WO2024023764A1

WO2024023764A1 - Increasing gene expression for increased protein content in plants

Info

Publication number: WO2024023764A1
Application number: PCT/IB2023/057646
Authority: WO
Inventors: Matthew Brett Begemann; Emma Elizabeth JANUARY; Erin ZESS
Original assignee: Benson Hill, Inc.
Priority date: 2022-07-27
Filing date: 2023-07-27
Publication date: 2024-02-01

Abstract

Provided herein are plants and plant parts comprising increased activity of a protein-related polypeptide and compositions and methods of producing such plants and plant parts. The protein-related polypeptide can be xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane-localized polyol/cyclitol/monosaccharide-H+-symporter (PMT), or PMT5. The plants and 5 plant parts can have a genetic mutation or transgene that increases the protein-related polypeptide activity, and can have increased protein content. The mutation can be located at least partially in a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or its homolog or in its regulatory region, e.g., promoter region. Also provided are a population of plants or plant parts comprising increased protein-related polypeptide activity and high protein content, and plant products (e.g., seed composition, protein 10 composition) produced from the plants or plant parts provided herein.

Description

INCREASING GENE EXPRESSION FOR INCREASED PROTEIN CONTENT IN PLANTS

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 protein content, and associated methods and compositions.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/392,718 filed on July 27, 2022, 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 July 26, 2023, is named “B88552_1550_SL.xml” and its size is 69,210 bytes. The entire contents of the Sequence Listing file are incorporated by reference herein.

BACKGROUND OF THE INVENTION

With the ever-increasing world population and the dwindling supply of arable land available for agriculture, nutrient rich, resilient plants are desired. High protein content is an exemplary desirable trait for plants and seeds. As the majority of the human population and livestock relies on a plant-based diet for their protein uptake, generating plants with increased protein content can help efficiently feed the global population. Further, different protein compositions (e.g., protein concentrates, protein extracts, protein isolates) are processed from plants and seeds for use in various industrial purposes. For instance, soy protein is valued for its high nutritional quality for humans and livestock, as well as for its functional properties, such as gel and foam formation. 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, and industrial materials. However, high protein content is often associated with negative effects on plant growth or yield. Accordingly, providing plants and seeds that possess high protein content, without negatively affecting plant growth or yield could offer important commercial advantages.

SUMMARY OF THE INVENTION

Plants and plant parts comprising increased protein-related polypeptide activity are provided. The protein-related polypeptide can be xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane-localized polyol/cyclitol/monosaccharide-H⁺-symporter (PMT), or PMT5. 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 genetic mutation that increases activity of the protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), e.g., one or more mutations in at least one protein-related gene (s. .,XTH16, XTH16-A, bF4, PMT, PMT5) or homolog or in its regulatory region (e.g., promoter, 5’UTR), increased expression levels of the protein-related gene, increased levels or activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), and/or increased protein content compared to a control plant or plant part.

In one aspect, the present disclosure provides a plant or plant part comprising increased activity of a protein-related polypeptide compared to a control plant or plant part, wherein said plant or plant part comprises a genetic mutation and/or transgene that increases the activity of said protein-related polypeptide, and wherein said protein-related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane-localized polyol/cyclitol/monosaccharide-H⁺-symporter (PMT), and PMT5. In some embodiments, said protein- related polypeptide is XTH16. In some embodiments, the plant or plant part comprises 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 protein-related XTH16, XTH16-A, bF4, PMT, or PMT 5 gene or homolog thereof, or in a regulatory region thereof in said plant or plant part, wherein said at least one protein-related gene or homolog encodes said protein-related polypeptide, and wherein an expression level of said at least one protein-related gene or homolog thereof is increased compared to an expression level of the gene or homolog thereof in a plant or plant part without said mutation. In some embodiments, the mutation comprises one or more insertions, substitutions, or deletions in at least one protein-related XTH16, XTH16- A, bF4, PMT, or PMT5 gene or homolog thereof or in a regulatory region thereof in said plant or plant part, wherein said at least one protein-related gene or homolog encodes said protein-related polypeptide, and wherein said mutation increases level of activity of said protein-related polypeptide compared to level or activity of a copy of said protein-related polypeptide in a plant or plant part without said mutation. In some embodiments, the mutation is located at least partially in the regulatory region of said at least one protein- related gene or homolog thereof, wherein said at least one protein-related gene comprises XTH16, XTH16-A, bF4, PMT, or PMT5. In some embodiments, the mutation is located at least partially in a promoter or 5 ’ untranslated region (5’UTR) of said at least one copy o XTH16, XTH16-A, BF4, PMT, or PMT 5. In some embodiments, at least one protein-related gene is XTH16.

In some embodiments, the mutation is located in a.XTH 16. XTH 16-A. bF4, PMT, or PMT5 gene or homolog thereof: (i) comprising a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) comprising a nucleic acid sequence of any one of SEQ ID NOs: 7-9; (iii) encoding 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: 4-6, wherein said polypeptide retains protein-related activity; (iv) encoding a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4-6; (v) said protein-related gene including said regulatory region thereof comprising a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-3, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; and/or (vi) said protein-related gene including said regulatory region thereof comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

In some embodiments, said protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) said protein-related gene comprises the nucleic acid sequence of SEQ ID NO: 7; (iii) said protein-related gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 4, wherein said polypeptide retains protein-related activity; (iv) said protein-related gene encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 4; (v) said protein-related gene including said regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; and/or (vi) said protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of SEQ ID NO: 1.

In some embodiments, at least one of said one or more mutations is located at least partially in the nucleic acid sequence of a Glycine max XTH16, XTH16-A, bF4, PMT, or PMT5 gene. In some embodiments, at least one of said one or more mutations is located at least partially in the promoter region of a Glycine maxXTH16, XTH16-A, bF4, PMT, or PMT5.

In some embodiments, the plant or plant part comprises a deletion of about 2-12 nucleotides at least partially in the promoter region of the Glycine max XTH16. XTH16-A, bF4, PMT, or PMT 5 gene. In some embodiments, the plant or plant part comprises: (i) a nucleic acid sequence of SEQ ID NO: 16, or a deletion of nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; (ii) a nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; (iii) a nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; (iv) a first allele comprising the nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, and a second allele comprising the nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter; (v) a nucleic acid sequence of SEQ ID NO: 19, or a deletion of nucleotides 2140 through 2141 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter; (vi) a nucleic acid sequence of SEQ ID NO: 20, or a deletion of nucleotides 2138 through 2144 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter; (vii) a nucleic acid sequence of SEQ ID NO: 21, or a deletion of nucleotides 1371 through 1374 of SEQ ID NO: 3 or 12 in the Glycine maxPMT5 promoter; (viii) a nucleic acid sequence of SEQ ID NO: 22, or a deletion of nucleotides 1367 through 1377 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter; or (ix) a nucleic acid sequence of SEQ ID NO: 23, or a deletion of nucleotides 1371 through 1378 of SEQ ID NO: 3 or 12 in the Glycine max PMT 5 promoter. In specific embodiments, (i) a nucleic acid sequence of SEQ ID NO: 16, or a deletion of nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; (ii) a nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; and/or (iii) a nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter.

In some embodiments, said mutation comprises an out-of-frame mutation of the at least one protein- related XTH16. XTH16-A, bF4, PMT, or PMT5 gene or homolog. In some embodiments, said mutation comprises a nonsense mutation of the at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof.

In some embodiments, said at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof is an endogenous copy of said gene. In some embodiments, said at least one protein- related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof is an exogenous copy of said gene.

In some embodiments, the plant or plant part comprises the transgene, said transgene comprising a nucleic acid sequence of at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT 5 gene or homolog thereof operably linked to a promoter, wherein said at least one protein-related gene or homolog thereof encodes said protein-related polypeptide, and wherein an expression level of said at least one protein-related gene or homolog thereof is increased compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said transgene, or level or activity of said protein-related polypeptide in a plant or plant part without said transgene. In specific embodiments, said protein-related polypeptide is XTH16, and said promoter is a native XTH16 promoter. In other specific embodiments, said protein-related polypeptide is XTH16, and said promoter is a heterologous promoter.

In some embodiments, said plant or plant part comprises 2-5 genes encoding a XTH16, XTH16-A, BF4, PMT, or PMT5 polypeptide. In some embodiments, said 2-5 genes have less than 100% sequence identity to one another.

In some embodiments, said plant or plant part is a legume. In some embodiments, said plant or plant part is selected from soybean (Glycine max)' , beans (Phaseolus spp., Vigna spp.), common bean (Phaseolus vulgaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), adzuki bean (Vigna angularis), fava bean (Vida faba), 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.). For example, a plant or plant part of the presence disclosure can be Glycine max or a part of Glycine max.

In some embodiments, said plant or plant part is 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 tin orius), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), 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 (Persea 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.

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 activity of said protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, or PMT5) and/or increased protein content compared to a control population. In some embodiments, said population is a population of seeds. In some embodiments, said plant or plant part is a seed.

In one aspect, the present disclosure provides a method for increasing protein content in a plant or plant part, said method comprising increasing level or activity of at least one endogenous gene encoding a protein-related polypeptide in said plant or plant part, wherein said protein-related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane-localized polyol/cyclitol/monosaccharide-H⁺-symporter (PMT), and PMT5.

In one aspect, the present disclosure provides a method for increasing protein content in a plant or plant part, said method comprising introducing a genetic mutation and/or a transgene that increases activity of a protein-related polypeptide into said plant or plant part, wherein said protein-related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane-localized polyol/cyclitol/monosaccharide-H⁺-symporter (PMT), and PMT5. In some aspects, said protein-related polypeptide is XTH16. In some embodiments, the method further comprises introducing the genetic mutation and/or transgene that increases activity of said protein- related polypeptide into a plant cell, and regenerating said plant or plant part from said plant cell. In some embodiments, the method comprises introducing the genetic mutation into at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof or in a regulatory region thereof in said plant or plant part, wherein the mutation comprises one or more insertions, substitutions, or deletions, wherein said at least one protein-related gene or homolog thereof encodes said protein-related polypeptide, and wherein an expression level of said at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof is increased compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said mutation; and/or level or activity of said protein-related polypeptide is increased compared to level or activity of said protein-related polypeptide in a plant or plant part without said mutation.

In some embodiments according to the methods provided herein, the mutation is introduced to locate at least partially in the regulatory region of said at least one protein-related gene or homolog thereof, wherein said at least one protein-related gene comprises XTH16, XTH16-A, bF4, PMT, or PMT5 gene. In some embodiments according to the methods provided herein, the mutation is introduced to locate at least partially in a promoter region or 5’ untranslated region (5’UTR) of said 77//6. XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof. In some embodiments, said at least one protein-related gene is at least one copy o XTH16.

In some embodiments according to the methods provided herein, the mutation is introduced at least partially into a protein-related gene (e.g., XTH 16, XTH16-A, bF4, PMT, or PMT 5) or regulatory region thereof: (i) comprising a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) comprising a nucleic acid sequence of any one of SEQ ID NOs: 7-9; (iii) encoding 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: 4-6, wherein said polypeptide retains protein-related activity; (iv) encoding a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4-6; (v) said protein-related gene including said regulatory region thereof comprising a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 1-3, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; and/or (vi) said protein-related gene including said regulatory region thereof comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

In some embodiments, said protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) said protein-related gene comprises the nucleic acid sequence of SEQ ID NO: 7; (iii) said protein-related gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 4, wherein said polypeptide retains protein-related activity; (iv) said protein-related gene encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 4; (v) said protein-related gene including said regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1, wherein said nucleic acid sequence encodes a polypeptide that retains protein- related activity; and/or (vi) said protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of SEQ ID NO: 1 .

In some embodiments of the methods provided herein, the mutation is introduced at least partially in the nucleic acid sequence of a Glycine max XTH 16, XTH 16-A, bF4, PMT, or PMT 5 gene. In some embodiments of the methods provided herein, the mutation is introduced at least partially in the promoter region of a Glycine max XTH16, XTH16-A, bF4, PMT, or PMT5 gene. In some embodiments of the methods provided herein, the mutation comprises a deletion of 2-12 nucleotides at least partially in the promoter region of a Glycine max XTH 16, XTH 16-A, bF4, PMT, or PMT5 gene.

In some embodiments of the methods provided herein, the mutation comprises a deletion of one or more nucleotides in the promoter region wherein: (i) the mutation comprises a deletion of nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 16 when said mutation is introduced; (ii) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine maxXTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 17 when said mutation is introduced; (iii) the mutation comprises a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine maxXTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced; (iv) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter in a first allele and a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine maxXTH16 promoter in a second allele, or said plant or plant part comprises the first allele comprising the nucleic acid sequence of SEQ ID NO: 17 and the second allele comprising the nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced; (v) the mutation comprises a deletion of nucleotides 2140 through 2141 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 19 when said mutation is introduced; (vi) the mutation comprises a deletion of nucleotides 2138 through 2144 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 20 when said mutation is introduced; (vii) the mutation comprises a deletion of nucleotides 1371 through 1374 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 21 when said mutation is introduced; (viii) the mutation comprises a deletion of nucleotides 1367 through 1377 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 22 when said mutation is introduced; or (ix) the mutation comprises a deletion of nucleotides 1371 through 1378 of SEQ ID NO: 3 or 12 in the Glycine maxPMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 23 when said mutation is introduced.

In some embodiments, the mutation comprises a deletion of one or more nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 16 when said mutation is introduced; (ii) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 17 when said mutation is introduced; and/or (iii) the mutation comprises a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced.

In some embodiments, introducing the mutation comprises introducing an out-of-frame mutation into said at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof.

In some embodiments, the method further 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 side in said at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof, or a regulatory region thereof in 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, or a Cmsl nuclease, or an ortholog of any thereof. In some embodiments, the CRISPR nuclease is a Casl2a nuclease or an ortholog thereof.

In some embodiments, the editing reagents comprise one or more guide RNAs (gRNAs). In some embodiments, the one or more gRNAs comprise a nucleic acid sequence complementary to a region of a genomic DNA sequence encoding said protein-related polypeptide or regulating transcription or translation of said protein-related polypeptide, in said plant or plant part, wherein said protein-related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (bF4), plasma membrane -localized polyol/cyclitol/monosaccharide-EF-symporter (PMT), and PMT5. In some embodiments, at least one of the one or more gRNAs binds a nucleic acid region corresponding to a promoter region of a Glycine max XTH16. XTH16-A, bF4, PMT, or PMT 5 gene in said plant or plant part. In some embodiments, at least one of the one or more gRNAs comprises a nucleic acid sequence encoded by: (i) a nucleic acid sequence that shares at least 80% sequence identity with a nucleic acid sequence of any one of SEQ ID NOs: 10-15; or (ii) the nucleic acid sequence of any one of SEQ ID NOs: 10-15. In some embodiments, at least one of the one or more gRNAs comprises a nucleic acid sequence encoded by: (i) a nucleic acid sequence that shares at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 13; or (ii) the nucleic acid sequence of SEQ ID NO: 13.

