Compositions and methods comprising plants with modified seed composition

Abstract

Provided herein are plants, plant parts, a population of plants or plant parts, and plant products (e.g., seed compositions, protein compositions, oil compositions, feed, food, and beverage products) comprising reduced sucrose efflux transporter activity, and compositions and methods of producing such plants and plant parts. The plants, plant parts, population of plants or plant parts, or plant products can have one or more genetic mutations that reduces the Sugars Will Eventually Be Exported Transporter (SWEET) activity, which can be located at least partially in a SWEET39 gene or its homolog or its regulatory region, and can have increased protein content, altered seed composition, and / or altered seed size.

Description

[0001] Atorney Docket No.: B88552 1710WO (00456)

[0002] COMPOSITIONS AND METHODS COMPRISING PLANTS WITH MODIFIED SEED COMPOSITION

[0003] RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 745,206, filed on January 14, 2025, the content of which is incorporated herein by reference in its entirety.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing which is submitted herewith in electronically readable format. The Sequence Listing file was created on January 13, 2026, is named “B88552_1710_SL.xml” and its size is 28,758 bytes. The entire contents of the Sequence Listing in the XML file are incorporated by reference herein.

[0007] FIELD OF THE INVENTION

[0008] The present disclosure relates to the field of agricultural biotechnology. More specifically, this disclosure relates to plants and plant parts having modified protein content, seed composition, or seed size, and associated methods and compositions.

[0009] BACKGROUND OF THE INVENTION

[0010] 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, feed, food, or beverage products, and industrial materials. Providing plants and seeds that possess high protein content could offer important commercial advantages.

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[0013] SUMMARY OF THE INVENTION

[0014] Plants and plant parts comprising decreased sucrose efflux transporter activity are provided. Compositions and methods for producing such plants and plant parts, and products (e.g., seed compositions, protein compositions, oil compositions, feed, food, beverage products) 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 decreases Sugars Will Eventually be Exported Transporter (SWEET) activity, e.g., one or more mutations in at least one SWEET39 gene (e.g., SWEET39A gene (e.g., Glyma.l5G049200), SWEET39B gene (e.g., Glyma.08G183500 ) or its homolog or in its regulatory region (e.g., promoter, 5’UTR); decreased expression levels of at least one SWEET39 gene; decreased SWEET39 activity; decreased protein content; decreased oil content; decreased sucrose content; altered seed composition; and / or altered seed size compared to a control plant or plant part.

[0015] In one aspect, provided herein is a plant or plant part comprising decreased sucrose efflux transporter activity compared to a control plant or plant part, wherein said plant or plant part comprises one or more genetic mutations that decreases sucrose efflux transporter activity, e.g., Sugars Will Eventually be Exported Transporter (SWEET) activity, e.g., SWEET39 activity.

[0016] In some embodiments of the plants or plant parts provided herein, the plant or plant part comprises increased protein content compared to a control plant or plant part.

[0017] In some embodiments of the plants or plant parts provided herein, the one or more mutations comprise one or more insertions, substitutions, or deletions in at least one SWEET39 gene or homolog thereof or regulatory region thereof in said plant or plant part. An expression level of said at least one SWEET39 gene or homolog thereof is reduced compared to a corresponding native SWEET39 gene or homolog thereof without said mutation, and / or level or activity of SWEET39 encoded by said at least one SWEET39 gene or homolog thereof is reduced compared to SWEET39 encoded by a corresponding native SWEET39 gene or homolog thereof without said mutation.

[0018] In some embodiments of the plants or plant parts provided herein, the one or more mutations comprise one or more insertions, substitutions, or deletions in two or more SWEET39 genes or homologs thereof or regulatory regions thereof in said plant or plant part. An expression level of said two or more SWEET39 genes or homologs thereof is reduced compared to a corresponding native SWEET39 gene or homolog thereof without said mutation, and / or level or activity of SWEET39 encoded by said two or more SWEET39 genes or homologs thereof is reduced compared to SWEET39 encoded by a corresponding native SWEET39 gene or homolog thereof without said mutation.

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[0021] In some embodiments of the plants or plant parts provided herein, the at least one SWEET39 gene is a SWEET39A gene or a SWEET39B gene.

