Microencapsulation in the Food Industry: A Practical Implementation Guide § 20.5). Similarly, the methods can increase the protein content of soybean seeds or a population of soybean seeds to at least 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% or more by dry weight, wherein seed protein content of typical soybean cultivars ranges approximately 36-46% in dry weight (Rizzo & Baroni 2018 Nutrients 10( 1) :43 ; Grieshop & Fahey 2001 J Agric Food Chem 49(5):2669-73; Garcia et al. 1997 Crit Rev Food Set Nutr 37(4):361 -91). Protein content in a plant sample can be measured by standard methods, for example by protein extraction and quantitation (e.g., BCA protein assay, Lowry protein assay, Bradford protein assay), spectroscopy, near-infrared reflectance (NIR) (e.g., analyzing 700 - 2500 nm), and nuclear magnetic resonance spectrometry (NMR).

In specific embodiments, the methods provided herein can increase organ (e.g., seed) size, biomass, yield (e.g., seed yield) as well as protein content in a plant, plant part, population of plants or plant parts, or plant product, as compared to a control plant, plant part, population, or plant product. In specific embodiments, the methods provided herein can increase GIF1 activity and optionally decrease BIG SEEDS activity in a population of seeds and increase seed size, seed yield, and/or seed protein content as compared to a control population.

D. Plants, plant parts, population, and plant products produced by present methods

The present disclosure provides plants, plant parts, a population of plants or plant parts, and plant products produced according to the methods provided herein. Such plants, plant parts, population of plants or plant parts, and plant products can have increased GIF 1 activity compared to a control plant, plant part, population, or plant product. A “plant part” produced according to the methods described herein can include any part of a plant, including seeds (e.g., a representative sample of seeds), plant cells, embryos, pollen, ovules, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, juice, pulp, nectar, stems, branches, and bark. A “plant product”, as used herein, refers to any product or composition produced from the plant, including any oil products, sugar products, fiber products, protein products (such as protein concentrate, protein isolate, flake, or other protein product), seed hulls, meal, or flour, for a food, feed, aqua, or industrial product, plant extract (e.g., sweetener, antioxidants, alkaloids, etc.), plant concentrate (e.g., whole plant concentrate or plant part concentrate), plant powder (e.g., formulated powder, such as formulated plant part powder (e.g., seed flour)), plant biomass (e.g., dried biomass, such as crushed and/or powdered biomass), grains, plant protein composition, plant oil composition, and food and beverage products containing plant compositions (e.g., plant parts, plant extract, plant concentrate, plant powder, plant protein, plant oil, and plant biomass) described herein. Plant parts and plant products provided herein can be intended for human or animal consumption.

Plant protein compositions obtained from the plants or plant parts produced according to the methods provided herein can be a concentrated protein solution (e.g. As used herein, a “protein product” or “protein composition” refers to any protein composition or product isolated, extracted, and/or produced from plants or plant parts (e.g., seed) and includes isolates, concentrates, and flours, e.g., soy/pea protein composition, soy/pea protein concentrate (SPC/PPC), soy/pea protein isolate (SPI/PPI), soy/pea flour, flake, white flake, texturized vegetable protein (TVP), or textured soy/pea protein (TSP/TPP)). The protein composition can comprise multiple proteins as a result of the extraction or isolation process. A plant protein composition can be a concentrated protein solution (e.g., soybean protein concentrate solution) in which the protein is in a higher concentration than the protein in the plant from which the protein composition is derived. The protein composition can comprise multiple proteins as a result of the extraction or isolation process. The plant protein composition can further comprise stabilizers, excipients, drying agents, desiccating agents, anti-caking agents, or any other ingredient to make the protein fit for the intended purpose. The protein composition can be a solid, liquid, gel, or aerosol and can be formulated as a powder. The protein composition can be extracted in a powder form from a plant and can be processed and produced in different ways, such as: (i) as an isolate - through the process of wet fractionation, which has the highest protein concentration; (ii) as a concentrate - through the process of dry fractionation, which are lower in protein concentration; and/or (Hi) in textured form - when it is used in food products as a substitute for other products, such as meat substitution (e.g. a “meat” patty). In specific embodiments, the plant protein compositions provided herein are obtained from a legume plant (e.g., Pisum sativum, Glycine max)' or plant part produced according to the methods of the present disclosure, e.g., by introducing into the plant or plant part a mutation (e.g., in the regulatory region, non-coding region, and/or coding region of at least one GIF1 gene) or an exogenous copy of a GIF1 gene that increases GIF1 activity, and optionally by introducing into the plant or plant part a mutation that decreases BIG SEEDS activity (e.g., in at least one native BS gene or homolog or regulatory region thereof). Also provided herein are food and/or beverage products produced from the plants, plant parts, or plant compositions (e.g., seed composition, plant protein compositions) produced according to the methods of the present disclosure. Such food and/or beverage products can be meant for human or animal consumption, and can include animal feed, shakes (e.g., protein shakes), health drinks, alternative meat products (e.g., meatless burger patties, meatless sausages), alternative egg products (e.g., eggless mayo), non-dairy products (e.g., non-dairy whipped toppings, non-dairy milk, non-dairy creamer, non-dairy milk shakes, non-diary ice cream), energy bars (e.g., protein energy bars), infant formula, baby foods, cereals, baked goods, edamame, tofu, and tempeh. A food and/or beverage product that contains plant compositions obtained from the plants or plant parts produced by the methods of the present disclosure can have desired traits (e.g., increased GIF activity, decreased BS activity, increased protein content) compared to a similar or comparable food and/or beverage product that contains plant compositions obtained from a control plant or plant part.

Plant parts (e.g., seeds) and plant products (e.g., plant biomass, seed compositions, protein compositions, food and/or beverage products) produced by the methods provided herein can be meant for consumption by agricultural animals or for use as feed in an agriculture or aquaculture system. In specific embodiments, plant parts and plant products produced according to the methods provided herein include animal feed (e.g., roughages - forage, hay, silage; concentrates - cereal grains, soybean cake) intended for consumption by bovine, porcine, poultry, lambs, goats, or any other agricultural animal. In some embodiments, plant parts and plant products produced according to the methods include aquaculture feed for any type of fish or aquatic animal in a farmed or wild environment including, without limitation, trout, carp, catfish, salmon, tilapia, crab, lobster, shrimp, oysters, clams, mussels, and scallops.

The plants, plant parts, and plant products, including plant protein compositions and plant-based food/beverage products produced according to the methods of the present disclosure can contain a mutation that increases GIF1 activity, e.g., one or more insertions, substitutions, or deletions in at least one native GIF1 gene or homolog or in a regulatory region of such GIF1 gene or homolog, e.g., a substitution of 1-10 nucleotides or a deletion of about 4-12 nucleotides at least partially in the G-box region of a Glycine max GIF1 gene. Additionally, the plant parts, population of plant parts, and plant products produced by the methods of the present disclosure can have one or more exogenous copies of a native or mutated GIF1 gene. The mutation can be located at least partially in the regulatory region, coding region, or non-coding region of the exogenous copy of the GIF1 gene. The plant parts, population of plant parts, and plant products produced by the methods of the present disclosure can have increased GIF1 activity, increased expression level of the GIF1 gene or homolog, increased expression level of the GIF1 protein, increased function or activity of the GIF1 protein, increased expression or activity of GIF1 downstream target molecules that regulate cell and organ growth and development (e.g., GRF5, GRF3, COL5, ARR4, RA2, CLE4a), increased seed size, increased biomass, increased yield, and/or increased protein content as compared to a control plant part, population, or plant product, e.g., comprising wild-type GIF1 level or activity. The plant parts, population of plant parts, and plant products according to the methods of the present disclosure can further contain a mutation that decreases BIG SEEDS activity, e.g., one or more insertions, substitutions, or deletions in at least one native BS gene or homolog or regulatory region thereof, e.g., a deletion of about 4-8 nucleotides at least partially in the nucleic acid region of exon 1 or 2 of a Glycine max BS1 gene or exon 1 or 2 of a Glycine max BS2 gene. The plant parts, population of plant parts, and plant products produced according to the methods of the present disclosure can further have decreased BIG SEEDS activity, decreased expression level of the BS gene, the BIG SEEDS protein, or homolog, decreased function or activity of the BIG SEEDS protein, and/or increased activity of one or more target molecules regulated by BIG SEEDS and regulating organ growth or size (e.g., GRF, GRF1, GRF5, GIF, GIF1, GIF2, cyclin D3;3, histone 4) compared to a control plant parts, population of plant parts, and plant products. Modifications that increase GIF1 activity and modifications that decrease BIG SEEDS activity can have additive or synergistic effects on organ (e.g., seed) size, biomass, yield, or protein content in the plant parts, population of plant parts, and plant products produced by the methods of the present disclosure, such that they have greater organ (e.g., seed) size, greater biomass or yield (e.g., seed yield), and/or greater protein content compared to a control plant part, population of plant parts, or plant product having modifications that increase GIF1 activity but not modifications that decrease BIG SEEDS activity, or having modifications that decrease BIG SEEDS activity but not modifications that increase GIF1 activity beyond the increase in GIF1 activity caused by the decreased BIG SEEDS activity.