In some embodiments of the methods provided herein, the at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof is an endogenous copy of said gene. In some embodiments of the methods provided herein, the at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof is an exogenous copy of said gene.

In some embodiments, the method further comprises introducing the transgene into said plant, plant part, or plant cell, wherein the transgene comprises a nucleic acid sequence of at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof operably linked to a promoter, wherein said at least one protein-related gene or homolog thereof encodes said protein-related polypeptide, and wherein the method increases: an expression level of said at least one protein-related XTH16, XTH16-A, bF4, PMT, or PMT5 gene or homolog thereof compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said transgene; or level or activity of said protein-related polypeptide compared to level of activity of said protein-related polypeptide in a plant or plant part without said transgene. In some embodiments, said protein-related polypeptide is XTH16, and said promoter is a native XTH16 promoter. In some embodiments, said protein-related polypeptide is XTH16, and said promoter is a heterologous promoter.

In some embodiments of the methods provided herein, said plant or plant part is a legume. In some embodiments, said plant or plant part is selected from soybean (Glycine max), beans (Phaseolus spp., Vigna spp.), common bean (Phaseolus vulgaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), adzuki bean (Vigna angularis), fava bean (Vida faba), 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 of the methods provided herein, said plant or plant part is 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 methods provided herein, wherein said plant or plant part comprises increased activity of said protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, or PMT5) compared to a control plant or plant part. In some embodiments, the plant or plant part comprises increased protein content compared to a control plant or plant part. In some embodiments, said plant or plant part is a seed.

In some embodiments, the present disclosure provides a population of plants or plant parts produced by the methods provided herein, wherein the population comprises increased activity of said protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, or PMT5), 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 or plant part, or a population of plants or plant parts provided herein.

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

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

In one aspect, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence of a mutated protein-related gene or coding sequence thereof, wherein said nucleic acid sequence comprises any one of altered SEQ ID NOs: 1-3 or 7-9 comprising one or more insertions, substitutions, or deletions therein. In some embodiments, the present disclosure provides a DNA construct comprising, in operable linkage: (i) a promoter that is functional in a plant cell; and (ii) the nucleic acid molecule comprising a nucleic acid sequence of a mutated protein-related gene or coding sequence thereof, wherein said nucleic acid sequence comprises any one of altered SEQ ID NOs: 1-3 or 7-9 comprising one or more insertions, substitutions, or deletions therein. In one aspect, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence of a mutated promoter of a protein-related gene (e.g., XTH 16, XTH16-A, bF4, PMT, or PMT5), wherein said nucleic acid sequence comprises: (i) a nucleic acid sequence of any one of SEQ ID NOs: 10-12 comprising one or more insertions, substitutions, or deletions therein; or (ii) a nucleic acid sequence of any one of SEQ ID NOs: 16-23. In some embodiments, the present disclosure provides a DNA construct comprising, in operable linkage: (i) the nucleic acid molecule comprising a nucleic acid sequence of a mutated promoter of a protein-related gene (e.g., XTH 16, XTH16-A, bF4, PMT, or PMT5), wherein said nucleic acid sequence comprises a nucleic acid sequence of any one of SEQ ID NOs: 10-12 comprising one or more insertions, substitutions, or deletions therein, or a nucleic acid sequence of any one of SEQ ID NOs: 16-23; and (ii) a polynucleotide of interest.

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

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts XTH16 expression levels in the R3 leaves in Plants A-C, wildtype (WT), and null (being introduced the gene editing reagents but resulted in no mutation) soybean plants according to the embodiments of the present disclosure. Plant A contains a 6 bp deletion in the promoter region of GmXTH16, resulting in the sequence of SEQ ID NO: 18. Plant B contains a 12 bp deletion in the promoter region of GmXTH16, resulting in the sequence of SEQ ID NO: 17. Plant C contains a 10 bp deletion in the promoter region of GmXTH16, resulting in the sequence of SEQ ID NO: 16. * indicates p < 0.05.

FIG. 2 depicts XTH 16 expression levels in the pod walls in Plants A-C, WT, and null soybean plants described above. ** indicates p < 0.01. *** indicates p < 0.001.

FIG. 3 depicts seed protein content of Plants A-C, WT, and null soybean plants described above, as measured by near infrared spectrometry (NIR).

FIG. 4 depicts seed protein content of five transgenic plants overexpressing GmXTH16, WT, and null soybean plants, as measured by near infrared spectrometry (NIR). Plants GM:C2 and GM:C3 express GmXTH16 under a CHS7 promoter.

Plants GM:N1, GM:N2, and GM:N3 express GmXTH16 under the native GmXTHl 6 promoter.

FIG. 5 depicts correlation between the GmXTHl 6 expression levels in V3 leaves and seed protein content of GmXTH 1 <5-overexpressing plants generated either by gene editing (gene edited plants) or gene modification (transgenic plants).

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 protein-related gene or a regulatory region of a protein-related 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 “a protein-related gene” includes the coding region of the protein-related gene, and may also include the regulatory region (e.g., promoter, 5’UTR) of the protein-related gene. Further, a “a protein-related gene” as used herein includes a homolog of a known a protein-related 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, plant part, 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 protein-related gene or a regulatory region of a protein-related 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 a protein-related gene or a regulatory region of the protein-related gene has been deliberately mutated such that the plant, plant part or plant cell expresses a mutated (e.g., truncated) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) or have an increased expression level of the protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or protein-related polypeptide. The mutated protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can have altered function, e.g., increased function, compared to a corresponding wildtype, or control, protein-related polypeptide 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(l-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., estimating quantitative trait locus (QTL) effects. Most commonly, the terms relate to 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 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. Modifying the native sequence of a protein-related gene or its regulatory region (e.g., promoter, 5’UTR) to enhance level or activity of protein-related polypeptide can be one approach to generate advantageous traits, such as increased protein content. For example, introducing mutation to a transcriptional regulator (e.g., transcriptional repressor) binding domain in the regulatory region (e.g., promoter, 5’UTR) of a protein- related gene can alter (e.g., decrease) the affinity of transcriptional regulator (e.g., transcriptional repressor) binding, thereby altering (e.g., increasing) level or activity of the protein-related gene. Provided herein are exemplary protein-related polypeptides, e.g., xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane-localized polyol/cyclitol/monosaccharide-H⁺-symporter (PMT), and PMT5, and genes encoding such protein-related polypeptides, e.g., XTH 16, XTH 16-A, bF4, PMT, and PMT5.

Disclosed herein are plants or plant parts comprising a genetic mutation that increases activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) compared to a control plant or plant part, as well as methods for making the plants or plant parts with increased protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity. Such plants or plant parts can have one or more insertions, substitutions, or deletions in at least one native (e.g., wild-type) protein-related gene (e.g., XTH 16, XTH 16-A, bF4, PMT, PMT5) or homolog thereof or in its regulatory region. The plants or plant parts can have an increased expression level of the protein-related gene or homolog thereof, increased level or activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the protein-related gene (s. .,XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof, altered (e.g., increased) expression or activity of the protein-related gene’s downstream target molecules that regulate protein content, 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 protein content by introducing a genetic mutation that increases protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity. The methods disclosed herein can include introducing one or more insertions, substitutions, or deletions in at least one a protein-related gene (e .g., XTH 16, XTH 16-A, bF4, PMT, PMT5) 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 protein-related gene (c. g., XTH 16, XTH 16-A, bF4, PMT, PMT5) or homolog thereof is increased, level or activity ofa protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof is increased, 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 protein- related 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 a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) 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.

Also disclosed herein are a population of plants or plant parts (e.g., seeds) having increased activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) and/or an 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.

Further provided herein are nucleic acid molecules comprising a mutated protein-related gene or its regulatory region (e.g., mutated promoter or 5' UTR), a DNA construct comprising (i) the mutated protein- related gene operably linked to a functional promoter or (ii) the mutated regulatory region of the protein- related gene operably linked to a polynucleotide of interest, and cells comprising the nucleic acid molecule or the DNA construct of the present disclosure.

III. Plants with Increased Protein Content

Plants and plant parts are provided herein having altered (e.g., increased) protein-related polypeptide level or activity as compared to a control plant or plant part. As used herein, a “protein-related polypeptide” refers to a polypeptide that has activity to directly or indirectly regulate (e.g., increase) protein level or content in plants or plant parts (e.g., seeds). In some embodiments, a protein-related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane-localized polyol/cyclitol/monosaccharide-H⁺-symporter (PMT), and PMT5.

“Protein-related polypeptide activity” refers to the ability of a protein-related polypeptide to regulate protein content by, e.g., regulating downstream target genes. “Protein-related polypeptide activity” can also refer to the activity of the respective native (e.g., wild-type) protein-related polypeptide activity. For example, in some embodiments, the protein-related polypeptide is XTH16 or XTH16-A, and the protein- related polypeptide activity includes XTH16 or XTH16-A activity, e.g., hydrolase activity on xyloglucan, xyloglucan endotransglucosylase (XET) activity (i.e., activity to cut and re-join hemicellulose chains in the plant cell wall, contributing to wall assembly and growth regulation), or activity to stimulate growth of hypocotyls.

In some embodiments, the protein-related polypeptide is BF4, and the protein-related polypeptide activity includes BF4 activity, e.g., vacuolar invertase activity, or activity to regulate sugar metabolism.

In some embodiments, the protein-related polypeptide is PMT or PMT5, and the protein-related polypeptide activity includes PMT or PMT5 activity, e.g., activity to transport (symport) polyol, cyclitol, monosaccharide, and H⁺ across plasma membrane, activity to catalyze the energy-dependent membrane passage of linear polyols, cyclic polyols, and monosaccharides (e.g., pyranose ring-forming and furanose ring -forming hexoses and pentoses).

In particular aspects, plants and plant parts (e.g., seeds, leaves) disclosed herein have a genetic mutation that alters (e.g., increases) the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity. The plants or plant parts described herein having altered protein-related polypeptide level or activity can comprise a genetic mutation or transgene that alters (e.g., increases) protein-related polypeptide level or activity, altered (e.g., increased) expression levels of at least one a protein-related gene encoding protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), altered (e.g., increased) protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) levels or activity, and/or altered (e.g., increased) protein content 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) protein-related polypeptide level or activity. In such population of plants or plant parts, having altered protein-related polypeptide level or activity relative to a control population, not all individual plants or plant parts need to have altered (e.g., increased) protein- related polypeptide level or activity, genetic mutation that cause altered (e.g., increased) protein-related polypeptide level or activity, or phenotypes caused by the altered (e.g., increased) activity of the protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) (e.g., increased protein content, altered protein metabolism). 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 protein-related polypeptide 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., Vigna spp.), common bean (Phaseolus vulgaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), adzuki bean (Vigna angularis), fava bean (Vicia faba), 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.). 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 protein-related polypeptide

Provided herein are plants or plant parts (e.g., seeds) comprising altered (e.g., increased) protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity compared to a control plant or plant part. In specific embodiments, plants or plant parts provided herein comprise increased protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity compared to a control plant or plant part. Also provided herein is a population of plants or plant parts (e.g., seeds) comprising altered (e.g., increased) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity compared to a control population provided herein.

The plants and plant parts can have genetic mutation that alters (e.g., increases) the activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), which comprise one or more insertions, substitutions, or deletions in at least one native protein-related gene (e.g., XTH 16, XTH 16-A, bF4, PMT, PMT5) or homolog thereof, or in a regulatory region of at least one native a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof. The genetic mutation that alters (e.g., increases) the activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can be located in at least one native a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof; in a regulatory region (e.g., promoter, 5’UTR) of the native a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT 5) 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 protein-related “gene”, as used herein, refers to any polynucleotide that encodes a polypeptide having protein-related polypeptide activity. In some embodiments, a protein-related gene is XTH 16, XTH 16-A, bF4, PMT, or PMT5. A protein-related gene, as used herein, can refer to a polynucleotide including a regulatory region (e.g., promoter, 5’UTR) of the protein-related gene. A protein-related gene can also include a homolog, ortholog, or variant, that retains protein-related polypeptide activity (e.g., XTH16, XTH16-A, BF4, PMT, or PMT5 activity), of a known a protein-related 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 a protein-related gene located at a genomic site or in a plant in a non-naturally occurring matter is a “native” a protein-related 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. 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 protein-related 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., wildtype) a protein-related 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 a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof, altered (e.g., increased) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) level or activity, and/or altered (e.g., increased) protein content 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.

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity at least partially in a regulatory region of at least one (e.g., one, more than one but not all, or all) protein-related gene. In specific embodiments, the protein-related polypeptide is XTH16. The protein-related gene with mutation can be an endogenous copy of the gene, and/or an exogenous copy of the gene that was introduced into the plants or plant parts. 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) protein-related 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) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5). 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 protein-related 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 protein-related 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 protein-related 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) protein-related 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) protein-related 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) protein-related gene at or near one or more transcriptional regulator (e.g., transcriptional repressor) binding domains. Without wishing to be bound by theory, a mutation in the binding domains of transcriptional regulator (e.g., transcriptional repressor) in the regulatory region (e.g., promoter region or 5’UTR) of the protein-related gene can de-repress and increase the level or activity of the protein-related gene (c.g.. A77//6. XTH16-A, bF4, PMT, PMT5). In specific embodiments, the protein- related gene is XTH16.

In some embodiments, the mutation is located in the regulatory region of a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5), 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: 10-12, wherein the regulatory region retains transcription initiation activity; (ii) the regulatory region comprises a nucleic acid sequence of any one of SEQ ID NOs: 10-12; (iii) the protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (iv) the protein-related gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 7-9; (v) the protein-related 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: 4-6, wherein the polypeptide retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (vi) the protein-related gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4-6; (vii) the protein-related 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: 1-3, wherein the nucleic acid sequence encodes a polypeptide that retains protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; and/or (viii) the protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

In specific embodiments, the mutation is located in the regulatory region of XTH16, and (i) the protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) the protein-related gene comprises the nucleic acid sequence of SEQ ID NO: 7; (iii) the protein-related gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 4, wherein the polypeptide retains protein- related activity; (iv) the protein-related gene encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 4; (v) the protein-related gene including the regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related activity; and/or (vi) the protein- related gene including said regulatory region thereof comprises the nucleic acid sequence of SEQ ID NO: 1.

In some embodiments, the mutation is located at least partially in a promoter region or 5’UTR of a gene selected from the group consisting of Glycine maxXTH16-A, bF4, or PMT 5 gene. In some embodiments, the mutation is located at least partially in a promoter region (e.g., SEQ ID NO: 10) of a Glycine maxXTH16-A gene, a promoter region (e.g., SEQ ID NO: 11) of a Glycine max bF4 gene, or a promoter region (e.g., SEQ ID NO: 12) of a Glycine max PMT5 gene. In some embodiments, the plant or plant part of the present disclosure comprises a deletion of one or more nucleotides, e.g., about 2-12 or more nucleotides at least partially in the promoter and/or 5’UTR of a Glycine maxXTH16-A gene, a Glycine max bF4 gene, or a Glycine max PMT5 gene.