[0022] In some embodiments of the plants or plant parts provided herein, the mutation is located at least partially in a SWEET39 gene or homolog thereof: (i) comprising a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1 or 2, wherein said nucleic acid sequence encodes a polypeptide that retains sucrose efflux transporter activity; (ii) comprising the nucleic acid sequence of SEQ ID NO: 1 or 2; (iii) encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 3 or 4, wherein said polypeptide retains sucrose efflux transporter activity; (iv) encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or 4; and / or in a regulatory region of said SWEET39 gene or homolog thereof.

[0023] In some embodiments of the plants or plant parts provided herein, the plant or plant part comprises a deletion of one or more nucleotides of SEQ ID NO: 1 in the Glycine max SWEET39A gene and / or SEQ ID NO: 2 in the Glycine max SWEET39B gene.

[0024] In some embodiments of the plants or plant parts provided herein, the plant or plant part comprises (i) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8, or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene; (ii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene; (iii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene; (iv) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene; (v) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene; and / or (vi) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene.

[0025] In some embodiments of the plants or plant parts provided herein, the plant or plant part comprises (i) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or (ii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or a polynucleotide comprising a nucleic acid

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[0028] sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene.

[0029] In some embodiments of the plants or plant parts provided herein, said mutation comprises an out-of-frame mutation of the at least one SWEET39 gene or homolog thereof. In some embodiments, said mutation comprises a nonsense mutation of the at least one SWEET39 gene or homolog thereof.

[0030] In some embodiments of the plants or plant parts provided herein, said plant or plant part comprises 2 or more genes encoding SWEET39. In some embodiments, said 2 or more genes have at least 80% sequence identity (e.g., less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 93%, 82%, 81%, or 80%; or 80-85%, 80-90%, 80-99%, 85-90%, 85-95%, 85-99%, 90-95%, 90-99%, or 95-99%) to one another and retain sucrose efflux transporter activity.

[0031] In some embodiments of the plants or plant parts provided herein, the plant or plant part comprises decreased oil content, decreased sucrose content, altered seed composition, and / or decreased seed size compared to a control plant or plant part. In some embodiments, (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis in the plant or plant part as compared to a control plant or plant part.

[0032] In some embodiments of the plants or plant parts provided herein, said plant or plant part is a legume. In some embodiments of the plants or plant parts provided herein, said plant or plant part is soybean (Glycine max'). In some embodiments of the plants or plant parts provided herein, said plant or plant part is pea (Pisum sativum).

[0033] In some embodiments of the plants or plant parts provided herein, 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 (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 (Cer atonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.).

[0034] In some embodiments of the plants or plant parts provided herein, said plant or plant part is selected from the group consisting of corn (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),

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[0037] foxtail millet (Setaria italica), finger millet Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aesliviim). tobacco (Nicotiana labacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsulum), sweet potato (Ipomoea balalus), 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 integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[0038] In some embodiments of the plants or plant parts provided herein, said plant or plant part is a seed.

[0039] In one aspect, provided herein is a population of plants or plant parts comprising the plant or plant part provided herein, wherein the population comprises decreased sucrose efflux transporter activity, increased protein content, decreased oil content, decreased sucrose content, and / or decreased seed size compared to a control population.

[0040] In some embodiments of the population of plants or plant parts provided herein, said plant or plant part is a seed, and said population is a population of seeds. In some embodiments, (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis in the population of plants or plant parts as compared to a control population.

[0041] In one aspect, provided herein is a method of increasing protein content in a plant or plant part, said method comprising reducing level or activity of a Sugars Will Eventually be Exported Transporter (SWEET) in said plant or plant part.

[0042] In some embodiments of the methods provided herein, the method comprises introducing one or more genetic mutations that decrease SWEET activity into said plant or plant part.

[0043] In some embodiments of the methods provided herein, the method further comprises introducing the one or more genetic mutation into a plant cell, and regenerating said plant or plant part from said plant cell.

[0044] In some embodiments of the methods provided herein, the one or more mutations comprise one or more insertions, substitutions, or deletions in at least one SWEET39 gene or homolog thereof or in a regulatory region thereof in said plant or plant part. An expression level of said at least one SWEET39 gene or homolog thereof is reduced compared to a corresponding native SWEET39 gene or homolog thereof without said mutation, and / or level or activity of SWEET39

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[0047] encoded by said at least one SWEET39 gene or homolog thereof is reduced compared to SWEET39 encoded by a corresponding native SWEET39 gene or homolog thereof without said mutation.