E. Transformation of plants

Provided herein are methods for transforming plants or plant parts by introducing into the plants or plant parts one or more mutations (e.g., insertions, substitutions, and/or deletions) in at least one GIF1 gene and optionally at least one BS gene (e.g., in the regulatory region, non-coding region, and/or a coding region), or introducing into the plants or plant parts a polynucleotide encoding a GIF1 protein or a functional fragment thereof. The methods can comprise introducing a system (e.g., a gene editing system), reagents (e.g., editing reagents), or a construct for introducing mutations at the target site of interest. The methods can also comprise introducing a construct containing a transgene (e.g., encoding a GIF1 protein or a functional fragment thereof) into the plant or plant part.

The term “transform” or “transformation” as used herein refers to any method used to introduce genetic mutations (e.g., insertions, substitutions, or deletions in the genome), polypeptides, or polynucleotides into plant cells. For purpose of the present disclosure, the transformation can be “stable transformation”, wherein the one or more mutations (e.g., in at least one GIF1 gene, a regulatory region of the GIF1 gene, at least one BS gene, a regulatory region of the BS gene) or the transformation constructs (e.g., a construct comprising a nucleic acid molecule encoding a gRNA and/or a nuclease for use in the methods of the present invention, or a construct comprising a polynucleotide encoding a GIF1 protein or functional fragment thereof) are introduced into a host (e.g., a host plant, plant part, plant cell, etc.), integrate into the genome of the host, and are capable of being inherited by the progeny thereof; or “transient transformation”, wherein the one or more mutations (e.g., in at least one GIF1 gene, a regulatory region of the GIF1 gene, at least one BS gene, a regulatory region of the BS gene) or the transformation constructs (e.g., a construct comprising a gRNA and/or a gene encoding a nuclease for use in the methods of the present invention, or a construct comprising a polynucleotide encoding a GIF 1 protein or functional fragment thereof) are introduced into a host (e.g., a host plant, plant part, plant cell, etc.) and expressed temporarily. The methods disclosed herein can also be used for insertion of heterologous genes and/or modification of native plant gene expression to achieve desirable plant traits, e.g., increased seed size, increased biomass, increased yield, and/or increased protein content and/or increased disease tolerance.

Any mutation or any polynucleotide of interest (e.g., editing reagents, e.g., a nuclease and a guide RNA; a polynucleotide encoding a GIF1 protein or functional fragment thereof) can be introduced into a plant cell, organelle, or plant embryo by a variety of means of transformation, including microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S. Patent No. 5,563,055 and U.S. Patent No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) E/V/BO J. 3:2717-2722), and ballistic particle acceleration [see, for example, U.S. Patent Nos. 4,945,050; U.S. Patent No. 5,879,918; U.S. Patent No. 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923- 926); and Uecl transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. ITAl' t- 477; Sanford et al. (1987) Particulate Science and Technology 5-. 1 -37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Patent Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae),' De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Eongman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250- 255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens)],’ all of which are herein incorporated by reference.

The embodiments disclosed herein are not limited to certain methods of introducing nucleic acids into a plant, and are not limited to certain forms or structures that the introduced nucleic acids take. Any method of transforming a cell of a plant described herein with nucleic acids are incorporated into the teachings of this innovation. Agrobacterium-and biolistic-mediated transformation remain the two predominantly employed approaches. However, one of ordinary skill in the art will realize that the use of particle bombardment (e.g. using a gene-gun), infection by other bacterial species capable of transferring DNA into plants (e.g., Ochrobactrum sp., Ensifer sp., Rhizobium sp.) or virus (e.g., Caulimoriviruses, Geminiviruses, RNA plant viruses) optionally with Agrobacterium infection, transfection, microinjection, electroporation, microprojection, electroporation, silica/carbon fibers, ultrasound mediated, PEG mediated, calcium phosphate co-precipitation, polycation DMSO technique, DEAE dextran procedure, liposome mediated and other techniques can be used to deliver nucleic acid sequences into a plant described herein. Methods disclosed herein are not limited to any size of nucleic acid sequences that are introduced, and thus one could introduce a nucleic acid comprising a single nucleotide (e.g. an insertion) into a nucleic acid of the plant and still be within the teachings described herein. Nucleic acids introduced in substantially any useful form, for example, on supernumerary chromosomes (e.g. B chromosomes), plasmids, vector constructs, additional genomic chromosomes (e.g. substitution lines), and other forms is also anticipated. It is envisioned that new methods of introducing nucleic acids into plants and new forms or structures of nucleic acids will be discovered and yet fall within the scope of the claimed invention when used with the teachings described herein.

More than one polynucleotides of interest can be introduced into the plant, plant cell, plant organelle, or plant embryo simultaneously or sequentially. For example, different editing reagents, e.g., nuclease polypeptides (or encoding nucleic acid), guide RNAs (or DNA molecules encoding the guide RNAs), donor polynucleotide(s), and/or repair templates can be introduced into the plant cell, organelle, or plant embryo simultaneously or sequentially. The amount or ratio of more than one polynucleotides of interest, or molecules encoded therein, can be adjusted by adjusting the amount or concentration of the polynucleotides and/or timing and dosage of introducing the polynucleotides into the plant or plant part. For example, the ratio of the nuclease (or encoding nucleic acid) to the guide RNA(s) (or encoding DNA) to be introduced into plants or plant parts generally will be about stoichiometric such that the two components can form an RNA-protein complex with the target DNA. In one embodiment, DNA encoding a nuclease and DNA encoding a guide RNA are delivered together within a plasmid vector.

Alteration of the GIF1 level or activity, and optionally BIG SEEDS level or activity, in plants, plant parts, or plant cells may also be achieved through the use of transposable element technologies to alter gene expression. It is well understood that transposable elements can alter the expression of nearby DNA (McGinnis et al. (1983) Cell 34:75-84). Alteration of the GIF1 or BIGSEEDS level or activity may be achieved by inserting a transposable element into at least one GIF1 gene, a regulatory region of the GIF1 gene, at least one BS gene, and/or a regulatory region of the BS gene.