In some embodiments, the plant or plant part provided herein comprises: (i) a nucleic acid sequence of SEQ ID NO: 16, or a deletion of nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine maxXTH16 promoter; (ii) a nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; (iii) a nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; (iv) the first allele comprising the nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, and the second allele comprising the nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; (v) a nucleic acid sequence of SEQ ID NO: 19, or a deletion of nucleotides 2140 through 2141 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter; (vi) a nucleic acid sequence of SEQ ID NO: 20, or a deletion of nucleotides 2138 through 2144 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter; (vii) a nucleic acid sequence of SEQ ID NO: 21, or a deletion of nucleotides 1371 through 1374 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter; (viii) a nucleic acid sequence of SEQ ID NO: 22, or a deletion of nucleotides 1367 through 1377 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter; or (ix) a nucleic acid sequence of SEQ ID NO: 23, or a deletion of nucleotides 1371 through 1378 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter.

In specific embodiments, the plant or plant part comprises: (i) a nucleic acid sequence of SEQ ID NO: 16, or a deletion of nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine maxXTH16 promoter; (ii) a nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; and/or (iii) a nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine maxXTH16 promoter.

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 protein-related 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 protein-related gene, e.g., by increasing transcription initiation activity of the protein-related gene promoter. In some embodiments, the mutation modifies or inserts transcription factor binding sites or enhancer elements that regulates a protein-related gene expression into the regulatory region of the protein- related gene. In some embodiments, the mutation inserts a part or whole of one or more positive regulatory elements of the protein-related 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 protein-related 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 protein-related genes (e.g., XTH16, XTH16, bF4, PMT, PMT5) can also include upstream open reading frames (uORFs). In some instances, a positive regulatory element can be inserted in a region upstream of the protein-related 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 a protein-related gene, or other regulatory region of a protein-related 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 a protein-related gene, or its homolog, ortholog, or variant

In some aspects, the plants and plant parts of the present disclosure comprise increased protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity and a genetic mutation that increases the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 a protein-related gene (e.g., coding region, non-coding region, exons, introns, and/or regulatory region thereof) or homolog thereof. The protein-related gene with mutation can be an endogenous copy of the gene, and/or an exogenous copy of the gene that was introduced into the plants or plant parts. 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 a protein-related gene, e.g., XTH16, bF4, or PMT5 genes, each encoding a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). In particular, a plant or plant part described herein can comprise at least 2 genes encoding a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), 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 a protein-related gene or homolog; in a regulatory region of one a protein-related gene or homolog; in more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homologs; in regulatory regions of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homologs; in all protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homologs; and/or in regulatory regions of all protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homologs in the plant or plant part. In one embodiment, the coding region of the protein-related gene does not comprise a mutation (e.g., only the regulatory region of the protein-related gene comprises a mutation). In another embodiment, the coding region of the protein-related 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 protein-related 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 protein-related gene or its regulatory region, and

(i) the protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity;

(ii) the protein-related gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 7-9; (iii) the protein-related 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: 4-6, wherein the polypeptide retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (iv) the protein- related gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4-6; (v) the protein-related 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: 1-3, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; and/or (vi) the protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In specific embodiments, the mutation that increases the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity is located in one or two alleles of one or more (e.g., one, more than one but not all, or all) copies of Glycine max XTH16 gene, Glycine max bF4 gene, a Glycine max PMT5 gene, and/or a regulatory region thereof.

In some embodiments, the mutation is located at least partially in the coding region of Glycine max XTH16, bF4, or PMT5 gene.

The mutation that increases the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) 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) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof. A plant or plant part of the present disclosure can have a genetic mutation that increases the protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity in a gene that is a homolog, ortholog, or variant of a protein-related gene disclosed herein and expresses a functional protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), or in a regulatory region of such homolog, ortholog, or variant of a protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity.

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 a protein-related polypeptide can be identified and used in the methods of the present disclosure. The variant sequences will retain the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity.

In certain instances, mutations in any a protein-related 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 detection methods may comprise use of primers for detecting mutation in a protein-related gene. For example, a forward primer (e.g., SEQ ID NO: 24) and a reverse primer (e.g., SEQ ID NO: 25) can be used for detection of a mutation in the Glycine max XTH16 promoter near the binding site of the GmXTHl 6 guide RNA (e.g., SEQ ID NO: 13), e.g., a mutation generated by introducing GmXTHl 6 guide RNA (e.g., SEQ ID NO: 13) into the plant or plant part, such as a deletion mutation comprising a nucleic acid sequence of any one of SEQ ID NOs: 16-18. A forward primer (e.g., SEQ ID NO: 26) and a reverse primer (e.g., SEQ ID NO: 27) can be used for detection of a mutation in Glycine max bF4 regulatory region near the binding site of the GmbF4 guide RNA (e.g., SEQ ID NO: 14), for example a mutation generated by introducing the GmbF4 guide RNA (e.g., SEQ ID NO: 14) into the plant or plant part, such as a deletion mutation comprising a nucleic acid sequence of SEQ ID NO: 19 or 20. A forward primer (e.g., SEQ ID NO: 28) and a reverse primer (e.g., SEQ ID NO: 29) can be used for detection of a mutation in Glycine max PMT5 regulatory region near the binding site of the GmPMT5 guide RNA (e.g., SEQ ID NO: 15), for example a mutation generated by introducing the GmPMT5 guide RNA (e.g., SEQ ID NO: 15) into the plant or plant part, such as a deletion mutation comprising a nucleic acid sequence of any one of SEQ ID NOs: 21-23. In certain instances, a kit comprising a set of primers can be used for detecting mutation of protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) in plants, plant parts, or plant product (e.g., seed composition, plant protein composition). For example, a kit comprising a forward primer (e.g., SEQ ID NO: 24) and a reverse primer (e.g., SEQ ID NO: 25) can be used for detection of mutation in GmXTH16 in plants, plant parts, or plant products (e.g., seed composition, plant protein compositions) near the binding site of the GmXTHl 6 guide RNA (e.g., SEQ ID NO: 13). A kit comprising a forward primer (e.g., SEQ ID NO: 26) and a reverse primer (e.g., SEQ ID NO: 27) can be used for detection of mutation in GmbF4 in plants, plant parts, or plant products (e.g., seed composition, plant protein compositions) near the binding site of the GmbF4 guide RNA (e.g., SEQ ID NO: 14). A kit comprising a forward primer (e.g., SEQ ID NO: 28) and a reverse primer (e.g., SEQ ID NO: 29) can be used for detection of mutation in GmPMT5 in plants, plant parts, or plant products (e.g., seed composition, plant protein compositions) near the binding site of the GmPMT5 guide RNA (e.g., SEQ ID NO: 15).

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity described herein.

One or more insertions, substitutions, or deletions located in at least one a protein-related gene or homolog or in a regulatory region of the protein-related gene or homolog in the plant or plant part provided herein can increase the expression levels of the protein-related gene or homolog, increase level or activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the protein-related gene or homolog, increase protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, and/or increase protein content in the plant or plant part (e.g., seeds) 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. Transgenic plants overexpressins, protein-related polypeptide

The plants, plant parts (e.g., seeds, leaves), or plant products (e.g., seed composition, plant protein composition) of the present disclosure can comprise a transgene that increases the activity of said protein- related polypeptide, and wherein said protein-related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane- localized polyol/cyclitol/monosaccharide-H+-symporter (PMT), and PMT5. In specific embodiments, the protein-related polypeptide is XTH16. “Transgene” as used herein refers to an exogenous copy of a gene, e.g., a protein-related gene, e .g., XTH16, XTH16-A, bF4, PMT, or PMT5. One or more exogenous copies of the protein-related gene (e.g., XTH 16) can be native, i.e., without mutation. Alternatively, one or more exogenous copies of the protein-related gene can have a mutation that increases protein-related polypeptide level or activity. The plant or plant part can comprise a transgene comprising a nucleic acid sequence of at least one native protein-related gene or homolog thereof operably linked to a promoter, and have an increased expression level of said at least one protein-related gene or homolog thereof compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said transgene. The promoter can be the native promoter of the protein-related gene (e.g., the transgene comprises XTH16 operably linked to a native XTH16 promoter) or a heterologous promoter (e.g., the transgene comprises XTH16 operably linked to a heterologous promoter, e.g., CHS7 promoter). The transgene can be introduced into the plant or plant part by standard methods, including transformation, e.g., Agrobacterium transformation.

4. Plants with increased protein-related polypeptide 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 protein-related polypeptide 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity compared to a control (e.g., wild-type) population of plants or plant parts.

In particular, the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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.

Activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can be measured by measuring protein content in the plant or plant part (e.g., seeds) by standard methods for measuring protein in a plant sample, 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). Protein content can also be measured by the Dumas method, by combusting samples at a high temperature in the presence of high- purity oxygen, analyzing the gas from combustion for nitrogen content using a thermal conductivity detector, and calculating the amount of protein present in the sample using a conversion factor. The industry standard conversion factor for soybean is 6.25. Activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can also be measured by measuring activity of the respective protein-related polypeptide. For example, activity of XTH16 or XTH16-A can be measured by standard methods for measuring hydrolase activity on xyloglucan (e.g., enzymatic assay), xyloglucan endotransglucosylase (XET) activity (e.g., enzymatic assay), or activity to stimulate growth of hypocotyls (e.g., growth assay). Activity of BF4 can be measured by standard methods for measuring vacuolar invertase activity (e.g., enzymatic assay), or standard methods for measuring total or specific sugar content (e.g., total sugar, sucrose, glucose, fructose, galactose, maltose, lactose) in plant samples [e.g., spectroscopy (near infrared spectroscopy), refractometry, solid-phase extraction (SPE), solid-phase micro-extraction (SPME), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GCMS), and/or enzymatic assay] . Activity of PMT or PMT5 can be measured by standard methods for measuring activity to transport (symport) polyol, cyclitol, monosaccharide, and H+ across plasma membrane (e.g., enzymatic assay), or activity to catalyze the energy-dependent membrane passage of linear polyols, cyclic polyols, and monosaccharides (e.g., enzymatic assay).

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 protein-related gene(s) or homolog as compared to the expression level of the protein-related 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 a protein-related gene(s) or protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) compared to a control (e.g., wild-type) population of plants or plant parts.

In some embodiments, the expression levels of the endogenous protein-related gene(s) or homolog are increased by, e.g., genetic mutation or other mechanisms to up-regulate the expression of the endogenous a protein-related 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 a protein-related gene or homolog or in a regulatory region thereof can have increased total expression levels of the protein- related 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) of at least one endogenous protein-related gene, e.g., at or near transcriptional repressor binding sites, that increases expression of the protein-related gene.

Alternatively or additionally, the expression levels of the protein-related gene(s) or homolog are increased by introduction of one or more exogenous copies of a protein-related gene (e.g., transgene) into the plant or plant part. One or more exogenous copies of the protein-related gene can be native, i.e., without mutation. Alternatively, one or more exogenous copies of the protein-related gene can have a mutation that increases protein-related polypeptide level or activity. The plant or plant part can comprise a transgene comprising a nucleic acid sequence of at least one native protein-related gene or homolog thereof operably linked to a promoter, and have an increased expression level of said at least one protein-related gene or homolog thereof compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said transgene.

In particular, the expression levels of a protein-related 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 a protein-related gene or homolog that contributes to an increased expression (e.g., up-regulation, overexpression) of the protein-related gene or homolog is an endogenous or exogenous copy of a Glycine max XTH16-A gene, a Glycine max bF4 gene, and/or a Glycine max PMT5 gene. Expression levels of the protein-related 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 protein-related 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, or dot blot analysis 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the protein-related gene.

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), as compared to the expression level of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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” protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), as used herein, refers to a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) comprising the complete amino acid sequence of a wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), e.g., encoded by a native a protein-related gene, and having the complete function of a wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). Additionally or alternatively, the expression levels of a functional fragment, variant, or ortholog of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the endogenous a protein-related gene(s) or homolog are increased by, e.g., genetic mutation or other mechanisms to up-regulate the expression of the endogenous protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). 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 protein-related gene or homolog (e.g., in the regulatory region, coding region, and/or noncoding region) can have increased expression level of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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) of at least one endogenous protein- related gene, e.g., at or near transcriptional repressor binding sites, e.g., a RAVI domain and/or a ERF domain, that increases expression of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). Alternatively or additionally, the levels of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can be increased by introduction of one or more exogenous copies of a protein-related gene (e.g., transgene) into the plant or plant part. The plant or plant part can comprise a transgene comprising a nucleic acid sequence of at least one native protein-related gene or homolog thereof operably linked to a promoter, and have an increased level or activity of the protein-related polypeptide compared to level of activity of said protein-related polypeptide in a plant or plant part without said transgene. One or more exogenous (e.g., transgenic) copies of the protein-related gene can be from the same, related, or different plant species. One or more exogenous copies of the protein-related gene can be native, i.e., without mutation; alternatively, one or more exogenous copies of the protein-related gene can have a mutation (e.g., in the regulatory region, coding region, and/or non-coding region) that increases protein-related polypeptide level or activity.

In the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure, expression of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), e.g., encoded by endogenous and/or exogenous copies of the protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) in a control plant, plant part, a population of plants or plant parts, or plant product. In certain embodiments, the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) is encoded by endogenous or exogenous copies of the Glycine max XTH 16 gene, Glycine max bF4 gene, and/or Glycine max PMT 5 gene. Expression of a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can be determined by western blot analysis, ELISA, or dot blot analysis 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5).

6. Plants with enhanced function of protein-related polypeptide

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), e.g., increased protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, as compared to the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) compared to a control (e.g., wild-type) 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 protein-related gene (e.g., XTH 16, XTH16-A, hF4, PMT, PMT5) or homolog thereof (e.g., in the regulatory, coding, and/or non-coding regions) that enhances function of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). Additionally or alternatively, the plant, plant part, population of plants or plant parts, or plant product can have an exogenous copy of a protein-related gene encoding protein-related polypeptide (e.g., XTH 16, XTH16-A, BF4, PMT, PMT5) 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 protein-related gene, or otherwise having wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity. The protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) with increased function can comprise a mutation compared to a wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) that causes enhanced protein-related polypeptide function. In some embodiments, the function or activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). In certain embodiments, the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) with enhanced function is encoded by an endogenous or exogenous copy of mutated Glycine maxXTH16 gene, Glycine max bF4 gene, and/or Glycine maxPMT5 gene. Function of a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) in a plant, plant part, a population of plants or plant parts, or plant product can be measured by measuring protein content in the plant or plant part (e.g., seeds) by standard methods for measuring protein in a plant sample, 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). Protein content can also be measured by the Dumas method, by combusting samples at a high temperature in the presence of high-purity oxygen, analyzing the gas from combustion for nitrogen content using a thermal conductivity detector, and calculating the amount of protein present in the sample using a conversion factor. The industry standard conversion factor for soybean is 6.25. Function of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can also be measured by measuring activity of the respective protein-related polypeptide. For example, function of XTH16 or XTH16-A can be measured by standard methods for measuring hydrolase activity on xyloglucan (e.g., enzymatic assay), xyloglucan endotransglucosylase (XET) activity (e.g., enzymatic assay), or activity to stimulate growth of hypocotyls (e.g., growth assay). Function of BF4 can be measured by standard methods for measuring vacuolar invertase activity (e.g., enzymatic assay), or standard methods for measuring total or specific sugar content (e.g., total sugar, sucrose, glucose, fructose, galactose, maltose, lactose) in plant samples [e.g., spectroscopy (near infrared spectroscopy), refractometry, solid-phase extraction (SPE), solid-phase micro-extraction (SPME), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GCMS), and/or enzymatic assay]. Function of PMT or PMT5 can be measured by standard methods for measuring activity to transport (symport) polyol, cyclitol, monosaccharide, and H+ across plasma membrane (e.g., enzymatic assay), or activity to catalyze the energy-dependent membrane passage of linear polyols, cyclic polyols, and monosaccharides (e.g., enzymatic assay). 7. Plants with 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, can have 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 protein as compared to a control population.