[0048] In some embodiments of the methods provided herein, the one or more mutations comprise one or more insertions, substitutions, or deletions in two or more SWEET39 genes or homologs thereof or regulatory regions thereof in said plant or plant part. An expression level of said two or more SWEET39 genes or homologs thereof is reduced compared to a corresponding native SWEET39 gene or homolog thereof without said mutation, and / or level or activity of SWEET39 encoded by said two or more SWEET39 genes or homologs thereof is reduced compared to SWEET39 encoded by a corresponding native SWEET39 gene or homolog thereof without said mutation.

[0049] In some embodiments of the methods provided herein, the at least one SWEET39 gene is a SWEET39A gene or a SWEET39B gene.

[0050] In some embodiments of the methods provided herein, the one or more mutations are introduced at least partially into a SWEET39 gene or homolog thereof: (i) comprising a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1 or 2, wherein said nucleic acid sequence encodes a polypeptide that retains sucrose efflux transporter activity; (ii) comprising the nucleic acid sequence of SEQ ID NO: 1 or 2; (iii) encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 3 or 4, wherein said polypeptide retains sucrose efflux transporter activity; (iv) encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or 4; and / or in a regulatory region of said SWEET39 gene or homolog thereof.

[0051] In some embodiments of the methods provided herein, the one or more mutations comprise a deletion of one or more nucleotides of SEQ ID NO: 1 in the Glycine max SWEET39A gene and / or SEQ ID NO: 2 in the Glycine max SWEET39B gene.

[0052] In some embodiments of the methods provided herein, (i) the one or more mutations comprise a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39 gene, or said plant or plant part comprises SEQ ID NO: 8 when said deletion is introduced; (ii) the one or more mutations comprise a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part comprises SEQ ID NO: 9 when said deletion is introduced; (iii) the one or more mutations comprise a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part comprises SEQ ID NO: 10 when said deletion is introduced; (iv) the one or more mutations comprise a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part

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[0055] comprises SEQ ID NO: 11 when said deletion is introduced; (v) the one or more mutations comprise a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NO: 9 when said deletion is introduced; (vi) the one or more mutations comprise a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39 gene, or said plant or plant part comprises SEQ ID NO: 10 when said deletion is introduced; (vii) the one or more mutations comprise or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 10 and 13 when said deletions are introduced, and / or (viii) the one or more mutations comprise a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 11 and 12 when said deletions are introduced.

[0056] In some embodiments of the methods provided herein, introducing the one or more mutations comprise introducing an out-of-frame mutation into said at least one SWEET39 gene or homolog thereof.

[0057] In some embodiments of the methods provided herein, the method further comprises introducing editing reagents or a nucleic acid construct encoding said editing reagents into said plant, plant part, or plant cell.

[0058] In some embodiments of the methods provided herein, said editing reagents comprise at least one nuclease. The nuclease cleaves a target site in said at least one SWEET39 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.

[0059] In some embodiments of the methods provided herein, the at least one nuclease comprises a CRISPR nuclease. In some embodiments, the CRISPR nuclease is a Type II CRISPR system nuclease, a Type V CRISPR system nuclease, a Cas9 nuclease, a Casl2a (Cpfl) nuclease, a Cmsl nuclease, or an ortholog of any thereof.

[0060] In some embodiments of the methods provided herein, the editing reagents comprise one or more guide RNAs (gRNAs).

[0061] In some embodiments of the methods provided herein, the one or more gRNAs comprise a nucleic acid sequence complementary to a region of a genomic DNA sequence encoding the SWEET39 or regulating transcription or translation of the SWEET39 in said plant or plant part.

[0062] In some embodiments of the methods provided herein, at least one of the one or more gRNAs comprises a nucleic acid sequence encoded by: (i) a nucleic acid sequence that shares at

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[0065] least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 7; or (ii) the nucleic acid sequence of SEQ ID NO: 7.

[0066] In some embodiments of the methods provided herein, said plant or plant part is a legume. In some embodiments of the methods provided herein, said plant or plant part is soybean (Glycine max). In some embodiments of the methods provided herein, said plant or plant part is pea (Pisum sativum).

[0067] In some embodiments of the methods provided herein, 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 (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 truncatuld), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.).

[0068] In some embodiments of the methods provided herein, said plant or plant part is selected from the group consisting of com (Zea mays), Brassica species, Brassica napus, Brassica rapa, Brassica juncea, rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet, pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Per sea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[0069] In one aspect, provided herein is a plant or plant part produced by the method provided herein, wherein said plant or plant part comprises reduced sucrose efflux transporter activity compared to a control plant or plant part.