The cells that have been transformed may be grown into plants (i.e., cultured) in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. In this manner, the present invention provides transformed plants or plant parts, transformed seed (also referred to as “transgenic seed”) or transformed plant progenies having a nucleic acid modification stably incorporated into their genome. The present invention may be used for transformation of any plant species, e.g., both monocots and dicots (including legumes). Plants or plant parts to be transformed according to the methods disclosed herein can be a legume, i.e., a plant belonging to the family Fabaceae (or Leguminosae), or a part (e.g., fruit or seed) of such a plant. When used as a dry grain, the seed of a legume is also called a pulse. Examples of legume include, without limitation, soybean (Glycine max), beans (Phaseolus spp.), common bean (Phaseolus vulgaris), fava bean (Vida faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut Arachis hypogaea), lentils (Lens culinaris, Lens esculenta), lupins (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.). In specific embodiments, a plant or plant part to be transformed according to the methods of the present disclosure is Glycine max or a part of Glycine max. Additionally, a plant or plant part to be transformed according to the methods present disclosure can be a crop plant or part of a crop plant, including legumes. Examples of crop plants include, but are not limited to, com (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), camelina (Camelina sativa), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), quinoa (Chenopodium quinoa), chicory (Cichorium intybus), lettuce (Lactuca sativa), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana spp., e.g., Nicotiana tabacum, Nicotiana sylvestris), potato (Solanum tuberosum), tomato (Solanum lycopersicum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), grapes (Vitis vinifera, Vitis riparia), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integri folia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oil palm (Elaeis guineensis), poplar (Populus spp.), pea (Pisum sativum), eucalyptus (Eucalyptus spp.), oats (Avena sativa), barley (Hordeum vulgare), vegetables, ornamentals, and conifers. Additionally, a plant or plant part of the present disclosure can be an oilseed plant (e.g., canola (Brassica napus), cotton (Gossypium sp.), camelina (Camelina sativa) and sunflower (Helianthus sp.)), or other species including wheat (Triticum sp., such as Triticum aestivum L. ssp. aestivum (common or bread wheat), other subspecies of Triticum aestivum, Triticum turgidum L. ssp. durum (durum wheat, also known as macaroni or hard wheat), Triticum monococcum L. ssp. monococcum (cultivated einkom or small spelt), Triticum timopheevi ssp. timopheevi, Triticum turgigum L. ssp. dicoccon (cultivated emmer), and other subspecies of Triticum turgidum (Feldman)), barley (Hordeum vulgare), maize (Zea mays), oats (Avena sativa), or hemp (Cannabis sativa). Additionally, a plant or plant part of the present disclosure can be a forage plant or part of a forage plant. Examples of forage plants include legumes and crop plants described herein as well as grass forages including Agrostis spp., Lolium spp., Festuca spp., Poa spp., and Bromus spp.

The present disclosure provides plants and plant parts transformed according to the methods of the present disclosure. Transformed plant parts of the invention include plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, grains, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the introduced mutations, polynucleotides, or polypeptides.

F. Breeding of Plants

Also disclosed herein are methods for breeding a plant, such as a plant which contains (i) a mutation that increases the GIF1 activity, e.g., one or more insertions, substitutions, or deletions in at least one native GIF1 gene or homolog or in a regulatory region of such GIF1 gene or homolog, (ii) editing reagents, e.g., a polynucleotide encoding a guide RNA specific to at least one GIF1 gene or homolog or in a regulatory region of such GIF1 gene or homolog, and/or (iii) a polynucleotide comprising a wild-type, mutated, native, or heterologous GIF1 gene (e.g., including regulatory region thereof). The plant can further contain (i) a mutation that decreases the BIG SEEDS activity, e.g., one or more insertions, substitutions, or deletions in at least one native BS gene or homolog or in a regulatory region of such BS gene or homolog, (ii) editing reagents, e.g., a polynucleotide encoding a guide RNA specific to at least one BS gene or homolog or in a regulatory region of such BS gene or homolog, and/or (iii) a polynucleotide comprising a wild-type, mutated, native, or heterologous BS gene (e.g., including regulatory region thereof). A plant containing the one or more mutations or the polynucleotide (e.g., transgene) of the present disclosure may be regenerated from a plant cell or plant part, wherein the genome of the plant cell or plant part is modified to contain the one or more mutations or the polynucleotide of the present disclosure. Using conventional breeding techniques or self-pollination, one or more seeds may be produced from the plant that contains the one or more mutations or the polynucleotide of the present disclosure. Such a seed, and the resulting progeny plant grown from such a seed, may contain the one or more mutations or the polynucleotide of the present disclosure, and therefore may be transgenic. Progeny plants are plants having a genetic modification to contain the one or more mutations or the polynucleotide of the present disclosure, which descended from the original plant having modification to contain the one or more mutations or the polynucleotide of the present disclosure. Seeds produced using such a plant of the invention can be harvested and used to grow generations of plants having genetic modification to contain the one or more mutations or the polynucleotide of the present disclosure, e.g., progeny plants, of the invention, comprising the polynucleotide and optionally expressing a gene of agronomic interest (e.g., herbicide resistance gene).

Descriptions of breeding methods that are commonly used for different crops can be found in one of several reference books, see, e.g., Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, Calif., 50-98 (1960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding Perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

Methods disclosed herein include conferring desired traits (e.g., high protein content) to plants, for example, by mutating sequences of a plant, introducing nucleic acids into plants, using plant breeding techniques and various crossing schemes, etc. These methods are not limited as to certain mechanisms of how the plant exhibits and/or expresses the desired trait. In certain nonlimiting embodiments, the trait is conferred to the plant by introducing a nucleic acid sequence (e.g. using plant transformation methods) that encodes production of a certain protein by the plant. In certain embodiments, the desired trait is conferred to a plant by causing a null mutation in the plant’s genome (e.g. when the desired trait is reduced expression or no expression of a certain trait). In some embodiments, the desired trait is conferred to a plant by introducing a mutation in the genome that cause overexpression or reduced expression of a gene related to the desired trait. In certain embodiments, the desired trait is conferred to a plant by causing a mutation into the GIF1 gene(s) or its regulatory region that causes increased activity of GIF1, and optionally further by causing a mutation into the BS gene(s) or its regulatory region that causes decreased activity of BIG SEEDS. In certain embodiments, the desired trait is conferred to a plant by transforming the plant with an exogenous copy of a GIF1 gene or a functional fragment thereof, operably linked to a functional promoter. In certain embodiments, the desired trait is conferred to a plant by crossing two plants to create offspring that express the desired trait. It is expected that users of these teachings will employ a broad range of techniques and mechanisms known to bring about the expression of a desired trait in a plant. Thus, as used herein, conferring a desired trait to a plant is meant to include any process that causes a plant to exhibit a desired trait, regardless of the specific techniques employed.

In certain embodiments, a user can combine the teachings herein with high-density molecular marker profiles spanning substantially the entire genome of a plant to estimate the value of selecting certain candidates in a breeding program in a process commonly known as genome selection.

V. Nucleic Acid Molecules, Constructs, and Cells Comprising GIF1 gene or Regulatory Region of GIF1 gene

A. Nucleic acid molecules

Nucleic acid molecules are provided herein comprising a polynucleotide sequence that alters (e.g., increases) GIF 1 activity in a plant or plant part. The nucleic acid molecule can comprise any nucleic acid sequence that alters (e.g., increases) GIF1 activity in a plant or plant part including those described herein, e.g., an altered (e.g., mutated, alternatively spliced) nucleic acid sequence of a GIF1 gene or homolog thereof or regulatory region thereof, an altered GIF1 gene transcript encoding an altered (e.g., mutated, alternatively spliced, truncated) GIF1 protein, or a wild-type sequence of a GIF1 gene or functional fragment thereof for overexpression in the plant or plant part. Such nucleic acid molecules may be present in, or obtained from, a plant cell, plant part, or plant of the present disclosure, or may be obtained by the methods described herein, e.g., by introducing one or more mutations into at least one GIF1 gene or a regulatory region of the GIF1 gene, introducing editing reagents targeting a site of interest in at least one GIF1 gene or a regulatory region of the GIF1 gene, or introducing a polynucleotide encoding a GIF1 protein or functional fragment thereof into a plant or plant part. The nucleic acid molecule described herein can contain a modified regulatory region (e.g., promoter, 5’UTR, binding site for a transcription modulator protein, enhancer sequence, or other genomic regions that contribute to regulation of transcription or translation) of a GIF1 gene that increases level or activity of an operably linked downstream gene. The nucleic acid molecule described herein can also encode an altered (e.g., mutated, truncated, alternatively spliced) GIF1 protein that has a different amino acid sequence from a native GIF1 protein (e.g., without mutations) and/or has increased GIF1 function or activity, e.g., the ability to regulate protein content , as compared to a native GIF1 protein (e.g., without mutations). The mutated sequence, e.g., altered nucleic acid sequence of the GIF1 gene and/or the regulatory region of the GIF1 gene can result in increased expression levels of the GIF1 gene or GIF1 protein (e.g., full-length GIF1 protein, functional GIF1 protein), as compared to a native GIF1 gene and/or a regulatory region of a native GIF1 gene e.g., without mutations.