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, and may express an endogenous (e.g., wild-type) a protein-related gene. A plant, plant part, a population of plants or plant parts, or plant product of the present disclosure can have 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, total protein content 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 amino acid or protein content.

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 a native protein-related polypeptide (XTH16, bG4, PMT5), 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 Sci 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). Protein content can also be measured by the Dumas method, by combusting samples at a high temperature in the presence of high-purity oxygen, analyzing the gas from combustion for nitrogen content using a thermal conductivity detector, and calculating the amount of protein present in the sample using a conversion factor. The industry standard conversion factor for soybean is 6.25.

In specific embodiments, the plant, plant part, or a population of plants or plant parts of the present disclosure have the trait of increased protein content as compared to a control plant, plant part, population of plants or plant parts, or plant product, without a significant decrease in yield. In some embodiments, a reduction in yield in the plant, plant part, or population of plants or plant parts of the present disclosure, having increased protein content, is no more than about 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or about 5.0%, 6%, 7%, 8%, 9%, or 10%, e.g., no more than about 0-5%, 0.5-4.5%, 0.5-4%, 1-5%, 1- 4%, 2-5%, 2-4%, 0.5-10%, 0.5-8%, 1-10%, 2-10%, 3-10%, 4-10%, 5-10%, 6-10%, 7-10%, or 8-10% reduction in yield as compared to a control plant, plant part, or population of plants or plant parts. Yield can be measured and expressed by any means known in the art. In specific embodiments, yield is measured by seed weight or volume of seeds, fruits, leaves, or whole plants harvested from a given harvest area.

In specific embodiments, provided herein are seeds and a population of seeds with increased protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity provided herein, having an 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 composition derived from the plant or plant part, 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., flake, white flake, soy/pea protein composition, soy/pea protein concentrate (SPC/PPC), soy/pea protein isolate (SPI/PPI), soy/pea flour, texturized vegetable protein (TVP), or textured soy/pea protein (TSP/TPP)). Plant protein compositions of the present disclosure 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, anticaking 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/pea 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”, which includes “textured soy/pea protein” (TSP/TPP), soy/pea meat, or soya/pea 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 protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, e.g., one or more insertions, substitutions, or deletions in at least one native a protein-related gene or homolog (e.g., in a regulatory region, a coding region, and/or a non-coding region), or an exogenous copy of a protein-related gene.

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.

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, e.g., one or more insertions, substitutions, or deletions in at least one native a protein-related gene or homolog or in a regulatory region of such a protein-related gene or homolog, e.g., a deletion of about 2-12 or more nucleotides at least partially in the promoter or 5’UTR of a Glycine max XTH 16 gene, a Glycine max bF4 gene, or a Glycine max PMT 5 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 a protein-related gene. The mutation can be at least partially located in the regulatory region, coding region, or non-coding region of the exogenous copy of the protein-related gene. The plant parts, population of plant parts, and plant products of the present disclosure can have increased protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, increased expression level of the protein-related gene or homolog, increased expression level of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), increased function or activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), increased expression or activity of protein-related gene downstream target molecules that regulate protein content, and/or increased protein content as compared to a control plant part, population, or plant product, e.g., comprising wild-type protein-related polypeptide level or activity.

IV. Increasing Protein Content in Plants

Methods are provided herein for altering (e.g., increasing) protein content in a plant or plant part. In some aspects, the methods comprise increasing protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity in the plant or plant part, by, e.g., increasing level or activity of a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). Level or activity of a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can be increased by increasing the expression level or activity of at least one endogenous gene encoding a protein-related polypeptide; or by introducing an exogenous (e.g., transgenic) copy of a protein- related 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 a protein-related polypeptide in said plant or plant part. In some aspects, the methods comprise introducing a genetic mutation that alters (e.g., increases) protein-related polypeptide (e.g., XTH16, XTH16- A, BF4, PMT, PMT5) activity into a plant or plant part. The method can further comprise introducing the genetic mutation that alters (e.g., increases) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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) protein-related polypeptide level or activity, alter (e.g., increase) expression levels of at least one a protein-related gene encoding protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), alter (e.g., increase) protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) levels or activity, alter (e.g., increase) activity of one or more target molecules regulated by the protein-related polypeptide and regulating protein content and/or disease, and/or alter (e.g., increase) 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 or a transgene (e.g., an exogenous copy of a protein-related 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) a protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, altered (e.g., increased) expression levels of at least one a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof, altered (e.g., increased) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) levels or activity, altered (e.g., increased) activity of one or more target molecules regulated by the protein-related polypeptide and regulating protein content, 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. In the population of plants or plant parts, having altered protein-related polypeptide level or activity relative to a control population, not all individual plants or plant parts need to have altered (e.g., increased) protein- related polypeptide level or activity, genetic mutation that cause altered (e.g., increased) protein-related polypeptide level or activity, or phenotypes caused by the altered (e.g., increased) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity (e.g., 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 protein-related polypeptide level or activity.

A. Altering expression or function of protein-related gene or polypeptide 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 or a transgene that alters (e.g., increases) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity into a plant or plant part. The method can further comprise introducing the genetic mutation that alters (e.g., increases) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity into a plant cell, and regenerating a plant or plant part from the plant cell (e.g., transformed plant cell). In specific embodiments, the protein-related polypeptide is XTH16. 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity can be introduced into at least one native a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof; a regulatory region (e.g., promoter, 5’UTR) of the native a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) 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. A “native” gene 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, including a gene that does not naturally occur within the plant, plant part, or plant cell comprising the gene. For example, a transgenic a protein-related gene located at a genomic site or in a plant in a non-naturally occurring matter is a “native” a protein-related gene if its nucleic acid sequence can be found in a plant existing in nature.

1. Introducing regulatory modifications

The methods described herein can comprise introducing a mutation that increases the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5). 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 fortranscription 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) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5), to confer to the plant or plant part an increased protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5). The one or more insertions, substitutions, and/or deletions in the promoter region of the protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT 5) 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 protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5)\, 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 protein-related gene (e.g., XTH 16, XTH16-A, bF4, PMT, PMT5) 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) protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) and alter (e.g., increase) translation regulation activity.

The promoter or 5’UTR activity to regulate transcription or translation of one or more protein- related genes (Q.g., XTH16,XTH16-A, bF4, PMT, PMT 5) 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 protein-related genes (Q.g.,XTH16, XTH16-A, bF4, PMT, PMT5) 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 protein-related genes (e.g., XTH 16, XTH16-A, bF4, PMT, PMT5) 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 specific embodiments, the protein- related gene is XTH16.

In some instances, the promoter sequence of one or more protein-related genes (e.g., XTH 16, XTH16-A, bF4, PMT, PMT5) 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 protein-related genes (e.g., XTH 16, XTH16-A, bF4, PMT, PMT5) 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 protein- related genes (c.a.. XTHI6. XTHI6-A. bF4, PMT, PMT5) 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) protein-related gene (e.g., XTH16. XTH16-A, bF4, PMT, PMT5) at or near one or more transcriptional regulator (e.g., transcriptional repressor) binding domains. Introducing mutation into a transcriptional regulator (e.g., transcriptional repressor) binding domain in the regulatory region (e.g., promoter, 5’UTR) of a protein-related gene can alter (e.g., decrease) the affinity of transcriptional regulator (e.g., transcriptional repressor) binding, thereby altering (e.g., increasing) level or activity of the protein- related gene.

In some embodiments, the methods includes introducing a mutation to locate at least partially into the regulatory region of a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5), 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: 10-12, wherein the regulatory region retains transcription initiation activity; (ii) the regulatory region comprises a nucleic acid sequence of any one of SEQ ID NOs: 10-12; (iii) the protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (iv) the protein-related gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 7-9; (v) the protein-related 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: 4-6, wherein the polypeptide retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (vi) the protein- related gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4-6; (vii) the protein-related 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: 1-3, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; and/or (viii) the protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

In some embodiments, the methods includes introducing a mutation to locate at least partially into the regulatory region of the XTH16 gene, and (i) the protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) the protein-related gene comprises the nucleic acid sequence of SEQ ID NO: 7; (iii) the protein-related gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 4, wherein the polypeptide retains protein-related activity; (iv) the protein-related gene encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 4; (v) the protein-related gene including the regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1, wherein the nucleic acid sequence encodes a polypeptide that retains protein- related activity; and/or (vi) the protein-related gene including the regulatory region thereof comprises the nucleic acid sequence of SEQ ID NO: 1.

In some embodiments, the methods include introducing a mutation to locate at least partially in a promoter region or 5’UTR of a gene selected from the group consisting of Glycine max XTH 16, bF4, and PMT5 genes. In some embodiments, the mutation is located at least partially in a promoter region (e.g., SEQ ID NO: 10) of a Glycine maxXTH16 gene, a promoter region (e.g., SEQ ID NO: 11) of a Glycine max bF4 gene, or a promoter region (e.g., SEQ ID NO: 12) of a Glycine max PMT5 gene. In some embodiments, the methods include introducing a deletion of about 2-12 or more nucleotides to locate at least partially in the promoter and/or 5’UTR of a Glycine max XTH 16 gene, a Glycine max bF4 gene, or a Glycine max PMT5 gene. In some embodiments according to the methods provided herein: (i) the mutation comprises a deletion of nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 16 when said mutation is introduced; (ii) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 17 when said mutation is introduced; (iii) the mutation comprises a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine maxXTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced; (iv) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter in the first allele and a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine maxXTH16 promoter in the second allele, or said plant or plant part comprises the first allele comprising the nucleic acid sequence of SEQ ID NO: 17 and the second allele comprising the nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced; (v) the mutation comprises a deletion of nucleotides 2140 through 2141 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 19 when said mutation is introduced; (vi) the mutation comprises a deletion of nucleotides 2138 through 2144 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 20 when said mutation is introduced; (vii) the mutation comprises a deletion of nucleotides 1371 through 1374 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 21 when said mutation is introduced; (viii) the mutation comprises a deletion of nucleotides 1367 through 1377 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 22 when said mutation is introduced; or (ix) the mutation comprises a deletion of nucleotides 1371 through 1378 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 23 when said mutation is introduced. In specific embodiments, the methods include introducing a mutation to locate at least partially in a promoter region or 5’UTR of the XTH16 acnc (i) the mutation comprises a deletion of one or more nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 16 when said mutation is introduced; (ii) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 17 when said mutation is introduced; and/or (iii) the mutation comprises a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced.

Function and/or expression of the one or more protein-related genes (e.g., XTH 16, XTH 16, bF4, PMT, PMT5) can also 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 protein-related gene, e.g., by increasing transcription initiation activity of the protein-related gene promoter. In some embodiments, the methods include introducing a mutation that modifies or inserts transcription factor binding sites or enhancer elements that regulates a protein-related gene expression into the regulatory region of the protein-related gene.

Function and/or expression of the one or more protein-related genes (e.g., XTH 16, XTH16-A, bF4, PMT, PMT5) 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 protein-related genes (e.g., XTH 16, XTH16-A, bF4, PMT, PMT 5). Alternatively, modification and/or engineering of existing transcription factor binding sites or enhancer elements can increase function and/or expression of protein-related genes (e.g., XTH 16, XTH16-A, bF4, PMT, PMT5).

Function and/or expression of the one or more protein-related genes (e.g., XTH 16, XTH16-A, bF4, PMT, PMT5) 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 protein-related gene, a part or whole of one or more positive regulatory elements of the protein-related 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 protein-related genes (e.g., XTH 16, XTH16-A, bF4, PMT, PMT5) 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 protein-related gene in order to increase the expression and/or function of the gene.

The insertion, substitution, or deletion that is introduced at least partially into the promoter, 5 ’ UTR, the gene encoding (or regulating expression of) one or more transcription factors that regulates expression of a protein-related gene, or other regulatory region of a protein-related 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. Introducing mutation to a protein-related sene, or its homolog, ortholo ., or variant

In some aspects, the methods of the present disclosure comprise introducing a genetic mutation that increases the protein-related polypeptide (e.g., XTH16, XTH16, BF4, PMT, PMT5) 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 a protein-related gene (Q. .,XTHl 6, XTH16-A, bF4, PMT, PMT5) 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 a protein-related gene, Q. ., XTH16-A, bF4, or PMT5 genes, each encoding a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), 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 a protein-related gene or homolog; into a regulatory region of one a protein-related gene or homolog; into more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homologs; into regulatory regions of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homologs; into all protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homologs; and/or into regulatory regions of all protein-related genes (e.g.. A77//6. XTH16-A, bF4, PMT, PMT5) 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 protein-related gene (e.g., introducing a mutation only into the regulatory region of the protein-related gene). In another embodiment, the methods include including a mutation at least partially into the coding region of the protein-related 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 into 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 protein-related 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 protein-related gene or its regulatory region, and (i) the protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (ii) the protein-related gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 7-9; (iii) the protein-related 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: 4-6, wherein the polypeptide retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (iv) the protein- related gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4-6; (v) the protein-related 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: 1-3, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; and/or (vi) the protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-3. In specific embodiments, the mutation that increases the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity is located in one or two alleles of one or more (e.g., one, more than one but not all, or all) copies of Glycine maxXTH16-A gene, Glycine max bF4 gene, Glycine max PMT 5 gene, and/or a regulatory region thereof.

In specific embodiments, the mutation is introduced into the XTH16 gene or its regulatory region, and (i) the XTH16 gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) the XTH16 gene comprises the nucleic acid sequence of SEQ ID NO: 7; (iii) the XTH16 gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 4, wherein the polypeptide retains protein-related activity; (iv) the XTH16 acnc encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 4; (v) the XTH16 gene including the regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related activity; and/or (vi) the XTH16 gene including the regulatory region thereof comprises the nucleic acid sequence of SEQ ID NO: 1.

In some embodiments, the mutation is introduced at least partially into the coding region of Glycine maxXTH16-A, bF4, or P 'MI '5 gene.

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) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) 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) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or homolog thereof.