[0070] In some embodiments, the plant or plant part produced by the method provided herein comprises increased protein content, decreased oil content, decreased sucrose content, altered seed composition, and / or decreased seed size compared to a control plant or plant part. In some

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[0073] embodiments, (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis in the plant or plant part compared to a control plant or plant part. In some embodiments of the plant or plant part produced by the method provided herein, said plant or plant part is a seed.

[0074] In one aspect, provided herein is a population of plants or plant parts produced by the method provided herein, wherein the population comprises decreased sucrose efflux transporter activity, increased protein content, decreased oil content, decreased sucrose content, and / or decreased seed size compared to a control population.

[0075] In some embodiments, the population of plants or plant parts is a population of seeds. In some embodiments, (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis in the population compared to a control population.

[0076] In one aspect, provided herein is a plant composition produced from the plant, plant part, or population plants or plant parts provided herein, wherein the plant composition is a seed composition, a protein composition, and / or an oil composition.

[0077] In some embodiments, provided herein is a plant product comprising the plant, plant part, or population of plants or plant parts or the seed composition provided herein, wherein the plant product is a feed, food, or beverage product.

[0078] In some embodiments of the plant composition or plant product provided herein, (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis in the plant composition or plant product compared to a control plant composition or plant product.

[0079] In one aspect, provided herein is a soybean composition comprising protein content of 50% dry basis or more and oil content of 15% dry basis or less, wherein the soybean composition is a seed composition, a protein composition, an oil composition, or a feed, food, or beverage product produced from soybean plants or seeds.

[0080] In some embodiments, the soybean composition comprises sucrose content of 5% dry basis or less.

[0081] In some embodiments, the soybean composition comprises a SWEET39 gene, homolog thereof, regulatory region thereof, or fragment thereof comprising a mutation.

[0082] In some embodiments, the soybean composition comprises: (i) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8, or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene; (ii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max

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[0085] SWEET39A gene; (iii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene; (iv) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene; (v) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene; (vi) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene; (vii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, and / or a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or a fragment of any thereof comprising the mutation.

[0086] In one aspect, provided herein is a nucleic acid molecule comprising a nucleic acid sequence of a mutated SWEET39 gene or coding sequence thereof, wherein said nucleic acid sequence comprises any one of SEQ ID NOs: 1, 2, 5, and 6 comprising one or more insertions, substitutions, or deletions therein.

[0087] In some embodiments of the nucleic acid molecule provided herein, the nucleic acid sequence of the mutated SWEET39 gene or coding sequence comprises any one of SEQ ID NOs: 8-13.

[0088] In one aspect, provided herein is a DNA construct comprising, in operable linkage: (i) a promoter that is functional in a plant cell; and (ii) the nucleic acid molecule provided herein.

[0089] In one aspect, provided herein is a cell comprising the nucleic acid molecule or the DNA construct provided herein. In some embodiments, the cell is a plant cell or a bacteria cell.

[0090] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts a box plot of seed protein content, as measured by near-infrared reflectance (NIR) in transformed soybean plants and controls. Plant A contains mutated Glyma.l5G049200 (SEQ ID NO: 8). Plant B contains mutated Glyma.08G 183500 (SEQ ID NO: 9). Plant C contains mutated Glyma.08G183500 (SEQ ID NO: 10). “WT” represents a control plant to which editing reagents have not been introduced.

[0091] FIGs. 2A and 2B depict seed protein and oil content, respectively, as measured by NIR in 10

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[0093] soybean plants with a SWEET39 mutation seeds and controls.

[0094] FIGs. 3 A and 3B depict seed protein and oil content (% dry basis), respectively, in SWEET39 double knockout soybean plants and controls grown in the field.

[0095] FIGs. 4A and 4B depict seed protein and oil content (% dry basis), respectively, in SWEET39 single knockout soybean plants and controls grown in the field.

[0096] DETAILED DESCRIPTION OF THE INVENTION

[0097] 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.

[0098] I. Definitions

[0099] 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.

[0100] 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.

[0101] 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.”

[0102] The term “about” or “approximately” usually means within 5%, or more preferably within 1%, of a given value or range.

[0103] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0104] 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,

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[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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 12

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[0113] 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 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 SWEET39 gene or a regulatory region of a SWEET39 gene.