The nucleic acid molecule provided herein can comprise a sequence of a mutated GIF1 gene and/or regulatory region thereof containing one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions that increases the GIF1 activity. The nucleic acid molecule can comprise a mutated regulatory region of a GIF1 gene, e.g., a mutated promoter region, 5’ untranslated region (5’UTR), binding site (e.g., a G-box region, an enhancer sequence) for a transcription modulator protein (e.g., repressor complex, transcription factor), or other genomic regions that contribute to regulation of transcription or translation of a GIF1 gene. The nucleic acid molecule can comprise a mutated GIF1 promoter having a mutation at or near the G-box region.

In some embodiments, the nucleic acid molecule comprises a mutated regulatory region of a GIF1 gene, and (i) the regulatory region, before the mutation is located, comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 13-16, wherein the regulatory region retains transcription initiation activity; (ii) the regulatory region, before the mutation is locate, comprises a nucleic acid sequence of any one of SEQ ID NOs: 13-16; (iii) the GIF1 gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; (iv) the GIF1 gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-12; (v) the GIF1 gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein the polypeptide retains GIF1 activity; (vi) the GIF1 gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5-8; (vii) the GIF1 gene including the regulatory region thereof, before the mutation is located, comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein the nucleic acid sequence encodes a polypeptide that retains GIF1 activity; and/or (viii) the GIF1 gene including said regulatory region thereof, before the mutation is located, comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4. The mutation can increase GIF1 activity.

In some embodiments, the nucleic acid molecule comprises a mutated promoter region or 5’UTR of a Glycine max GIF1 gene (e.g., Glyma.03G249000, Glyma.19G246600, Glyma.l0G164100, Glyma.20G226500, Glyma.18G121100, Glyma.14G122500, Glyma.OlGl 13500), e.g., a nucleic acid sequence of any one of SEQ ID NOs: 13-16 with a mutation. In some embodiments, the nucleic acid molecule comprises a mutated G-box region of a GmGIFl gene, e.g., a nucleic acid sequence of any one of SEQ ID NOs: 17-19 in the Glycine max GIF1 gene with a mutation. In some embodiments, the nucleic acid molecule comprises a mutated promoter, G-box region, and/or 5’UTR of a Glycine max GIF1 gene with a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of a mutated G-box region of a GIF1 (i) having at least 80% identity to a nucleic acid sequence of any one of SEQ ID NOs: 20-25 or (ii) comprising the nucleic acid sequence of any one of SEQ ID NOs: 20-25.

The nucleic acid molecule may comprise an in-frame mutation, a frameshift (out-of-frame) mutation, a missense mutation, or a nonsense mutation of the GIF1 gene or homolog. The nucleic acid molecule described herein can comprise the regulatory region (e.g., promoter region) of the GIF1 gene as well as the exon/intron region of the GIF1 gene, one or both of which has one or more insertions, substitutions, and/or deletions that increase level or activity of GIF 1.

Nucleic acid molecules provided herein comprising a mutated genomic sequence that decreases BIG SEEDS activity in a plant or plant part. The nucleic acid molecules having a mutated sequence that alters (e.g., increases) GIF1 activity can further comprise a mutated genomic sequence that decreases BIG SEEDS activity; alternatively, separate nucleic acid molecules can include a mutated sequence that alters (e.g., increases) GIF1 activity and a mutated genomic sequence that decreases BIG SEEDS activity separately. The nucleic acid molecule can comprise any nucleic acid sequence that decreases BIG SEEDS activity in a plant or plant part including those described herein, e.g., an altered (e.g., mutated, alternatively spliced) nucleic acid sequence of a BS gene, a regulatory region of the BS gene, or a BS gene transcript, encoding an altered (e.g., mutated, alternatively spliced, truncated) BIG SEEDS protein relative to a corresponding native BS gene or BIG SEEDS protein. Such nucleic acid molecules may be present in, or obtained from, a plant cell, plant part, or plant of the present disclosure, or may be obtained by the methods described herein, e.g., by introducing one or more mutations into at least one BS gene or a regulatory region of the BS gene and/or by introducing editing reagents targeting a site of interest in at least one BS gene or a regulatory region of the BS gene in a plant or plant part. The nucleic acid molecule described herein can encode an altered (e.g., mutated, truncated, alternatively spliced) BIG SEEDS protein that can comprise a different amino acid sequence from a native BIG SEEDS protein (e.g., without mutations). The nucleic acid molecule described herein can encode a BIG SEEDS protein with reduced function or loss-of-fimction of BIG SEEDS, e.g., the ability to regulate organ (e.g., seed) size or protein content, or the ability to regulate molecules that regulate organ size or growth (e.g., GRF1, GRF5, GIF1, GIF2, CYCD3;3, H4), as compared to a native BIG SEEDS protein (e.g., without mutations). The mutated sequence, e.g., altered nucleic acid sequence of the BS gene and/or the regulatory region of the BS gene can result in reduced expression levels of the BS gene or BIG SEEDS protein (e.g., full-length BIG SEEDS protein, functional BIG SEEDS protein), as compared to a native BS gene and/or a regulatory region of a native BS gene e.g., without mutations.

The nucleic acid molecule provided herein can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions in BS gene or homolog and/or a regulatory region of the BS gene or homolog compared to a corresponding native a BS gene or homolog and/or a regulatory region of the native BS gene or homolog. The nucleic acid molecule may comprise an in-frame mutation, a frameshift (out-of-frame) mutation, a missense mutation, or a nonsense mutation of the BS gene or homolog.

The mutation in the nucleic acid molecule provided herein can be located in Glycine max BS genes, such as a Glycine max BS1 gene, a Glycine max BS2 gene, and/or a regulatory region of such one or more Glycine max BS genes. In some embodiments, the mutation is located in a BS gene or homolog thereof comprising a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 26, 27, or 49 and encoding a polypeptide that retains BIG SEEDS activity, for example the nucleic acid sequence of SEQ ID NO: 26, 27, or 49; and/or a regulatory region of the BS gene or homolog thereof comprising such nucleic acid sequence. Additionally, the mutation can be located in a BS gene or homolog thereof encoding a polypeptide comprising an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 28, 29, or 41 and retaining BIG SEEDS activity, for example a polypeptide comprising the amino acid sequence of SEQ ID NO: 28, 29, or 41; and/or a regulatory region of the BS gene or homolog thereof encoding such polypeptide.

The mutation in the nucleic acid molecule provided herein can be at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertion, substitution, or deletion located at least partially in a nucleic acid region of exon 1 or 2 of a Glycine max BS1 gene (set forth as SEQ ID NOs: 30 and 32, respectively) or exon 1 or 2 of a Glycine max BS2 gene (set forth as SEQ ID NOs: 31 and 33, respectively). The mutation in the nucleic acid molecule provided herein can comprise a deletion of about 4- 8 nucleotides at least partially in the nucleic acid region of exon 1 or 2 of a Glycine max BS1 gene or exon 2 of a Glycine max BS2 gene. For example, the nucleic acid molecule of the present disclosure can comprise (i) a mutated Glycine max BS1 gene sequence with a deletion of nucleotides 98 through 101 of SEQ ID NO: 26, (ii) a mutated Glycine max BS1 gene sequence with a deletion of nucleotides 389 through 396 of SEQ ID NO: 26, or (iii) a mutated Glycine max BS2 gene sequence with a deletion of nucleotides 409 through 415 of SEQ ID NO: 26.