A genetic mutation that increases the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity can be introduced into a gene that is a homolog, ortholog, or variant of a protein- related gene disclosed herein and expresses a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) with protein-related polypeptide function, or in a regulatory region of such homolog, ortholog, or variant of a protein-related gene, according to the methods provided herein. For example, the mutation (e.g., one or more insertions, substitutions, or deletions that increase the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity) can be introduced into orthologs of protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5).

Variant sequences (e.g., homologs, orthologs) can be isolated by PCR. In this manner, variant sequences encoding a protein-related polypeptide can be identified and used in the methods of the present disclosure. The variant sequences will retain the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity.

In certain instances, mutations introduced into any a protein-related 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 detection method described herein. Such detection methods may comprise use of primers for detecting mutation in a protein-related gene. For example, a forward primer (e.g., SEQ ID NO: 24) and a reverse primer (e.g., SEQ ID NO: 25) can be used for detection of a mutation in the Glycine max XTH16 promoter near the binding site of the GmXTH16 guide RNA (e.g., SEQ ID NO: 13), e.g., a mutation generated by introducing GmXTHl 6 guide RNA (e.g., SEQ ID NO: 13) into the plant or plant part, such as a deletion mutation comprising a nucleic acid sequence of any one of SEQ ID NOs: 16-18. A forward primer (e.g., SEQ ID NO: 26) and a reverse primer (e.g., SEQ ID NO: 27) can be used for detection of a mutation in Glycine max bF4 regulatory region near the binding site of the GmbF4 guide RNA (e.g., SEQ ID NO: 14), for example a mutation generated by introducing the GmbF4 guide RNA (e.g., SEQ ID NO: 14) into the plant or plant part, such as a deletion mutation comprising a nucleic acid sequence of SEQ ID NO: 19 or 20. A forward primer (e.g., SEQ ID NO: 28) and a reverse primer (e.g., SEQ ID NO: 29) can be used for detection of a mutation in Glycine max PMT5 regulatory region near the binding site of the GmPMT5 guide RNA (e.g., SEQ ID NO: 15), for example a mutation generated by introducing the GmPMT5 guide RNA (e.g., SEQ ID NO: 15) into the plant or plant part, such as a deletion mutation comprising a nucleic acid sequence of any one of SEQ ID NOs: 21- 23.

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 a protein-related gene or homolog or in a regulatory region of such a protein-related gene or homolog in the genome of the plant or plant part can increase the expression levels of the protein-related gene or homolog, increase level or activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the protein-related gene or homolog, increase protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, and/or increase 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. Introducing a transgene

The method provided herein can comprise introducing a transgene into a plant, plant part, or plant cell. The transgene can comprise a nucleic acid sequence of at least one protein-related gene (e.g.. A77//6. XTH16-A, BF4, PMT, PMT5) or homolog thereof operably linked to a promoter, wherein said at least one protein-related gene or homolog thereof encodes said protein-related polypeptide, and wherein the method increases an expression level of said at least one protein-related gene or homolog thereof compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said transgene, or level or activity of said protein-related polypeptide compared to level of activity of said protein-related polypeptide in a plant or plant part without said transgene. In specific embodiments, the protein-related polypeptide is XTH16. “Transgene” as used herein refers to an exogenous copy of a gene, e.g., a protein-related gene, e .g., XTH16, XTH16-A, bF4, PMT, or PMT5. One or more exogenous copies of the protein-related gene (e.g., XTH 16) can be native, i.e., without mutation. Alternatively, one or more exogenous copies of the protein-related gene can have a mutation that increases protein-related polypeptide level or activity. The method can include introducing into the plant or plant part a transgene comprising a nucleic acid sequence of at least one native protein-related gene or homolog thereof operably linked to a promoter, and increasing expression level of said at least one protein-related gene or homolog thereof compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said transgene. The promoter can be the native promoter of the protein-related gene (e.g., the transgene comprises XTH16 operably linked to a native XTH16 promoter) or a heterologous promoter (e.g., the transgene comprises XTH 16 operably linked to a heterologous promoter, e.g., CHS7 promoter). The transgene can be introduced into the plant or plant part by standard methods, including transformation, e.g., Agrobacterium transformation, as described in this disclosure.

4. Increasing protein-related polypeptide activity

The methods of the present disclosure, e.g., introducing a mutation or a transgene that increases level or activity of a protein-related polypeptide in the plant or plant part, can increase activity of a protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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.

Activity of the protein-related polypeptide can be measured by measuring protein content in the plant or plant part (e.g., seeds) by standard methods for measuring protein in a plant sample, 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). Protein content can also be measured by the Dumas method, by combusting samples at a high temperature in the presence of high-purity oxygen, analyzing the gas from combustion for nitrogen content using a thermal conductivity detector, and calculating the amount of protein present in the sample using a conversion factor. The industry standard conversion factor for soybean is 6.25. Activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can also be measured by measuring activity of the respective protein-related polypeptide. For example, activity of XTH16 or XTH16- A can be measured by standard methods for measuring hydrolase activity on xyloglucan (e.g., enzymatic assay), xyloglucan endotransglucosylase (XET) activity (e.g., enzymatic assay), or activity to stimulate growth of hypocotyls (e.g., growth assay). Activity of BF4 can be measured by standard methods for measuring vacuolar invertase activity (e.g., enzymatic assay), or standard methods for measuring total or specific sugar content (e.g., total sugar, sucrose, glucose, fructose, galactose, maltose, lactose) in plant samples [e.g., spectroscopy (near infrared spectroscopy), refractometry, solid-phase extraction (SPE), solidphase micro-extraction (SPME), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GCMS), and/or enzymatic assay]. Activity of PMT or PMT5 can be measured by standard methods for measuring activity to transport (symport) polyol, cyclitol, monosaccharide, and H+ across plasma membrane (e.g., enzymatic assay), or activity to catalyze the energy-dependent membrane passage of linear polyols, cyclic polyols, and monosaccharides (e.g., enzymatic assay).

The methods of the present disclosure can increase expression level of the protein-related 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 protein-related 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 a protein-related gene(s) or homolog by, e.g., introducing a genetic mutation or additional mechanism to up-regulate the expression of the endogenous a protein-related gene(s). For example, the methods provided herein can introduce a mutation in the regulatory region (e.g., promoter, 5’UTR) of at least one endogenous a protein-related gene, e.g., at or near transcriptional repressor binding sites, e.g., a RAV 1 domain and/or a ERF domain, in the plant, plant part, population of plants or plant parts, or plant product to increase expression of the endogenous a protein-related gene(s).

Alternatively or additionally, the methods provided herein can include introducing one or more copies of a protein-related gene (transgene), e.g., a polynucleotide encoding a functional protein-related polypeptide or functional fragment thereof, into a plant or plant part. The methods can further include introducing a polynucleotide encoding a functional protein-related polypeptide into a plant cell, and regenerating a plant or plant part overexpressing the plant-related polypeptide from the plant cell. One or more exogenous (e.g., transgenic) copies of the protein-related gene can be from the same, related, or different plant species. One or more exogenous copies of the protein-related gene can be native, i.e., without mutation; alternatively, one or more exogenous copies of the protein-related gene can have a mutation (e.g., in the regulatory region, coding region, and/or non-coding region) that increases protein-related polypeptide level or activity. A polynucleotide comprising a sequence of a protein-related gene or encoding a functional protein-related polypeptide or functional fragment thereof can be assembled within a DNA construct with an operably-linked promoter molecule, which can be a homologous (native) protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) 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 protein-related gene or encoding a functional protein-related polypeptide or functional fragment thereof. The protein-related polypeptide-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 protein-related gene or a protein-related gene transcript, or a protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the polynucleotides, thereby increasing level of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) in the plant, plant part, or plant cell compared to a level of the protein-related gene or homolog thereof in a plant or plant part without the transgene.

In particular, the methods provided herein can increase the expression levels of a protein-related 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 a protein-related gene or homolog that contributes to an increased expression (e.g., up-regulation, overexpression) of the protein-related gene or homolog is an endogenous or exogenous copy of a Glycine max XTH16-A gene, a Glycine max bF4 gene, and/or a Glycine max PMT5 gene. Expression levels of the protein-related 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 protein-related 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, or dot blot analysis 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the protein-related gene.

The methods of the present disclosure can increase expression levels of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) (e.g., a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) having the complete amino acid sequence and function of a wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), e.g., encoded by a native a protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by the endogenous a protein-related gene(s) or homolog by, e.g., introducing genetic mutation into at least one endogenous a protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). For example, the method can include introducing a mutation in the regulatory region (e.g., promoter, 5’UTR) of at least one endogenous protein-related gene, e.g., at or near transcriptional repressor binding sites, e.g., a RAVI domain and/or a ERF domain, to increase expression of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). Alternatively or additionally, the methods can include increasing the levels of protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) by introducing one or more exogenous copies of a protein-related gene into the plant or plant part. The method can include introducing a transgene comprising a nucleic acid sequence of at least one native protein-related gene or homolog thereof operably linked to a promoter into a plant or plant part, to increase level or activity of the protein-related polypeptide compared to level of activity of said protein-related polypeptide in a plant or plant part without said transgene. One or more exogenous (e.g., transgenic) copies of the protein-related gene can be from the same, related, or different plant species. One or more exogenous copies of the protein-related gene can be native, i.e., without mutation; alternatively, one or more exogenous copies of the protein-related gene can have a mutation (e.g., in the regulatory region, coding region, and/or non-coding region) that increases protein- related polypeptide level or activity.

In particular, the methods provided herein can increase expression of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), e.g., encoded by endogenous and/or exogenous copies of the protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) encoded by endogenous or exogenous copies of the Glycine maxXTH16-A gene, Glycine max bF4 gene, and/or Glycine max PMT 5 gene. Expression of a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can be determined by western blot analysis, ELISA, or dot blot analysis 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). 6. Enhancing yrotein-related yolyyeytide function

The methods of the present disclosure can enhance function in the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT 5) or homolog thereof (e.g., in the regulatory, coding, and/or non-coding regions) to enhance function of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related gene encoding protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) (e.g., with enhanced function) into the plant, plant part, or population of plants or plant parts, such that the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related gene, or otherwise having wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity. The protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) with increased function can comprise a mutation compared to a wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) that causes enhanced protein-related polypeptide function. In some embodiments, the methods increase the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) function (e.g., by introducing a mutation into the protein-related gene or homolog or its regulatory region, or by introducing an exogenous copy of a protein-related gene encoding a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). In certain embodiments, the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) with enhanced function is encoded by an endogenous or exogenous copy of mutated Glycine maxXTH16 gene, Glycine max hF4 gene, and/or Glycine max PMT 5 gene.

Function of a protein-related polypeptide (e.g., XTH16, XTH16A, BF4, PMT, PMT5) in a plant, plant part, a population of plants or plant parts, or plant product can be measured by measuring protein content in the plant or plant part (e.g., seeds) by standard methods for measuring protein in a plant sample, 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). Protein content can also be measured by the Dumas method, by combusting samples at a high temperature in the presence of high-purity oxygen, analyzing the gas from combustion for nitrogen content using a thermal conductivity detector, and calculating the amount of protein present in the sample using a conversion factor. The industry standard conversion factor for soybean is 6.25. Function of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) can also be measured by measuring activity of the respective protein-related polypeptide. For example, function of XTH16 or XTH16-A can be measured by standard methods for measuring hydrolase activity on xyloglucan (e.g., enzymatic assay), xyloglucan endotransglucosylase (XET) activity (e.g., enzymatic assay), or activity to stimulate growth of hypocotyls (e.g., growth assay). Function of BF4 can be measured by standard methods for measuring vacuolar invertase activity (e.g., enzymatic assay), or standard methods for measuring total or specific sugar content (e.g., total sugar, sucrose, glucose, fructose, galactose, maltose, lactose) in plant samples [e.g., spectroscopy (near infrared spectroscopy), refractometry, solid-phase extraction (SPE), solid-phase micro-extraction (SPME), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GCMS), and/or enzymatic assay]. Function of PMT or PMT5 can be measured by standard methods for measuring activity to transport (symport) polyol, cyclitol, monosaccharide, and H+ across plasma membrane (e.g., enzymatic assay), or activity to catalyze the energydependent membrane passage of linear polyols, cyclic polyols, and monosaccharides (e.g., enzymatic assay).

B. Introducing mutations into the genome of plant cells

Introducing one or more mutations into the plant genome, e.g., into at least one a protein-related gene (e.g., Glycine max XTH 16, bF4, or PMT 5) or its regulatory region, and modulating the level or activity of a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related gene (e.g., Glycine max XTH 16, bF4, or PMT5) or its regulatory region 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 protein-related gene 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]; Natronobacterium gregoryi Argonaute -mediated DNA insertion (Gao et al. 2016 Nat Biotechnol doi: 10.1038/nbt.3547); Cre-lox site-specific recombination (Dale et al. 1995

77:649-659; 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 (Y an et al. 2011 Plant 7701: 147- 166); zinc -finger mediated integration (Wright et al. 2005 Plant J 44: 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 a protein-related gene and/or a regulatory region of the protein-related 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 a protein- related gene and/or a regulatory region of the protein-related 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, Cas 12a, 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. The CRISPR nuclease can be a Type II CRISPR system nuclease, a Type V CRISPR system nuclease, a Cas9 nuclease, a Casl2a (Cpfl) nuclease, or a Cmsl nuclease, or an ortholog of any thereof.

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 a protein-related gene and/or a regulatory region of the protein-related 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, Casl2a, 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 a protein-related gene, thereby modulating the transcription of the protein-related gene (Piatek et al. 2015 Plant Biotechnol J 13:578-589). Site-specific 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 a tracrRNA (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 Cas 12a ortholog nucleases and crRNAs (CRISPR RNAs) for forming gRNAs that are coactive with the Cas 12a 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 a protein-related gene or the regulatory region of the protein-related gene. “Guide RNA” as used herein refers to a 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 deletion (e.g., deletion of 10 or more nucleotides) in the promoter or 5’UTR of one or both alleles of a protein-related gene, e.g., a Glycine max XTH16 gene, a Glycine max bF4 gene, or a Glycine max PMT5 gene. The one or more gRNAs can be designed to specifically target a regulatory region (e.g., promoter, 5’UTR) of a protein-related gene, or exons or introns of a protein-related gene. In some embodiments, the one or more gRNAs are specific to a region of a binding site of a transcriptional regulator (e.g., transcriptional suppressor), e.g., a region containing one or more RAVI motif and/or one or more ERF motif in the 5’ regulatory region (e.g., promoter, 5’UTR) of a protein-related gene (c.a.. XTH 16. XTH 16-A. bF4, PMT, PMT 5). An exemplary RAV motif includes CAACA and its reverse complement, TGTTG. An exemplary ERF motif includes TGACC and its reverse complement, GGTCA.