[0114] 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.

[0115] 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 13

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[0117] 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 SWEET39 gene” includes the coding region of the SWEET39 gene, and may also include the regulatory region (e.g., promoter, 5’UTR) of the SWEET39 gene. Further, a “a SWEET39 gene” as used herein includes a homolog of a known a SWEET39 gene.

[0118] 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.

[0119] 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.

[0120] 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

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[0123] expression profiles, protein sequences, and / or sequences encoding RNA elements such as tRNAs, ribozymes, ribosome components, and microRNAs.

[0124] 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 SWEET39 gene or a regulatory region of a SWEET39 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 SWEET39 gene or a regulatory region of the SWEET39 gene has been deliberately mutated such that the plant, plant part or plant cell expresses a mutated (e.g., truncated) SWEET39 or have a reduced expression level of the SWEET39 gene or homolog or SWEET39. The mutated SWEET39 can have altered function, e.g., reduced function or loss-of-function, compared to a corresponding wild-type, or control, SWEET39 comprising no mutation.

[0125] “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.

[0126] 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.

[0127] 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 15

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[0129] 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.

[0130] 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.

[0131] “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).

[0132] 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.

[0133] 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.

[0134] 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,

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[0137] alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0138] 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.

[0139] 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.

[0140] The term “isolated” refers to at least partially separated from the natural environment e.g., from a plant cell.

[0141] 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.

[0142] 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.

[0143] 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

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[0146] heterologous gene that is from another plant species or another location within the genome of the modified cell.

[0147] 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 of 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.

[0148] “Homolog” or “homologous sequence” may refer to both orthologous and paralogous sequences. Paralogous sequence relates to gene-duplications within the genome of a species.

[0149] 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

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[0152] 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.

[0153] 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 function(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.

[0154] Homology (e.g., percent homology, sequence identity+sequence similarity) can be determined using any homology comparison software computing a pairwise sequence alignment.

[0155] 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.

[0156] 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth.

[0157] Enzymol.266: 131-141; Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402); Zhang et al. (2000), J. Comput. Biol. 7(1 -2):203- 14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word size=3; gap opening penalty=-ll; 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 19

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[0159] 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 Sci 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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 20

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[0165] 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.

[0166] 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 white flake 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).

[0167] 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,

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[0170] 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.

[0171] 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.

[0172] 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.

[0173] “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.

[0174] “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.

[0175] 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 22

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[0177] 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 extracellularly 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.

[0178] 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 23

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[0180] 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.

[0181] 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.

[0182] 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.

[0183] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

[0184] II. Overview of the Invention

[0185] Increased protein content in plants, plant parts (e.g., seeds), and plant products is an advantageous trait in the growing markets of feed, food, or beverages (e.g., plant-based food), and industrial use. “Protein content” of plants, plant parts, or plant products as used herein includes total protein content, white flake protein content, total amino acid content, or content of specific amino acids in the plants, plant parts, or plant products.

[0186] The SWEET family (Sugars Will Eventually Be Exported Transporter), also known as the PQ-loop, Saliva, or MtN3 family (TC# 2. A.123), is a family of sugar transporters and a member of the TOG superfamily. A SWEET is involved in the transport of sucrose in plant tissues. Sucrose produced during photosynthesis in the mesophyll cells of apoplasmic loaders is transported from cell to cell through the plasmodesmata to the phloem parenchyma cells and then is exported into the phloem apoplasm by the SWEET, which may be localized in the plasma membrane of phloem parenchyma cells. Subsequently sucrose is taken up and concentrated in the sieve element / companion cell complex by an H+-coupled sucrose symporter SUT / SUC energized by proton pump ATPase. More than 50 putative SWEET genes are expressed in various tissues in plants. For example, SWEET39 is highly expressed in seeds (e.g., soybean seeds; see Table 1). Without wishing to be bound by theory, SWEET39 may be associated with seed protein content, seed oil content, seed composition, and / or seed size. Altering level or activity of a SWEER39 gene or the sucrose efflux transporter encoded by the SWEET39 gene may alter the distribution of sugar,

[0187] 24

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[0189] protein, oil, and carbohydrate in the plant tissues, leading to altered seed composition and / or seed size.