In some embodiments, the nucleic acid molecules described herein do not comprise a regulatory region (e.g., a promoter region) of a BS gene or homolog. Alternatively, the nucleic acid molecules can comprise the regulatory region (e.g., promoter region) of the BS gene or homolog. The regulatory region (e.g., promoter regions) in the nucleic acid molecule can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertions, substitutions, and/or deletions. The one or more insertions, substitutions, and/or deletions in the regulatory region of the BS gene or homolog can alter expression level or manner of the BS gene or homolog. For example, the one or more insertions, substitutions, and/or deletions in the promoter region of the BS gene or homolog can alter the transcription initiation activity of the promoter. The modified promoter can alter (e.g., reduce) transcription of the operably linked nucleic acid molecule, initiate transcription in a developmentally-regulated manner, initiate transcription in a cell-specific, cell-preferred, tissue-specific, or tissue-preferred manner, or initiate transcription in an inducible manner. The modified promoter can comprise a deletion, a substitution, or an insertion, e.g., introduction of a heterologous promoter sequence, a cis-acting factor, a motif or a partial sequence from any promoter, including those described elsewhere in the present disclosure, to confer an altered (e.g., reduced) transcription initiation function to the promoter region of the BS gene according to the present disclosure.

The nucleic acid molecule described herein can comprise one or more insertions, substitutions, and/or deletions in the regulatory region (e.g., promoter region) of the BS gene as well as in the exon/intron region of the BS gene.

B. DNA constructs, vectors, and cells

The nucleic acid molecules encoding molecules of interest of the present invention can be assembled within a DNA construct with an operably-linked promoter. When transiently or stably transformed with such DNA construct, a plant, plant part, or plant cell can express or accumulate polynucleotides comprising the native or mutated sequence of a GIF1 gene or a GIF1 gene transcript, or a native or mutated GIF1 protein encoded by the polynucleotides; and optionally, polynucleotides comprising the native or mutated sequence of a BS gene or a BS gene transcript, or a native or mutated BIG SEEDS protein encoded by the polynucleotides. For example, the nucleic acid molecules described herein can be provided in expression cassettes or expression constructs along with a promoter sequence of interest, a native or heterologous promoter sequence, for expression in the plant of interest. By “heterologous promoter sequence” is intended a sequence that is not naturally operably linked with the nucleic acid molecule of interest. For instance, a 2x35 s promoter, a native promoter, or a promoter (native or heterologous) comprising an exogenous or synthetic motif sequence may be operably linked to the nucleic acid sequences comprising a sequence of a native or mutated GIF1 gene or a GIF1 gene transcript, and optionally, a sequence of a native or mutated BS gene or a BS gene transcript. The GIF 1 -encoding nucleic acid sequences or the promoter sequence (and optionally the BS-encoding nucleic acid sequences or the promoter sequence) may each be homologous, native, heterologous, or foreign to the plant host. It is recognized that the heterologous promoter may also drive expression of its homologous or native nucleic acid sequence. In this case, the transformed plant will have a change in phenotype. Accordingly, the present disclosure provides DNA constructs comprising, in operable linkage, a regulatory region of a GIF1 gene that can be native (without mutation) or mutated (e.g., having at last 80% sequence identity with any one of SEQ ID NOs: 13-25, or having the sequence of any one of SEQ ID NOs: 13- 25), and a polynucleotide of interest (e.g., a GIF1 gene or a reporter gene, e.g., GFP, luciferase, HA tag). In specific embodiments, the DNA constructs comprise, in operable linkage, a GIF1 promoter comprising the nucleic acid sequence of any one of SEQ ID NOs: 20-25, and a polynucleotide of interest. Also provided herein are DNA constructs comprising, in operable linkage, a promoter that is functional in a plant cell, and a nucleic acid molecule comprising a native (wild-type) or altered nucleic acid sequence of a GIF1 gene or a GIF1 gene transcript. Additionally or alternatively, the DNA construct can contain a native or mutated BS gene (e.g., native or mutated GmBSl or GmBS2 gene provided herein) and an operably linked promoter, or a native or mutated BS gene and an operably linked polynucleotide of interest. One DNA construct can contain both (i) the nucleic acid molecule comprising the native or mutated sequence of the GIF1 gene or regulatory region and (ii) the nucleic acid molecule comprising the native or mutated BS gene or regulatory region, or alternatively, separate DNA constructs can contain (i) and (ii) separately.

When the DNA construct or nucleic acid molecule provided herein, comprising a native or mutated GIF1 gene or regulatory region, is introduced in a plant, plant part, or plant cell, GIF1 activity can be increased, expression levels of the GIF1 gene can be increased, GIF1 protein level or activity can be increased, activity of one or more target molecules regulated by GIF1 and regulating cell or tissue growth or development can be increased, and protein content can be increased in the plant, plant part, or plant cell as compared to a control plant, plant part, or plant cell, e.g., a plant, plant part, or plant cell to which the construct or the nucleic acid molecule of the present disclosure are not introduced. When the DNA construct or nucleic acid molecule provided herein, comprising a native or mutated BS gene or regulatory region, is introduced in a plant, plant part, or plant cell, BIG SEEDS activity can be decreased, expression levels of the BS gene can be decreased, BIG SEEDS protein level or activity can be decreased, activity of one or more target molecules regulated by BIG SEEDS and regulating cell or tissue growth or development can be increased, and protein content can be increased in the plant, plant part, or plant cell as compared to a control plant, plant part, or plant cell, e.g., a plant, plant part, or plant cell to which the construct or the nucleic acid molecule of the present disclosure are not introduced. The DNA construct can comprise, in operable linkage with a promoter (e.g., a GIF1 promoter with a mutated G-box region) and/or a reporter / selectable marker construct (e.g., GFP, luciferase, HA tag). Any reporter or selectable marker can be used, including the reporters and selectable markers described elsewhere in the present disclosure.

Provided herein are vectors comprising the nucleic acid molecule and/or the DNA construct of the present disclosure comprising an altered or native nucleic acid sequence of the GIF1 gene, the regulatory region of the GIF1 gene, and/or the GIF1 gene transcript, and optionally an altered or native nucleic acid sequence of the BS gene, the regulatory region of the BS gene, and/or the BS gene transcript. Any vectors can be used, including the vectors described elsewhere in the present disclosure. Also provided herein are cells comprising the nucleic acid molecule, the DNA construct, and/or the vector of the present disclosure comprising an altered or native nucleic acid sequence of the GIF1 gene, the regulatory region of the GIF1 gene, and/or the GIF1 gene transcript, and optionally an altered or native nucleic acid sequence of the BS gene, the regulatory region of the BS gene, and/or the BS gene transcript. The cell can be a plant cell, a bacterial cell, and a fungal cell. The cell can be a bacterium, e.g., an Agrobacterium tumefaciens, containing the nucleic acid molecule, the DNA construct, or the vector of the present disclosure. The cell can be a plant cell. The cells of the present disclosure may be grown, or have been grown, in a cell culture.

Also provided herein are methods for generating a plant, plant part (e.g., seed), plant cell, or a population of plants or plant parts (e.g., seeds) comprising increased GIF1 activity (and optionally decreased BIG SEEDS activity), increased seed size, increased biomass, increased yield, and/or increased protein content, by introducing into the plant, plant part, or plant cell the nucleic acid molecule, the DNA construct, the vector, or the cell of the present disclosure. In some embodiments, the nucleic acid molecule, DNA construct, vector, or cell is introduced into the plant by stable transformation. In other embodiments, the nucleic acid molecule, DNA construct, vector, or cell is introduced into the plant by transient transformation. The present disclosure further provides plants, plant parts (seed, juice, pulp, fruit, flowers, nectar, embryos, pollen, ovules, leaves, stems, branches, bark, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, etc.), or plant products (e.g., seed compositions, plant protein, plant protein compositions, plant extract, plant concentrate, plant powder, plant biomass, and food and beverage products) generated by the methods described herein.