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: 1-3. The gRNA can be specific to the nucleic acid sequence of any one of SEQ ID NOs: 1-3 and/or can drive a deletion at least partially in the 5’ regulatory region (e.g., promoter, 5’UTR), exons, and/or introns of the Glycine max XTH16 gene, Glycine max bF4 gene, Glycine max PMT 5 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 a protein-related gene, a regulatory region of the protein-related gene, 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: 1-3, 7-12. The two or more gRNA can be specific to the nucleic acid sequence of any one of SEQ ID NOs: 1-3, 7-12 and/or can drive one or more deletions at least partially in the 5’ regulatory region (e.g., promoter, 5’UTR), exons, and/or introns of the Glycine maxXTH16 gene, Glycine max bF4 gene, Glycine max PMT5 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 a protein-related gene or a regulatory region of the protein-related 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 is encoded by a nucleic acid sequence comprising a nucleic acid sequence having at least 75% (e.g., 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: 1-3, 7-12. In particular instances, the targeting region of a gRNA for use in the method described herein is encoded by a nucleic acid sequence comprising the nucleic acid sequence of any one of SEQ ID NOs: 1-3, 7-12. 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 any one of SEQ ID NOs: 1-3, 7-12 or a nucleic acid sequence of any one of SEQ ID NOs: 1-3, 7-12. 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. At least one of the gRNAs provided herein can comprise a nucleic acid sequence encoded by: (i) a nucleic acid sequence that shares at least 80% sequence identity with a nucleic acid sequence of SEQ ID NOs: 10-15; or (ii) the nucleic acid sequence of SEQ ID NOs: 10-15. In specific embodiments, at least one of the one or more gRNAs comprises a nucleic acid sequence encoded by: (i) a nucleic acid sequence that shares at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 13; or (ii) the nucleic acid sequence of SEQ ID NO: 13.

The gRNA or a combination of two or more gRNAs provided herein can introduce a deletion of one or more, e.g., about 10, nucleotides at least partially in the 5’ regulatory region (e.g., promoter, 5’UTR) or the coding region (e.g., exons, introns) of a Glycine max XTH16 gene, a Glycine max bF4 gene, or a Glycine max PMT5 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 region (e.g., containing a transcriptional regulator (e.g., repressor) binding site) in the 5’ regulatory region (e.g., promoter, 5’UTR) of a Glycine maxXTH16 gene, a Glycine max bF4 gene, or a Glycine max PMT5 gene, and introduce into the plant, plant part, or plant cell: (i) a deletion of nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter, resulting in a nucleic acid sequence of SEQ ID NO: 10; (ii) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter, resulting in a nucleic acid sequence of SEQ ID NO: 17; (iii) the mutation comprises a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, resulting in a nucleic acid sequence of SEQ ID NO: 18; (iv) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter in the first allele and a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter in the second allele, resulting in the first allele comprising the nucleic acid sequence of SEQ ID NO: 17 and the second allele comprising the nucleic acid sequence of SEQ ID NO: 18; (v) the mutation comprises a deletion of nucleotides 2140 through 2141 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter, resulting in a nucleic acid sequence of SEQ ID NO: 19; (vi) the mutation comprises a deletion of nucleotides 2138 through 2144 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter, resulting in a nucleic acid sequence of SEQ ID NO: 20; (vii) the mutation comprises a deletion of nucleotides 1371 through 1374 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, resulting in a nucleic acid sequence of SEQ ID NO: 21; (viii) the mutation comprises a deletion of nucleotides 1367 through 1377 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, resulting in a nucleic acid sequence of SEQ ID NO: 22; or (ix) the mutation comprises a deletion of nucleotides 1371 through 1378 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, resulting in a nucleic acid sequence of SEQ ID NO: 23.

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%). In specific embodiments, the methods do not introduce mutations into at least one allele comprising at least one a protein-related gene and its regulatory region. In some embodiments, the methods introduce mutations into all alleles each comprising a protein-related gene and its regulatory region.

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 a protein-related gene or a regulatory region of the protein-related gene and alter the level or activity of the protein-related gene or protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5). 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 protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT 5) or a regulatory region of the protein-related 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. methanesulfonate, sodium azide, aminopurine, etc.), genome/gene editing techniques (e.g. CRISPR-like technologies, TALENs, zinc finger nucleases, and meganucleases), ionizing radiation (e.g. ultraviolet and/or gamma rays) 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 23-XT3Q),- 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 et aZ. (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 gRNA sequence specific for at least one a protein-related gene or a regulatory region of the protein-related 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 a protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or the regulatory region of the protein-related 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 a protein-related gene or the regulatory region of the protein-related 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 veinclearing 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 a protein-related gene and/or a regulatory region of the protein-related gene. The cell can be a plant cell, a bacterial cell, or a fungal cell. The cell can be a bacterium, e.g., an Agrobacterium tumefaciens, containing the gRNA targeting at least one a protein- related gene and/or a regulatory region of the protein-related 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 protein content in plants

The methods of the present disclosure, by introducing a mutation or an exogenous gene copy that increases protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity into plants, plant parts, or plant cells and/or regenerating plants from transformed cells, can increase protein content in the plants, plant parts (e.g., seeds, leaves), a 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. “Protein content” as used herein refers to total protein content, as well as content of any specific type of proteins or a combination of any types of proteins, including white flake content and specific amino acid content.

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, and may express an endogenous (e.g., wild-type) protein-related gene. A plant, plant part, a population of plants or plant parts, or plant product of the present disclosure can have 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 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 a native protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), 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 Sci 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). Protein content can also be measured by the Dumas method, by combusting samples at a high temperature in the presence of high-purity oxygen, analyzing the gas from combustion for nitrogen content using a thermal conductivity detector, and calculating the amount of protein present in the sample using a conversion factor. The industry standard conversion factor for soybean is 6.25.

In specific embodiments, the methods confer the plant, plant part, or a population of plants or plant parts the trait of increased protein content as compared to a control plant, plant part, population of plants or plant parts, or plant product, without a significant decrease in yield. In some embodiments, the methods cause a reduction in yield in the plant, plant part, or population of plants or plant parts by no more than about 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or about 5.0%, 6%, 7%, 8%, 9%, or 10%, e.g., no more than about 0-5%, 0.5-4.5%, 0.5-4%, 1-5%, 1-4%, 2-5%, 2-4%, 0.5-10%, 0.5-8%, 1-10%, 2-10%, 3- 10%, 4-10%, 5-10%, 6-10%, 7-10%, or 8-10%, while increasing protein content as compared to a control plant, plant part, or population of plants or plant parts. Yield can be measured and expressed by any means known in the art. In specific embodiments, yield is measured by seed weight or volume of seeds, fruits, leaves, or whole plants harvested from a given harvest area.

In specific embodiments, the methods increase protein content in seeds and a population of seeds as compared to control seeds or a population of seeds. 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 composition derived from the plant or plant part, 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.

A “protein product” or “protein composition” obtained from the plants or plant parts produced according to the methods provided herein can include 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)). White flake or white flake protein refers to a protein composition obtained by de-hulling, flaking, and defattening plants or plant parts (e.g., legume plants or plant parts) by solvent (e.g., hexane) extraction, with limited use of heat to run off the solvent (Lusas and Riaz, 1995). White flake protein is an intermediate product in the production of plant protein concentrates and isolates. In contrast to conventional toasted plant meal (e.g., soybean meal), white flakes contains undenaturated proteins due to the very mild heat treatment. Thus, little or no reduction of protease inhibitors would be expected. The undenaturated proteins in white flakes may be advantageous in supporting binding properties during production of the extruded compound feed. White flakes can be used for human and animal consumption, including as a source of protein in aquaculture feeds for any type of fish or aquatic animal in a farmed or wild environment.

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., 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 to a mutation (e.g., in the regulatory region, noncoding region, and/or coding region of at least one a protein-related gene) or an exogenous copy of a protein-related gene that increases protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity.

Also provided herein are food and/or beverage products obtained 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.

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, e.g., one or more insertions, substitutions, or deletions in at least one native a protein-related gene or homolog or in a regulatory region of such a protein-related gene or homolog, or a transgene (e.g., an exogenous copy of a protein-related gene) that increases protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity. The plants, plant parts, and plant products produced according to the methods of the present disclosure can have increased protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, increased expression level of the protein-related gene or homolog, increased expression level of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) or functional fragment thereof, increased function or activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), increased expression or activity of protein-related gene downstream target molecules that regulate protein content, increased protein content as compared to a control (e.g., wild-type) plant part or plant product comprising wild-type protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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) to at least one a protein- related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 a protein-related gene and/or a regulatory region of the protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 a protein- related gene and/or a regulatory region of the protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein content.

Any mutation or any polynucleotide of interest (e.g., editing reagents, e.g., a nuclease and a guide RNA; a polynucleotide encoding a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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) EMBO 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. 22:421-477 ; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/T echnology 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 lumefaciens)\ 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related polypeptide level or activity may be achieved by inserting a transposable element into at least one a protein- related gene and/or a regulatory region of the protein-related 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., Vigna spp.), common bean (Phaseolus vulgaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), adzuki bean (Vigna angularis), fava bean (Vida faba), 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 hicolor, 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 intyhus), lettuce (Lactuca sativa), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana spp., e.g., Nicotiana tahacum, 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.

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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, e.g., one or more insertions, substitutions, or deletions in at least one native protein-related gene or homolog or in a regulatory region of such a protein-related gene or homolog, (ii) editing reagents, e.g., a polynucleotide encoding a guide RNA specific to at least one protein-related gene or homolog or in a regulatory region of such a protein-related gene or homolog, and/or (iii) a polynucleotide comprising a wild-type, mutated, native, or heterologous protein-related 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 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 protein-related gene(s) or its regulatory region that causes increased activity of a protein-related polypeptide (e.g., XTH16, XTH16- A, BF4, PMT, PMT5). In certain embodiments, the desired trait is conferred to a plant by transforming the plant with an exogenous copy of a protein-related 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 a Protein-Related Gene or Regulatory Region of a Protein-Related Gene

A. Nucleic acid molecules

Nucleic acid molecules are provided herein comprising a polynucleotide sequence that alters (e.g., increases) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity in a plant or plant part. The nucleic acid molecule can comprise any nucleic acid sequence that alters (e.g., increases) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related gene or regulatory region thereof, an altered a protein-related gene transcript encoding an altered (e.g., mutated, alternatively spliced, truncated) protein-related polypeptide (e.g., XTH16, XTH16- A, BF4, PMT, PMT5), or a wild-type sequence of a protein-related 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 protein-related gene or a regulatory region of the protein-related gene, introducing editing reagents targeting a site of interest in at least one a protein-related gene or a regulatory region of the protein-related gene, or introducing a polynucleotide encoding a protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related 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) protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) that has a different amino acid sequence from a native protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) (e.g., without mutations) and/or has increased protein-related polypeptide function or activity, e.g., the ability to regulate protein content, as compared to a native protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) (e.g., without mutations). The mutated sequence, e.g., altered nucleic acid sequence of the protein-related gene and/or the regulatory region of the protein-related gene can result in increased expression levels of the protein-related gene or protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) (e.g., full-length protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), functional protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5)), as compared to a native a protein-related gene and/or a regulatory region of a native a protein-related gene e.g., without mutations.

The nucleic acid molecule provided herein can comprise a sequence of a mutated a protein-related 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 protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity. The nucleic acid molecule can comprise a mutated regulatory region of a protein-related gene, e.g., a mutated promoter region, 5’ untranslated region (5’UTR), 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 a protein-related gene. The nucleic acid molecule can comprise a mutated regulatory region of a protein-related gene having a mutation at or near a transcriptional modulator (e.g., transcriptional repressor) binding site.

In some embodiments, the nucleic acid molecule comprises a mutated regulatory region of a protein- related gene, and (i) the regulatory region (without mutation) comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 10-12, wherein the regulatory region retains transcription initiation activity; (ii) the regulatory region (without mutation) comprises a nucleic acid sequence of any one of SEQ ID NOs: 10-12; (iii) the protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein the nucleic acid sequence encodes a polypeptide that retains protein- related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (iv) the protein-related gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 7-9; (v) the protein-related 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: 4-6, wherein the polypeptide retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; (vi) the protein-related gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4-6; (vii) the protein-related 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: 1-3, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity; and/or (viii) the protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

In some embodiments, the nucleic acid molecule comprises a mutated regulatory region of XTH16, and (i) the XTH16 gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) the XTH16 gene comprises the nucleic acid sequence of SEQ ID NO: 7; (iii) the XTH16 gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 4, wherein the polypeptide retains protein-related activity; (iv) the XTH16 gene encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 4; (v) the XTH16 gene including the regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1, wherein the nucleic acid sequence encodes a polypeptide that retains protein-related activity; and/or (vi) the XTH16 gene including the regulatory region thereof comprises the nucleic acid sequence of SEQ ID NO: 1.

In some embodiments, the nucleic acid molecule comprises a mutated promoter region or 5’UTR of a gene selected from the group consisting of Glycine maxXTH16, bF4, and PMT5 genes, e.g., a mutated promoter (e.g., SEQ ID NO: 16, 17, 18) of a Glycine maxXTH16 gene, a mutated promoter (e.g., SEQ ID NO: 19, 20) of a Glycine max bF4 gene, or a mutated promoter (e.g., SEQ ID NO: 21, 22, 23) of a Glycine max PMT5 gene. In some embodiments, the nucleic acid molecule comprises a mutated regulatory site containing a transcriptional modulator (e.g., transcriptional repressor) binding site. In some embodiments, the nucleic acid molecule comprises a mutated promoter or 5’UTR sequence with a deletion of about 2-12 or more nucleotides of a Glycine max XTH 16 gene, a Glycine max bF4 gene, or a Glycine max PMT5 gene. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of a mutated GmXTHl 6 promoter or 5’UTR (i) comprising SEQ ID NOs: 10 with one or more insertions, substitutions, or deletions therein, or (ii) comprising the nucleic acid sequence of any one of SEQ ID NOs: 16-18. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of a mutated GmbF4 promoter or 5’UTR (i) comprising SEQ ID NOs: 11 with one or more insertions, substitutions, or deletions therein, or (ii) comprising the nucleic acid sequence of SEQ ID NO: 19 or 20. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of a mutated GmPMT5 promoter or 5’UTR (i) comprising SEQ ID NOs: 12 with one or more insertions, substitutions, or deletions therein, or (ii) comprising the nucleic acid sequence of any one of SEQ ID NOs: 21-23. The mutated promoter sequence in the nucleic acid molecule can increase level or activity of an operably-linked gene of interest as compared to a control promoter sequence (e.g., without mutation).

In some embodiments, the nucleic acid molecule comprises a mutated sequence of a protein-related gene selected from the group consisting of Glycine max XTH 16, bF4, and PMT5 genes (e.g., SEQ ID NOs: 1-3, respectively) or a mutated coding sequence of Glycine maxXTH16, bF4, or PMT5 genes (e.g., SEQ ID NOs: 7-9, respectively). In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of a mutated GmXTHl 6 gene having one or more insertions, substitutions, or deletions in a nucleic acid sequence of SEQ ID NO: 1 or 7. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of a mutated GmbF4 gene having one or more insertions, substitutions, or deletions in a nucleic acid sequence of SEQ ID NO: 2 or 8. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of a mutated GmPMT5 gene having one or more insertions, substitutions, or deletions in a nucleic acid sequence of SEQ ID NO: 3 or 9. The mutated protein-related gene can have increased level or activity of the protein-related gene or an encoded protein-related polypeptide relative to a control protein- related gene (e.g., without mutation).