[0190] Disclosed herein are plants or plant parts comprising one or more genetic mutations that decrease sucrose efflux transporter activity compared to a control plant or plant part, as well as methods for making the plants or plant parts with decreased sucrose efflux transporter activity. Such plants or plant parts can have one or more insertions, substitutions, or deletions in at least one native (e.g., wild-type) SWEET39A gene (e.g., Glyma.l5G049200) and / or native (e.g., wild-type) SWEET39B gene (e.g., Glyma.08G 183500) or in its regulatory region. The plants or plant parts can have a reduced expression level of the SWEET39 gene or homolog thereof, reduced level or sucrose efflux transporter activity encoded by the SWEET39 gene or homolog thereof, and / or increased protein content compared to a plant or plant part without the mutation.

[0191] 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 one or more genetic mutations that reduces sucrose efflux transporter activity. The methods disclosed herein can include introducing one or more insertions, substitutions, or deletions in at least one a SWEET39 gene or homolog thereof or in its regulatory region in the genome of a plant, plant part, or plant cell, such that an expression level of the SWEET39 gene or homolog thereof is reduced, level or sucrose efflux transporter activity encoded by the SWEET39 gene or homolog thereof is reduced, or protein content is increased in the plant, plant part (e.g., seed), or plant cell compared to a plant, plant part, or plant cell without the mutation. 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 SWEET39 gene or homolog thereof or in its regulatory region. Introducing two or more guide RNAs into a plant or plant part can increase sequence diversity of mutations generated in the plant genome.

[0192] Also disclosed herein are a population of plants or plant parts (e.g., seeds) having reduced sucrose efflux transporter activity and / or an increased protein content compared to a control population, and plant products (e.g., seed compositions, protein compositions, or feed, food, or beverage products) produced from the plants, plant parts, or population of plants or plant parts of the present disclosure.

[0193] Further provided herein are nucleic acid molecules comprising a mutated SWEET39 gene or its regulatory region (e.g., mutated promoter or 5’ UTR), a DNA construct comprising (i) the mutated SWEET39 gene operably linked to a functional promoter or (ii) the mutated regulatory region of the SWEET39 gene operably linked to a polynucleotide of interest, and cells comprising the nucleic acid molecule or the DNA construct of the present disclosure.

[0194] 25

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[0196] III. Plants with Increased Protein Content

[0197] Plants and plant parts are provided herein having altered (e.g., reduced) sucrose efflux transporter level or activity as compared to a control plant or plant part. “Sucrose efflux transporter activity”, “Sucrose Will Eventually be Exported Transporter activity”, “SWEET activity”, or “SWEET39 activity” refers to the enzymatic activity of a sucrose efflux transporter, such as a Sucrose Will Eventually be Exported Transporter (SWEET, e.g., SWEET39) to convert 2-phosphoglycerate to phosphoenol pyruvate. Sucrose efflux transporter activity”, “Sucrose Will Eventually be Exported Transporter activity”, “SWEET activity”, or “SWEET39 activity” also refers to the activities downstream of the enzymatic activity of the sucrose efflux transporter, such as activities to increase protein content or decrease oil content in plants, plant parts, and plant cells.

[0198] In particular aspects, plants and plant parts (e.g., seeds, leaves) disclosed herein have a genetic mutation that alters (e.g., increases) the sucrose efflux transporter activity. The plants or plant parts described herein having altered SWEET39 level or activity can comprise a genetic mutation or transgene that alters (e.g., reduces) SWEET39 level or activity, altered (e.g., reduced) expression levels of at least one a SWEET39A gene (e.g., Glyma.15G049200) or a SWEET39B gene (e.g., Glyma.08G183500 altered (e.g., increased) SWEET39 levels or activity, altered (e.g., increased) protein content, altered (e.g., decreased) oil and / or sucrose content, altered seed composition, and / or altered (e.g., decreased) seed size compared to a control plant or plant part.

[0199] Also provided herein is a population of plants and plant parts comprising the plants and plant parts described herein having altered (e.g., reduced) SWEET39 level or activity. In such population of plants or plant parts, having altered SWEET39 level or activity relative to a control population, not all individual plants or plant parts need to have altered (e.g., reduced) SWEET39 level or activity, genetic mutation that cause altered (e.g., reduced) SWEET39 level or activity, or phenotypes caused by the altered (e.g., reduced) sucrose efflux transporter activity (e.g., increased protein content, increased white flake protein content, altered protein metabolism, decreased oil content, decreased sucrose content, altered seed composition, decreased seed size). 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 SWEET39 level or activity.