It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. Having now described the invention in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. Unless otherwise noted, all parts and percentages are by dry weight.

EXAMPLES

EXAMPLE 1: Expression of GIF1 copies in wild-type soybean tissues

Transcript expression levels of four GIF1 copies in soybean, Glyma.03G249000, Glyma.19G246600, Glyma.l0G164100, and Glyma.20G226500 in various tissues of soybean were studied based on data available from a soy expression database. As shown in FIG. 1A, Glyma.03G249000 was expressed throughout various tissues of soybean, with highest expression in flowers. As shown in FIG. IB, Glyma.19G246600 was expressed throughout various tissues of soybean, with highest expression in flowers. As shown in FIG. 1C, Glyma.10G164100 was expressed throughout various tissues of soybean, with highest expression in embryo. As shown in FIG. ID, Glyma.20G226500 was expressed throughout various tissues of soybean, with highest expression in embryo.

EXAMPLE 2: GIF1 promoter modification upregulates GIF1 activity in soybean

Guide RNAs targeting at or near the G-box region of the GmGIFl (Glyma.03G 249000) promoter were designed according to standard methods of the art (Zetsche et al., Cell, Volume 163, Issue 3, Pages 759-771, 2015; Cui et al., Interdisciplinary Sciences: Computational Life Sciences, volume 10, pages 455- 465, 2018). Optimized gRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 and CRISPR-Casl2a have been extensively characterized (Nat Biotechnol 34, 184-191, doi: 10.1038/nbt.3437 (2016)). The CRISPR-Casl2a system described herein can be employed for targeting PAM sites such as TTN, TTV, TTTV, NTTV, TATV, TATG, TATA, YTTN, GTTA, and GTTC, utilizing corresponding gRNAs.

Soybean protoplasts were transformed with constructs comprising guide RNAs targeting a genomic site at or near the G-box region of the GmGIFl (Glyma.03G249000) promoter and a nuclease, as well as the GmGIFl coding sequence (Glyma.03G 249000) linked to a reporter, using Agrobacterium transformation. Amplicons were produced near the target sites, and were sequenced to detect mutations. A mutated read was recorded for any sequence with more than two reads containing a deletion at the predicted cleavage site. Editing efficiency was calculated based on the percentage of mutated reads to total aligned reads using next generation sequencing (NGS).

A number of mutants having mutations in the GmGIFl promoter at or near the G-box region of the GmGIFl (Glyma.03G249000) were generated using the gene editing system provided herein, and were screened for editing efficiency and effects on GIF1 level and activity. The minor groove width of the mutants were analyzed for binding affinity of the KIX-PPD-MY C repressor complex. The SNP3 mutant contains a mutation at the G-box region of the GIF1 promoter, set forth as SEQ ID NO: 22, that increases the binding of the repressor complex to the G-box region. The SNP4 and SNP5 mutants each contain a mutation at the G-box region of the GIF1 promoter, set forth as SEQ ID NOs: 23 and 24, respectively, that decreases the binding of the repressor complex to the G-box region. As shown in FIG. 2, the SNP3 mutant protoplast showed decreased GIF 1 expression compared to control (“WT”) protoplast without mutation, as measured by normalized fold change (NFC) of the reporter gene expression. The SNP4 and SNP5 mutant protoplasts showed increased GIF1 expression as compared to control (“WT”) protoplast, as measured by NFC of the reporter gene expression. The data demonstrate a correlation between a decreased binding of the repressor complex to the G-box region of the GIF1 promoter (e.g., due to a mutation in the G-box region) and an increased GIF1 expression.

Embryonic axes of mature seeds of soybean varieties are stably transformed with constructs comprising one, two, or multiple GmGIFl guide RNAs and a nuclease using Agrobacterium transformation. Transformed plants are identified by a selectable marker (e.g., resistance to glyphosate). Amplicons are produced of the genomic regions near the targeted GmGIFl promoter sites and sequenced to evaluate the presence of the mutation using a pair of primers to detect mutations introduced. Transgenic events are recorded, and the TO plants are assigned unique plant names and are subjected to molecular characterization and propagation. TO plants are self-pollinated and T1 plants are generated. Crosses are made to generate lines that are homozygous or heterozygous for the target mutation and lack the editing reagents.

EXAMPLE 3: Screening of plants with mutations

Transformed plants are screened using a variety of molecular tools to identify plants and genotypes that will result in the expected phenotype. For example, expression levels of GIF1 genes and levels and activities of GIF1 protein are measured in mutant plants (e.g., having a homozygous or heterozygous mutation in the GIF1 promoter). Expression levels of the GIF1 genes are measured by any standard methods for measuring mRNA levels of a gene, including quantitative RT-PCR, northern blot, and serial analysis of gene expression (SAGE). Expression levels of GIF1 protein (e.g., full-length GIF1 protein) are measured by any standard methods for measuring protein levels, including western blot analysis, ELISA, dot blot analysis, or a reporter assay (e.g., by transfecting a plant cell with a GIF1 gene - reporter gene (e.g., luciferase) construct and measuring expression of the reporter) of a protein sample obtained from the plant using an antibody directed to the GIF1 protein (e.g., full-length GIF1 protein). GIF1 activity can be assessed by measuring organ size (e.g., leaf size, seed size) or protein content. The plant with mutation may have increased GIF1 level or activity, increased expression levels of the GIF1 genes, increased seed size, increased leaf size, increased biomass, increased yield, and/or increased protein content as compared to a control plant (e.g., without the mutation) when grown under the same environmental conditions.

EXAMPLE 4: Plants with increased GIF1 activity and decreased BIG SEEDS activity

Plants having increased GIF1 activity and decreased BIG SEEDS activity are produced by introgressing plants having increased GIF1 activity with plants having decreased BIG SEEDS activity, or by introducing a mutation that increases GIF1 activity (e.g., mutations in a GmGIFl promoter comprising a nucleic acid sequence of any one of SEQ ID NOs: 22-26) and a mutation that decreases BIG SEEDS activity (e.g., a deletion of nucleotides 98 through 101 of SEQ ID NO: 26 in a Glycine max BS1 gene, a deletion of nucleotides 389 through 396 of SEQ ID NO: 26 in a Glycine max BS1 gene, and/or a deletion of nucleotides 409 through 415 of SEQ ID NO: 27 in a Glycine max BS2 gene). Introgressed or transformed plants are studied for phenotypes including GIF and BIG SEEDS activity, organ size, and protein content. The plant with increased GIF1 activity and decreased BIG SEEDS activity may have increased seed size, increased leaf size, increased biomass, increased yield, and/or increased protein content as compared to a control plant when grown under the same environmental conditions.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

While various aspects of the invention are described herein, it is not intended that the invention be limited by any particular aspect. On the contrary, the invention encompasses various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Furthermore, where feasible, any of the aspects disclosed herein may be combined with each other (e.g., the feature according to one aspect may be added to the features of another aspect or replace an equivalent feature of another aspect) or with features that are well known in the art, unless indicated otherwise by context.

TABLE 3. Sequence Descriptions

Claims

What is claimed is:

1. A plant or plant part comprising increased nuclear transcription factor GRF -interacting factor 1 (GIF1) activity compared to a control plant or plant part, wherein said plant or plant part comprises a genetic mutation that increases the GIF1 activity.

2. The plant or plant part of claim 1, comprising increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control plant or plant part.