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 protein-related gene or homolog. The nucleic acid molecule described herein can comprise the regulatory region (e.g., promoter region) of the protein- related gene as well as the exon/intron region of the protein-related gene, one or both of which has one or more insertions, substitutions, and/or deletions that increase level or activity of a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5).

B. DNA constructs, vectors, and cells

The nucleic acid molecules encoding molecules of interest (e.g., a mutated Glycine maxXTH16 gene, Glycine max bF4 gene, or Glycine max PMT5 gene) of the present invention can be assembled within a DNA construct with an operably-linked promoter. The mutated regulatory region of a gene (e.g., the mutated Glycine max XTH16 promoter, Glycine max bF4 promoter, or Glycine max PMT 5 promoter) of the present invention can be assembled within a DNA construct with an operably-linked polynucleotide of interest. When transiently or stably transformed with such DNA construct, a plant, plant part, or plant cell can express or accumulate polynucleotides comprising the sequence of a protein-related gene or a protein- related gene transcript, or a protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) 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 protein-related gene or a protein-related gene transcript. The protein-related polypeptide-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 protein-related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT 5) that can be native (without mutation, e.g., comprising the sequence of any one of SEQ ID NOs: 10-12) or mutated (e.g., comprising the sequence of any one of SEQ ID NOs: 10-12 with one or more insertions, substitutions, or deletions therein, or comprising the sequence of any one of SEQ ID NOs: 16-23), and a polynucleotide of interest [e.g., a protein- related gene (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) or a reporter gene, e.g., GFP, luciferase, HA tag] . For example, provided herein is a nucleic acid molecule comprising a nucleic acid sequence of a mutated promoter of a protein-related gene, wherein the nucleic acid sequence comprises: (i) a nucleic acid sequence of any one of SEQ ID NOs: 10-12 comprising one or more insertions, substitutions, or deletions therein; or (ii) a nucleic acid sequence of any one of SEQ ID NOs: 16-23. Also provided is a DNA construct comprising, in operable linkage: (i) the nucleic acid molecule comprising a nucleic acid molecule of a mutated promoter of a protein-related gene provided herein, and (ii) a polynucleotide of interest. In specific embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence of a mutated promoter of a XTH16 gene, wherein the nucleic acid sequence comprises: (i) a nucleic acid sequence of SEQ ID NO: 10 comprising one or more insertions, substitutions, or deletions therein; or (ii) a nucleic acid sequence of any one of SEQ ID NOs: 16-18. Also provided is a DNA construct comprising, in operable linkage: (i) the nucleic acid molecule comprising a nucleic acid molecule of a mutated promoter of the XTH16 gene provided herein, and (ii) 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 (e.g., wild-type) nucleic acid sequence (e.g., comprising the sequence of any one of SEQ ID NOs: 1-3 and 7-9) or mutated nucleic acid sequence (e.g., comprising the sequence of any one of SEQ ID NOs: 1-3 and 7-9 with one or more insertions, substitutions, or deletions) of a protein-related gene (s. .,XTH16, XTH16-A, bF4, PMT, PMT5), coding sequence thereof, or transcript thereof. The DNA construct can comprise, in operable linkage with a promoter (e.g., a protein- related gene (e.g., XTH16, bF4, PMT5 promoter), 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 protein-related gene, the regulatory region of the protein-related gene, and/or the protein-related 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 a nucleic acid sequence of the protein-related gene, the regulatory region of the protein-related gene, and/or the protein-related 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 protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) activity, 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 protein-related gene copies in wild-type soybean tissues

Transcript expression levels of the XTH 16, bF4, PMT5 genes in soybean, i.e., Glycine maxXTH16

(Glyma.l 1G253900), Glycine max bF4 (Glyma.01G211000) and Glycine max PMT5 (Glyma.09G 190900) in the SoyBase and Phytozome databases were studied. As shown in Tables 1 and 2, a protein-related gene transcripts were expressed across various tissues of soybean, including flowers, leaves, nodules, pods, roots, root hairs, seeds, shoot apical meristems, and stems.

TABLE 1. Expression of Protein-Related Gene Copies in Wild-Type Soybean Tissues According to Phytozome

TABLE 2. Expression of Protein-Related Gene Copies in Wild-Type Soybean Tissues According to Soybase

EXAMPLE 2: Protein-related gene promoter/5’UTR modification and expression of a downstream gene

Guide RNAs targeting a protein-related gene (e.g., GmXTH16, GmbF4, GmPMT5) 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 in the promoter and/or 5’ UTR of GmXTH16, GmbF4, and GmPMT5 and a nuclease using Agrobacterium transformation. Amplicons were produced near the target sites, and are 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 constructs having mutations in the promoter region or 5 ’UTR of the GmXTH16, GmbF4, and GmPMT5 genes were generated by introducing into protoplasts the gene editing system provided herein, including one or more guide RNAs (in specific embodiments, two or more guide RNAs) and were screened for editing efficiency and effects on expression levels of a downstream gene. A dual luciferase assay system was used to determine a change in downstream gene expression produced by introducing gRNAs in protoplasts. Briefly, expression cassettes comprising the (mutated or wild-type) promoter sequence of the GmXTHl 6. GmbF4, or GmPMT5 gene operably linked to a polynucleotide encoding firefly luciferase, and a constitutive promoter sequence operably linked to a polynucleotide encoding renilla luciferase were generated. The promoter sequence represented the native promoter or a promoter with a -lObp deletion at the expected cut site for a specific gRNA. The promoter-driven firefly luciferase and constructs were transfected into protoplast and luminescence was measured. Expression changes were calculated by comparing luminescence values produced by the construct comprising a modified promoter sequence to the construct comprising the native promoter sequence. The firefly luciferase luminescence used in this comparison were normalized to a ubiquitously expressed renilla luciferase luminescence to control for protoplast transformation variability. As summarized in Table 3, mutations in the GmXTH16, GmbF4, and GmPMT5 promoter sequences introduced by guide RNAs set forth as SEQ ID NOs: 13-15, respectively, produced increase in expression levels of the operably-linked gene (firefly luciferase) by 45%, 119%, and 79%, respectively, as compared to the expression levels obtained by the respective native promoter sequences.

TABLE 3. Effect of Mutated Promoters in Expression of Operably-Linked Gene in Protoplast

* Sequence corresponding to a 10 bp deletion in the respective gene target is underlined in bold

EXAMPLE 3: Generation of TO and T1 soybean plants with mutations

Embryonic axes of mature seeds of soybean varieties were stably transformed with constructs comprising one, two, or multiple GmXTH16, GmbF4, or GmPMT5 guide RNAs and a nuclease using Agrobacterium transformation. Transformed plants were identified by their resistance to glyphosate. Amplicons were produced of the genomic regions near the targeted GmXTH16, GmbF4, or GmPMT5 sites and sequenced to evaluate the presence of the mutation using a pair of primers to detect mutations introduced. For example, a pair of primers comprising the nucleic acid sequence of SEQ ID NOs: 24 and 25 can be used for detecting a mutation near the targeted site in the GmXTH16 regulatory region; a pair of primers comprising the nucleic acid sequence of SEQ ID NOs: 26 and 27 can be used for detecting a mutation near the targeted site in the GmbF4 regulatory region; and a pair of primers comprising the nucleic acid sequence of SEQ ID NOs: 28 and 29 can be used for detecting a mutation near the targeted site in the GmPMT5 regulatory region. Transgenic events were recorded, and the TO plants were assigned unique plant names (e.g., Plant A) and were subjected to molecular characterization and propagation. TO plants are selfpollinated and T1 plants were generated. Crosses were made to generate lines that are homozygous or heterozygous for the target mutation and lack the editing reagents. Mutated sequences in example plants (Plants A-H) are set forth as SEQ ID NOs: 16-23. Expression levels of the protein-related polypeptide, as well as seed protein content of transformed plants are analyzed, as described in Example 4.

EXAMPLE 4: 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 protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) and levels and activities of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) are measured in mutant plants (e.g., having a homozygous or heterozygous mutation in the protein-related gene promoter). Expression levels of the protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT5) 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 protein-related polypeptides (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) (e.g., full-length protein- related polypeptide) are measured by any standard methods for measuring protein levels, including western blot analysis, ELISA, or dot blot analysis of a protein sample obtained from the plant using an antibody directed to the protein-related polypeptide.

Activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) is assessed by measuring seed protein content by standard methods for measuring protein content in a plant sample, 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). Protein content can also be measured by the Dumas method, by combusting samples at a high temperature in the presence of high-purity oxygen, analyzing the gas from combustion for nitrogen content using a thermal conductivity detector, and calculating the amount of protein present in the sample using a conversion factor. The industry standard conversion factor for soybean is 6.25. Activity of the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5) is also measured by measuring activity of the respective protein-related polypeptide. For example, activity of XTH16 or XTH16- A is measured by standard methods for measuring hydrolase activity on xyloglucan (e.g., enzymatic assay), xyloglucan endotransglucosylase (XET) activity (e.g., enzymatic assay), or activity to stimulate growth of hypocotyls (e.g., growth assay). Activity of BF4 is measured by standard methods for measuring vacuolar invertase activity (e.g., enzymatic assay), or standard methods for measuring total or specific sugar content (e.g., total sugar, sucrose, glucose, fructose, galactose, maltose, lactose) in plant samples [e.g., spectroscopy (near infrared spectroscopy), refractometry, solid-phase extraction (SPE), solid-phase micro-extraction (SPME), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GCMS), and/or enzymatic assay]. Activity of PMT or PMT5 is measured by standard methods for measuring activity to transport (symport) polyol, cyclitol, monosaccharide, and H+ across plasma membrane (e.g., enzymatic assay), or activity to catalyze the energy-dependent membrane passage of linear polyols, cyclic polyols, and monosaccharides (e.g., enzymatic assay).

The plant with mutation and desirable phenotype is selected, e.g., having increased activity or function of protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), increased expression levels of the protein-related genes (e.g., XTH16, XTH16-A, bF4, PMT, PMT 5) or the protein-related polypeptide (e.g., XTH16, XTH16-A, BF4, PMT, PMT5), or increased protein content as compared to a control plant (e.g., without the mutation) when grown under the same environmental conditions.

EXAMPLE 5: Increased Seed Protein Content in XTH16 Promoter-Edited or Transgenic Soybean Plants

Embryonic axes of mature seeds of soybean varieties were stably transformed with constructs comprising a nuclease and a guide RNA targeting the GmXTH16 gene using Agrobacterium transformation. The targeting sequence of the GmXTHl 6 gRNA is encoded by SEQ ID NO: 13. Plants A-C, containing a 6 bp, 12 bp, and 10 bp deletion, respectively, in the GmXTHl 6 promoter and overexpressing GmXTHl 6 WCK generated. The mutated GmXTH16 promoter of Plant A-C contain SEQ ID NO: 18, 17, and 16, respectively.

As shown in FIGs. 1 and 2 and Table 3, significant increases in GmXTHl 6 level were detected in Plant B in the V3 leaf, R3 leaf, and 25 DAP pod wall, and in Plant C in the R3 leaf, 25 DAP embryo, and 25 DAP pod wall as compared to the null plants.

As shown in FIG. 3 and Table 4, Plants A-C demonstrated increased seed protein content relative to a WT or null soybean plant, as measured by near infrared spectrometry (NIR). The seed protein content in Plant B (with a 12 bp deletion) demonstrated a statistically significant increase relative to the null plant (p = 0.04) and the wildtype plant (p = 0.02).

Soybean plants were transformed with a construct comprising the GmXTH gene operably linked to a native GmXTHl 6 promoter or a CHS7 promoter. Three transgenic plants overexpressing GmXTH 16 under the native GmXTH16 promoter (Plants GM Nl, GM N2, and GM N3) and two transgenic plants overexpressing GmXTH 16 under the CHS7 promoter (Plants GM C2 and GM C3) were generated. As shown in FIG. 4 and Table 5, the five GmXTHl 6 transgenic plants exhibited increased seed protein content relative to a null or WT plant, as measured by NIR. Plants GM N2 and GM N3 exhibited statistically significant increase in seed protein content relative to a WT plant. The seed protein content in Plants N2 and N3 demonstrated a statistically significant increase relative to the wildtype plant (p = 0.08 and 0.04, respectively).

As shown in FIG. 5, the GmXTH 16 expression levels in V3 leaves correlated with seed protein content in the GmXTH 1 <5-overexpressing plants, generated either by gene editing (Plants A, B, C) or gene modification (Plants GM Nl, GM N2, GM N3, GM C2, GM C3).

TABLE 3. XTH16 Expression in Mutant and Wild-Type Lines Compared to Null Group

TABLE 4. Protein Content in XTH16-Edited Plants

* indicates p < 0.05 TABLE 5. Protein Content in Transgenic XTH16 Plants

* indicates p < 0.05

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 activity of a protein-related polypeptide compared to a control plant or plant part, wherein said plant or plant part comprises a genetic mutation and/or a transgene that increases the activity of said protein-related polypeptide, and wherein said protein- related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane -localized polyol/cyclitol/monosaccharide-H⁺- symporter (PMT), and PMT5.

2. The plant or plant part of claim 1, wherein said protein-related polypeptide is XTH16.

3. The plant or plant part of claim 1 or 2, comprising increased protein content compared to a control plant or plant part.

4. The plant or plant part of any one of claims 1-3, wherein the mutation comprises one or more insertions, substitutions, or deletions in at least one protein-related gene or homolog thereof or in a regulatory region thereof in said plant or plant part, wherein said at least one protein-related gene or homolog encodes said protein-related polypeptide , and wherein an expression level of said at least one protein-related gene or homolog thereof is increased compared to an expression level the gene or homolog thereof in a plant or plant part without said mutation.

5. The plant or plant part of any one of claims 1-4, wherein the mutation comprises one or more insertions, substitutions, or deletions in at least one protein-related gene or homolog thereof or in a regulatory region thereof in said plant or plant part, wherein said at least one protein-related gene or homolog encodes said protein-related polypeptide, and wherein said mutation increases level or activity of said protein-related polypeptide compared to level or activity of a copy of said protein-related polypeptide in a plant or plant part without said mutation.

6. The plant or plant part of claim 4 or 5, wherein the mutation is located at least partially in the regulatory region of said at least one protein-related gene or homolog thereof, wherein said at least one protein-related gene comprises XTH16. XTH16-A, bF4, PMT, or PMT5.

7. The plant or plant part of claim 5, wherein the mutation is located at least partially in a promoter region or 5’ untranslated region (5’UTR) of said A/77/6. A77//6-4. bF4, PMT, or PMT5 or homolog thereof.