3. The plant or plant part of claim 1 or 2, wherein the mutation comprises one or more insertions, substitutions, or deletions in at least one GIF1 gene or homolog thereof or regulatory region thereof in the plant or plant part, wherein: an expression level of said at least one GIF1 gene or homolog thereof is increased compared to an expression level a corresponding GIF1 gene or homolog thereof without said mutation; and/or level or activity of a GIF1 protein encoded by said at least one GIF1 gene or homolog thereof is increased compared to level or activity of a GIF1 protein encoded by a corresponding GIF1 gene or homolog thereof without said mutation.

4. The plant or plant part of claim 3, wherein the mutation is located at least partially in a promoter region or 5’ untranslated region (5’UTR) of said at least one GIF1 gene or homolog thereof.

5. The plant or plant part of claim 3 or 4, wherein the mutation is located at least partially in a G-box region in said regulatory region of said at least one GIF1 gene or homolog thereof.

6. The plant pr plant part of any one of claims 4 or 5, wherein the mutation decreases binding of a GIF1 repressor complex to the regulatory region of said at least one GIF1 gene or homolog thereof, thereby increasing level or activity of a GIF 1 protein encoded by said at least one GIF1 gene or homolog thereof.

7. The plant or plant part of any one of claims 3-6, wherein said at least one GIF1 gene or homolog thereof, before the mutation is located:

(i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein said nucleic acid sequence encodes a polypeptide that retains GIF1 activity;

(ii) comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-12;

(iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein said polypeptide retains GIF1 activity;

(iv) encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 5-8; (v) includes said regulatory region thereof, and comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein said nucleic acid sequence encodes a polypeptide that retains GIF 1 activity; and/or

(vi) includes said regulatory region thereof, and comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4.

8. The plant or plant part of any one of claims 3-7, wherein the mutation is located at least partially in a promoter region or 5’ untranslated region (5’UTR) of a Glycine max GIF1 gene.

9. The plant or plant part of claim 8, wherein the Glycine max GIF1 gene is selected from the group consisting of Glyma.03G249000, Glyma.19G246600, Glyma.10G 164100, Glyma.20G226500, Glyma.18G121100, Glyma.14G 122500, and Glyma.OlGl 13500.

10. The plant or plant part of claim 8 or 9, wherein the mutation is located at least partially in a G-box region for the Glycine max GIF1 gene.

11. The plant or plant part of claim 10, wherein the mutation is located at least partially in a nucleic acid sequence of any one of SEQ ID NOs: 17-19 in the G-box region of the Glycine max GIF1 gene.

12. The plant or plant part of any one of claims 8-11, wherein the mutation comprises a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides located at least partially in the G-box region for the Glycine max GIF1 gene.

13. The plant or plant part of claim 12, comprising:

(i) Glyma.03G249000 with a mutated G-box region comprising a nucleic acid sequence of any one of SEQ ID NOs: 20-24; and/or

(ii) Glyma.19G246600 with a mutated G-box region comprising a nucleic acid sequence of SEQ ID NO: 25.

14. The plant or plant part of any one of 1-13, further comprising a genetic mutation that decreases BIG SEEDS (BS) activity.

15. The plant or plant part of claim 14, wherein the mutation that decreases BIG SEEDS activity comprises one or more insertions, substitutions, or deletions in at least one BS gene or homolog thereof or regulatory region thereof, wherein: an expression level of said at least one BS gene or homolog thereof is increased compared to an expression level a corresponding BS gene or homolog thereof without said mutation; and/or level or activity of a BIG SEEDS protein encoded by said at least one BS gene or homolog thereof is increased compared to level or activity of a BIG SEEDS protein encoded by a corresponding BS gene or homolog thereof without said mutation.

16. The plant or plant part of claim 15, wherein said at least one BS gene or homolog thereof, before the mutation is located:

(i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 26, 27, or 49, wherein said nucleic acid sequence encodes a polypeptide that retains BIG SEEDS activity;

(ii) comprises the nucleic acid sequence of SEQ ID NO: 26, 27, or 49;

(iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 28, 29, or 41, wherein said polypeptide retains BIG SEEDS activity; and/or

(iv) encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, 29, or 41.

17. The plant or plant part of claim 15 or 16, wherein said mutation is located at least partially in a nucleic acid region of exon 1 or exon 2 of a Glycine max BS1 gene and/or exon 2 of a Glycine max BS2 gene.

18. The plant or plant part according to claim 17, comprising:

(i) a mutated Glycine max BS1 gene comprising a deletion of nucleotides 98 through 101 of SEQ ID NO: 26;

(ii) a mutated Glycine max BS1 gene comprising a deletion of nucleotides 389 through 396 of SEQ ID NO: 26; and/or

(iii) a mutated Glycine max BS2 gene comprising a deletion of nucleotides 409 through 415 of SEQ ID NO: 27.

19. The plant or plant part of any one of claims 1-18, wherein said plant or plant part is a legume.

20. The plant or plant part of claim 19, wherein said plant or plant part is selected from the group consisting of soybean (Glycine max)' , beans (Phaseolus spp.), common bean (Phaseolus vulgaris), fava bean (Vida faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentils (Lens culinaris, Lens esculenta), lupins (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.).

21. The plant or plant part of any one of claims 1-18, wherein said plant or plant part is selected from the group consisting of com (Zea mays), Brassica species, Brassica napus, Brassica rapa, Brassica juncea, rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet, pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbcidense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculentd), coffee (Coffea spp ), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp ), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp ), avocado (Per sea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integri folia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

22. The plant or plant part of any one of claims 1-21, wherein said plant or plant part is a seed.

23. A population of plants or plant parts comprising the plant or plant part of any one of claims 1-22, wherein the population comprises increased GIF1 activity, increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control population.

24. The population of plants or plant parts of claim 23, wherein said plant or plant part is a seed, and said population is a population of seeds.

25. A method for increasing seed size, biomass, protein content, and/or yield in a plant or plant part, said method comprising increasing level or activity of GRF -interacting factor 1 (GIF1) in said plant or plant part.

26. The method of claim 25, comprising introducing a genetic mutation that increases GIF1 activity into said plant or plant part.

27. The method of claim 26, further comprising introducing the genetic mutation that increases GIF1 activity into a plant cell, and regenerating said plant or plant part from said plant cell.

28. The method of claim 26, 27, or 49, wherein the mutation comprises one or more insertions, substitutions, or deletions in at least one GIF1 gene or homolog thereof or regulatory region thereof in said plant or plant part, wherein: an expression level of said at least one GIF1 gene or homolog thereof is increased by said mutation; and/or level or activity of a GIF1 protein encoded by said at least one GIF1 gene or homolog thereof is increased by said mutation.

29. The method of claim 28, comprising introducing the mutation to locate at least partially in a promoter region or 5’ untranslated region (5’UTR) in the regulatory region of said at least one native GIF1 gene or homolog thereof.

30. The method of claim 28, 29, or 41, comprising introducing the mutation to locate at least partially in a G-box region in the regulatory region of said at least one GIF1 gene or homolog thereof.

31. The method of claim 29 or 30, wherein the mutation decreases binding of a GIF1 repressor complex to the regulatory region of said at least one GIF1 gene or homolog thereof, thereby increasing level or activity of a GIF1 protein encoded by said at least one GIF1 gene or homolog thereof.

32. The method of any one of 28-31, wherein said at least one GIF1 gene or homolog thereof, before the mutation is introduced:

(i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 9-12, wherein said nucleic acid sequence encodes a polypeptide that retains GIF1 activity;

(ii) comprises the nucleic acid sequence of any one of SEQ ID NOs: 9-12;

(iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 5-8, wherein said polypeptide retains GIF1 activity;

(iv) encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 5-8;

(v) includes said regulatory region thereof, and comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-4, wherein said nucleic acid sequence encodes a polypeptide that retains GIF 1 activity; and/or

(vi) includes said regulatory region thereof, and comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-4.