8. The plant or plant part of claim 6 or 7, wherein said at least one protein-related gene is XTH16.

9. The plant or plant part of any one of claims 4-8, wherein the mutation is located at least partially in a protein-related gene or regulatory region thereof, wherein: (i) said protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity;

(ii) said protein-related gene comprises the nucleic acid sequence of any one of SEQ ID NOs: 7-9;

(iii) said protein-related 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: 4-6, wherein said polypeptide retains protein-related activity;

(iv) said protein-related gene encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4-6;

(v) said protein-related gene including said 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: 1-3, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; and/or

(vi) said protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of any one of SEQ ID NOs: 1-3.

10. The plant or plant part of claim 9, wherein:

(i) said protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity;

(ii) said protein-related gene comprises the nucleic acid sequence of SEQ ID NO: 7;

(iii) said protein-related gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 4, wherein said polypeptide retains protein-related activity;

(iv) said protein-related gene encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 4;

(v) said protein-related gene including said regulatory region thereof comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; and/or

(vi) said protein-related gene including said regulatory region thereof comprises the nucleic acid sequence of SEQ ID NO: 1.

11. The plant or plant part of any one of claims 4-10, wherein said mutation is located at least partially in: the nucleic acid sequence of a Glycine max XTH16. XTH16-A, bF4, PMT, or PMT 5 gene; or a promoter region of a Glycine max XTH16, XTH16-A, bF4, PMT, or PMT5 gene.

12. The plant or plant part of claim 11, wherein the mutation comprises a deletion of 2-12 nucleotides at least partially in the promoter region of the Glycine max XTH16, XTH16-A, bF4, PMT, or PMT 5 gene.

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

(i) a nucleic acid sequence of SEQ ID NO: 16, or a deletion of nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter;

(ii) a nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter;

(iii) a nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter;

(iv) a first allele comprising the nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, and a second allele comprising the nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter;

(v) a nucleic acid sequence of SEQ ID NO: 19, or a deletion of nucleotides 2140 through 2141 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter;

(vi) a nucleic acid sequence of SEQ ID NO: 20, or a deletion of nucleotides 2138 through 2144 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter;

(vii) a nucleic acid sequence of SEQ ID NO: 21, or a deletion of nucleotides 1371 through 1374 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter;

(viii) a nucleic acid sequence of SEQ ID NO: 22, or a deletion of nucleotides 1367 through 1377 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter; and/or

(ix) a nucleic acid sequence of SEQ ID NO: 23, or a deletion of nucleotides 1371 through 1378 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter.

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

(ii) a nucleic acid sequence of SEQ ID NO: 17, or a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter; and/or

(iii) a nucleic acid sequence of SEQ ID NO: 18, or a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter.

15. The plant or plant part of any one of claims 4-14, wherein said mutation comprises an out- of-frame mutation of the at least one protein-related gene or homolog thereof.

16. The plant or plant part of any one of claims 4-14, wherein said mutation comprises a nonsense mutation of the at least one protein-related gene or homolog thereof.

17. The plant or plant part of any one of claims 4-16, wherein said at least one protein-related gene or homolog thereof is an endogenous copy of said gene.

18. The plant or plant part of any one of claims 4-16, wherein said at least one protein-related gene or homolog thereof is an exogenous copy of said gene.

19. The plant or plant part of any one of claims 1-3, wherein the plant or plant part comprises the transgene, said transgene comprising a nucleic acid sequence of at least one protein-related gene or homolog thereof operably linked to a promoter, wherein said at least one protein-related gene or homolog thereof encodes said protein-related polypeptide, and wherein an expression level of said at least one protein-related gene or homolog thereof is increased compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said transgene, or level or activity of said protein-related polypeptide is increased compared to level of activity of said protein-related polypeptide in a plant or plant part without said transgene.

20. The plant or plant part of claim 19, wherein said protein-related polypeptide is XTH16, and said promoter is a native XTH16 promoter.

21. The plant or plant part of claim 19, wherein said protein-related polypeptide is XTH16, and said promoter is a heterologous promoter.

22. The plant or plant part of any one of claims 1-21, wherein said plant or plant part comprises 2-5 genes encoding said protein-related polypeptide.

23. The plant or plant part of claim 22, wherein said 2-5 genes have less than 100% sequence identity to one another.

24. The plant or plant part of any one of claims 1-23, wherein said plant or plant part is a legume.

25. The plant or plant part of claim 24, wherein said plant or plant part is selected from the group consisting of soybean (Glycine max), beans (Phaseolus spp., Vigna spp.), common bean (Phaseolus vulgaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), adzuki bean (Vigna angularis), fava bean (Vida faba), 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.).

26. The plant or plant part of any one of claims 1-23, 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 (Solcinum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbcidense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculentci), coffee (Coffeci spp ), coconut (Cocos nuciferci), 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.

27. The plant or plant part of any one of claims 1-26, wherein said plant or plant part is a seed.

28. A population of plants or plant parts comprising the plant or plant part of any one of claims 1-27, wherein the population comprises increased activity of said protein-related polypeptide and/or increased protein content compared to a control population.

29. The population of plants or plant parts of claim 28, wherein said plant or plant part is a seed, and said population is a population of seeds.

30. A method for increasing protein content in a plant or plant part, said method comprising increasing level or activity of at least one endogenous gene encoding a protein-related polypeptide in said plant or plant part, wherein said protein-related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane- localized polyol/cyclitol/monosaccharide-H⁺-symporter (PMT), and PMT5.

31. A method for increasing protein content in a plant or plant part, said method comprising introducing a genetic mutation or a transgene that increases activity of a protein-related polypeptide into said plant or plant part, wherein said protein-related polypeptide is selected from the group consisting of xyloglucan endotransglucosylase/hydrolase 16 (XTH16), XTH16-A, betaFruct4 (BF4), plasma membrane- localized polyol/cyclitol/monosaccharide-H⁺-symporter (PMT), and PMT5.

32. The method of claim 31, wherein said protein-related polypeptide is XTH16.

33. The method of claim 31 or 32, further comprising introducing the genetic mutation or the transgene that increases activity of said protein-related polypeptide into a plant cell, and regenerating said plant or plant part from said plant cell.

34. The method of any one of claims 31-33, wherein the method comprises introducing the genetic mutation into at least one protein-related gene or homolog thereof or in a regulatory region thereof in said plant or plant part or plant cell, wherein the mutation comprises one or more insertions, substitutions, or deletions, and wherein said at least one protein-related gene or homolog thereof encodes said protein-related polypeptide, and wherein: an expression level of said at least one protein-related gene or homolog thereof is increased compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said mutation; and/or level or activity of said protein-related polypeptide is increased compared to level or activity of said protein-related polypeptide in a plant or plant part without said mutation.

35. The method of claim 34, wherein the mutation is introduced to locate at least partially in the regulatory region of said at least one protein-related gene or homolog thereof, wherein said at least one protein-related gene comprises XTH16. XTH16-A, bF4, PMT, or PMT5.

36. The method of claim 35, wherein the mutation is introduced to locate at least partially in a promoter region or 5’ untranslated region (5’UTR) of

XTH 16. XTH 16-A. bF4, PMT, or PMT5 or homolog thereof.

37. The method of claim 35 or 36, wherein said at least one protein-related gene is at least one copy MXTH16.

38. The method of any one of 34-47, wherein the mutation is introduced at least partially into a protein-related gene or regulatory region thereof, wherein:

(i) said protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 7-9, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity;

39. The method of claim 38, wherein:

(i) said protein-related gene comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 7, wherein said nucleic acid sequence encodes a polypeptide that retains protein-related activity; (ii) said protein-related gene comprises the nucleic acid sequence of SEQ ID NO: 7;

40. The method of any one of 34-39, wherein the mutation is introduced at least partially in: the nucleic acid sequence of a Glycine max XTH16. XTH16-A, bF4, PMT, or PMT5 gene; or a promoter region of a Glycine max XTH16, XTH16-A, bF4, PMT, or PMT5 gene.

41. The method of claim 40, wherein the mutation comprises a deletion of 2- 12 nucleotides at least partially in the promoter region of the Glycine max XTH16, XTH16-A, bF4, PMT, or PMT 5 gene.

42. The method of claim 41, wherein:

(i) the mutation comprises a deletion of one or more nucleotides 1483 through 1492 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 16 when said mutation is introduced;

(ii) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 17 when said mutation is introduced;

(iii) the mutation comprises a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced;

(iv) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter in a first allele and a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter in a second allele, or said plant or plant part comprises a first allele comprising the nucleic acid sequence of SEQ ID NO: 17 and a second allele comprising the nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced;

(v) the mutation comprises a deletion of nucleotides 2140 through 2141 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 19 when said mutation is introduced; (vi) the mutation comprises a deletion of nucleotides 2138 through 2144 of SEQ ID NO: 2 or 11 in the Glycine max bF4 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 20 when said mutation is introduced;

(vii) the mutation comprises a deletion of nucleotides 1371 through 1374 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 21 when said mutation is introduced;

(viii) the mutation comprises a deletion of nucleotides 1367 through 1377 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 22 when said mutation is introduced; and/or

(ix) the mutation comprises a deletion of nucleotides 1371 through 1378 of SEQ ID NO: 3 or 12 in the Glycine max PMT5 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 23 when said mutation is introduced.

43. The method of claim 42, wherein:

(ii) the mutation comprises a deletion of nucleotides 1484 through 1495 of SEQ ID NO: 1 or 10 in the Glycine max XTH 16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 17 when said mutation is introduced; and/or

(iii) the mutation comprises a deletion of nucleotides 1486 through 1491 of SEQ ID NO: 1 or 10 in the Glycine max XTH16 promoter, or said plant or plant part comprises a nucleic acid sequence of SEQ ID NO: 18 when said mutation is introduced.

44. The method of any one of claims 34-43, wherein introducing the mutation comprises introducing an out-of-frame mutation into said at least one protein-related gene or homolog thereof.

45. The method of any one of claims 34-44, further comprising introducing editing reagents or a nucleic acid construct encoding said editing reagents into said plant, plant part, or plant cell.

46. The method of claim 45, wherein said editing reagents comprise at least one nuclease, wherein the nuclease cleaves a target site in said at least one protein-related gene or homolog thereof or a regulatory region thereof in said plant, plant part, or plant cell, and said mutation is introduced at said cleaved target site.

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

48. The method of claim 47, 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 an ortholog of any thereof.

49. The method of any one of claims 45-48, wherein the editing reagents comprise one or more guide RNAs (gRNAs).

50. The method of claim 49, wherein the one or more gRNAs comprise a nucleic acid sequence complementary to a region of a genomic DNA sequence encoding said protein-related polypeptide or regulating transcription or translation of said protein-related polypeptide in said plant or plant part.

51. The method of claim 49 or 50, wherein at least one of the one or more gRNAs binds a nucleic acid region corresponding to a promoter region of a Glycine max XTH16, XTH16, bF4, PMT, PMT5 gene in said plant or plant part.

52. The method of any one of claims 49-51, wherein at least one of the one or more gRNAs comprises a nucleic acid sequence encoded by:

(i) a nucleic acid sequence that shares at least 80% sequence identity with a nucleic acid sequence of SEQ ID NOs: 10-15; or

(ii) the nucleic acid sequence of SEQ ID NOs: 10-15.

53. The method of claim 52, wherein at least one of the one or more gRNAs comprises a nucleic acid sequence encoded by:

(i) a nucleic acid sequence that shares at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 13; or

(ii) the nucleic acid sequence of SEQ ID NO: 13.

54. The method of any one of claims 34-53, wherein said at least one protein-related gene or homolog thereof is an endogenous copy of said gene.

55. The method of any one of claims 34-53, wherein said at least one protein-related gene or homolog thereof is an exogenous copy of said gene.

56. The method of any one of claims 31-33, comprising introducing the transgene into said plant, plant part, or plant cell, wherein the transgene comprises a nucleic acid sequence of at least one protein-related gene or homolog thereof operably linked to a promoter, wherein said at least one protein- related gene or homolog thereof encodes said protein-related polypeptide, and wherein the method increases an expression level of said at least one protein-related gene or homolog thereof compared to an expression level of said at least one protein-related gene or homolog thereof in a plant or plant part without said transgene, or level or activity of said protein-related polypeptide compared to level of activity of said protein- related polypeptide in a plant or plant part without said transgene.

57. The method of claim 56, wherein said protein-related polypeptide is XTH16, and said promoter is a native XTH16 promoter.

58. The method of claim 56, wherein said protein-related polypeptide is XTH16, and said promoter is a heterologous promoter.

59. The method of any one of claims 30-58, wherein said plant or plant part is a legume.

60. The method of claim 59, wherein said plant or plant part is selected from the group consisting of soybean (Glycine max), beans (Phaseolus spp., Vigna spp.), common bean (Phaseolus vulgaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), adzuki bean (Vigna angularis), fava bean (Vida faba), 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.).

61. The method of any one of claims 30-58, 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 tindorius), 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.

62. A plant or plant part produced by the method of any one of claims 30-61, wherein said plant or plant part comprises increased activity of said protein-related polypeptide compared to a control plant or plant part.

63. The plant or plant part of claim 62, comprising increased protein content compared to a control plant or plant part.

64. The plant or plant part of claim 62 or 63, wherein said plant or plant part is a seed.

65. A population of plants or plant parts produced by the method of any one of claims 30-61, wherein the population comprises increased activity of said protein-related polypeptide and/or increased protein content compared to a control population.

66. The population of plants or plant parts of claim 65, wherein said population is a population of seeds.

67. A seed composition produced from the plant, plant part, or population of plants or plant parts of any one of claims 1-29 and 62-66.

68. A protein composition produced from the plant, plant part, or population of plants or plant parts of any one of claims 1-29 and 62-66, or the seed composition of claim 67.

69. A food or beverage product comprising the plant, plant part, or population of plants or plant parts of any one of claims 1-29 and 62-66, the seed composition of claim 67, or the protein composition of claim 68.

70. A nucleic acid molecule comprising a nucleic acid sequence of a mutated protein-related gene or coding sequence thereof, wherein said nucleic acid sequence comprises any one of SEQ ID NOs: 1- 3 and 7-9 comprising one or more insertions, substitutions, or deletions therein.

71. A DNA construct comprising, in operable linkage :

(i) a promoter that is functional in a plant cell; and

(ii) the nucleic acid molecule of claim 70.

72. A nucleic acid molecule comprising a nucleic acid sequence of a mutated promoter of a protein-related gene, wherein said nucleic acid sequence comprises:

(i) a nucleic acid sequence of any one of SEQ ID NOs: 10-12 comprising one or more insertions, substitutions, or deletions therein; or

(ii) a nucleic acid sequence of any one of SEQ ID NOs: 16-23.

73. A DNA construct comprising, in operable linkage:

(i) the nucleic acid molecule of claim 72; and

(ii) a polynucleotide of interest.

74. A cell comprising the nucleic acid molecule of claim 70 or 72, or the DNA construct of claim 71 or 73.

75. The cell of claim 74, wherein the cell is a plant cell.