33. The method of any one of 28-32, wherein the mutation is introduced at least partially into a promoter region or 5’ untranslated region (5’UTR) of a Glycine max GIF1 gene.

34. The method of claim 33, wherein the Glycine max GIF1 gene is selected from the group consisting of Glyma.03G249000, Glyma.19G246600, Glyma.10G 164100, Glyma.20G226500,

Glyma.18G121100, Glyma.14G 122500, and Glyma.OlGl 13500.

35. The method of claim 33 or 34, wherein the mutation is introduced at least partially into a G- box region for the Glycine max GIF1 gene.

36. The method of claim 35, wherein the mutation is introduced at least partially into a nucleic acid sequence of any one of SEQ ID NOs: 17-19 in the G-box region of the Glycine max GIF1 gene.

37. The method of claim 35 or 36, wherein the mutation comprises a substitution of about 1-10 nucleotides or a deletion of about 4-12 nucleotides introduced at least partially into the G-box region for the Glycine max GIF1 gene.

38. The method of claim 37, comprising:

(i) introducing a mutation into a G-box region of Glyma.03G249000, thereby producing a mutated G-box region comprising a nucleic acid sequence of any one of SEQ ID NOs: 20-24; and/or

(ii) introducing a mutation into a G-box region of Glyma.l9G246600, thereby producing a mutated G-box region comprising a nucleic acid sequence of SEQ ID NO: 25.

39. The method of any one of 26-38, further comprising introducing a genetic mutation that decreases BIG SEEDS (BS) activity into the plant or plant part.

40. The method of claim 39, wherein the mutation that decreases the BIG SEEDS activity comprises one or more insertions, substitutions, or deletions in at least one BS gene or homolog thereof or regulatory region thereof, wherein: an expression level of said at least one BS gene or homolog thereof is increased compared to an expression level a corresponding BS gene or homolog thereof without said mutation; and/or level or activity of a BIG SEEDS protein encoded by said at least one BS gene or homolog thereof is increased compared to level or activity of a BIG SEEDS protein encoded by a corresponding BS gene or homolog thereof without said mutation.

41. The method of claim 40, wherein said at least one BS gene or homolog thereof, before the mutation is introduced:

(i) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 26, 27, or 49, wherein said nucleic acid sequence encodes a polypeptide that retains BIG SEEDS activity;

(ii) comprises the nucleic acid sequence of SEQ ID NO: 26, 27, or 49;

(iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 28, 29, or 41, wherein said polypeptide retains BIG SEEDS activity; and/or

(iv) encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, 29, or 41.

42. The method of claim 40 or 41, wherein said mutation that decreases the BIG SEEDS activity is located at least partially in a nucleic acid region of exon 1 or exon 2 of a Glycine max BS1 gene and/or exon 2 of a Glycine max BS2 gene.

43. The method of claim 42, comprising:

(i) introducing a mutation into a Glycine max BS1 gene, thereby producing a mutated Glycine max BS1 gene comprising a deletion of nucleotides 98 through 101 of SEQ ID NO: 26;

(ii) introducing a mutation into Glycine max BS1 gene, thereby producing a mutated Glycine max BS1 gene comprising a deletion of nucleotides 389 through 396 of SEQ ID NO: 26; and/or (iii) introducing a mutation into Glycine max BS2 gene, thereby producing a mutated Glycine max BS2 gene comprising a deletion of nucleotides 409 through 415 of SEQ ID NO: 27.

44. The method of any one of claims 26-43, comprising introducing editing reagents or a nucleic acid construct encoding said editing reagents into said plant, plant part, or plant cell.

45. The method of claim 44, wherein said editing reagents comprise at least one nuclease, wherein the nuclease cleaves a target site in a genome of said plant, plant part, or plant cell, and said mutation is introduced at said cleaved target site.

46. The method of claim 45, wherein the at least one nuclease comprises a CRISPR nuclease.

47. The method of claim 46, wherein the CRISPR nuclease is a Type II CRISPR system nuclease, a Type V CRISPR system nuclease, a Cas9 nuclease, a Casl2a (Cpfl) nuclease, a Cmsl nuclease, or ortholog of any thereof.

48. The method of any one of claims 44-47, wherein the editing reagents comprise one or more guide RNAs (gRNAs).

49. The method of claim 48, wherein the one or more guide RNAs comprise a nucleic acid sequence complementary to a region of a GIF1 gene or homolog thereof or regulatory region thereof in the plant or plant part.

50. The method of claim 48 or 49, wherein at least one of the one or more guide RNAs binds a promoter region or a 5’ untranslated region (5’UTR) of a Glycine max GIF1 gene in said plant or plant part.

51. The method of claim 50, wherein at least one of the one or more guide RNAs binds G-box region of the Glycine max GIF1 gene at or adjacent to a nucleic acid sequence of any one of SEQ ID NO: 17-19.

52. The method of any one of claims 48-51, wherein the editing reagents comprise two or more gRNAs.

53. The method of any one of claims 25-52, wherein said plant or plant part is a legume.

54. The method of claim 53, wherein said plant or plant part is selected from the group consisting of: soybean (Glycine max)' , beans (Phaseolus spp.), common bean (Phaseolus vulgaris), fava bean (Vida faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentils (Lens culinaris, Lens esculenta), lupins (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.).

55. The method of any one of claims 25-52, wherein said plant or plant part is selected from the group consisting of: com (Zea mays), Brassica species, Brassica napus, Brassica rapa, Brassica juncea, rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet, pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp ), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp ), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp ), avocado (Per sea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integri folia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

56. A plant or plant part produced by the method of any one of claims 25-55, wherein said plant or plant part comprises increased GIF 1 activity compared to a control plant or plant part.

57. The plant or plant part of claim 56, comprising increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control plant or plant part.

58. The plant or plant part of claim 56 or 57, wherein said plant or plant part is a seed.

59. A population of plants or plant parts produced by the method of any one of claims 25-55, wherein the population comprises increased GIF1 activity, increased seed size, increased biomass, increased yield, and/or increased protein content compared to a control population.

60. The population of plants or plant parts of claim 59, wherein said population is a population of seeds.

61. A seed composition produced from the plant, plant part, or population of plants or plant parts of any one of claims 1-24 and 56-60.

62. A protein and/or oil composition produced from the plant, plant part, or population of plants or plant parts of any one of claims 1-24 and 56-60, or the seed composition of claim 61.

63. A food or beverage product comprising the plant, plant part, or population of plants or plant parts of any one of claims 1-24 and 56-60, or the composition of claim 61 or 62.

64. A nucleic acid molecule comprising a nucleic acid sequence of a mutated GIF1 regulatory region comprising a GIF1 promoter and/or a 5’ untranslated region (5’UTR), wherein said nucleic acid sequence:

(i) has at least 80% identity to a nucleic acid sequence of any one of SEQ ID NOs: 20-25; or (ii) comprises the nucleic acid sequence of any one of SEQ ID NOs: 20-25, wherein binding of a

GIF 1 repressor complex to the mutated GIF1 regulatory region is decreased as compared to a corresponding GIF1 regulatory region without said mutation.

65. A DNA construct comprising, in operable linkage:

(i) the nucleic acid molecule of claim 64; and (ii) a polynucleotide of interest.

66. A DNA construct of claim 65, further comprising, in operable linkage:

(i) a nucleic acid sequence comprising a mutated Glycine max BS1 gene comprising a deletion of nucleotides 98 through 101 of SEQ ID NO: 26, a mutated Glycine max BS1 gene comprising a deletion of nucleotides 389 through 396 of SEQ ID NO: 26, or a mutated Glycine max BS2 gene comprising a deletion of nucleotides 409 through 415 of SEQ ID NO: 27; and

(ii) a promoter functional in a plant cell.

67. A cell comprising the nucleic acid molecule of claim 64, or the DNA construct of claim 65 or 66.

68. The cell of claim 67, wherein the cell is a plant cell or a bacterial cell.