[0200] 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.,

[0201] 26

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[0203] 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 saliva), 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 a part of Glycine max. 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, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago saliva , rice (Oryza saliva , rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgar e), camelina (Camelina saliva), 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 saliva , 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 saliva), 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 saliva) 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 27

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[0205] emmer), and other subspecies of Triticum turgidum (Feldman)), barley (Hordeum vulgare), maize (Zea mays), oats (Avena saliva), 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 Brom us spp.

[0206] A. Plants with altered level or activity of SWEET39

[0207] Provided herein are plants or plant parts (e.g., seeds) comprising altered (e.g., decreased) sucrose efflux transporter 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., reduced) sucrose efflux transporter activity compared to a control population provided herein.

[0208] The genetic mutation that alters (e.g., decreases) the sucrose efflux transporter activity in the plants and plant parts provided herein can comprise one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog thereof, or in a regulatory region of at least one native SWEET39 gene or homolog thereof. The genetic mutation that alters (e.g., decreases) the sucrose efflux transporter activity can be located in at least one native SWEET39 gene or homolog thereof; in a regulatory region of the native SWEET39 gene or homolog thereof; a coding region, a non-coding region, or a regulatory region of any other gene; or at any other site in the genome of the plant or plant part, a SWEET39 “gene”, as used herein, refers to any polynucleotide that encodes a polypeptide having sucrose efflux transporter activity. In some embodiments, a SWEET39 gene is a SWEET39A gene, e.g., Glyma.15G049200. In some embodiments, a SWEET39 gene is a SWEET39B gene, e.g., Glyma.08G183500. A SWEET39 gene, as used herein, can refer to a polynucleotide including a regulatory region (e.g., promoter, 5’UTR) of the SWEET39 gene, a SWEET39 gene can also include a homolog, ortholog, or variant of a known SWEET39 gene, that retains sucrose efflux transporter activity.

[0209] 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 SWEET39 gene located at a genomic site or in a plant in a non-naturally occurring matter is a “native” SWEET39 gene if its nucleic acid sequence can be found in a plant existing in nature.

[0210] 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 and interact to control mRNA synthesis of the gene, such as promoter regions, binding sites for transcription 28

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[0212] modulator proteins, and other genomic regions that contribute to regulation of transcription of the gene. A regulatory region of the gene can be located in the 5’ untranslated region of the gene.

[0213] A control plant or plant part can be a plant or plant part to which a mutation provided herein has not been introduced, e.g., by methods of the present disclosure. Thus, a control plant or plant part (e.g., seeds, leaves) may express a native (e.g., wild-type) SWEET39 gene endogenously or transgenically. A control plant of the present disclosure may be grown under the same environmental conditions (e.g., same or similar temperature, humidity, air quality, soil quality, water quality, and / or pH conditions) as a plant 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., decreased) expression levels of at least one SWEET39 gene or homolog thereof, altered (e.g., decreased) SWEET39 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.

[0214] 1. Plants with one or more mutations in at least one a SWEET39 sene, or its homolog, ortholog, or variant

[0215] In some aspects, the plants and plant parts of the present disclosure comprise decreased sucrose efflux transporter activity and a genetic mutation that decreases the sucrose efflux transporter 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 SWEET39 gene or homolog thereof and / or in a regulatory region of said at least one native SWEET39 gene or homolog 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 SWEET39 gene or homolog, each encoding SWEET39. In particular, a plant or plant part described herein can comprise at least 2 genes encoding SWEET39, such as 2, 3, 4, or 5 genes that have at least 80% (e.g., less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 93%, 82%, 81%, or 80%; or 80-85%, 80-90%, 80-99%, 85-90%, 85-95%, 85-99%, 90-95%, 90-99%, or 95-99%) sequence identity to one another, and retain sucrose efflux transporter (e.g., SWEET) activity. In specific embodiments, a plant or plant part has a SWEET39A gene and a SWEET39B gene. 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 SWEET39 gene or homolog; in a regulatory region of one SWEET39 gene or homolog; in more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all SWEET39 genes or homologs; in regulatory regions of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9,

[0216] 29

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[0218] 10), but not all SWEET39 genes or homologs; in all SWEET39 genes or homologs; and / or in regulatory regions of all SWEET39 genes or homologs in the plant or plant part.

[0219] 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 SWEET39 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.

[0220] In some embodiments, the mutation is located in a SWEET39 gene or its regulatory region, and (i) the SWEET39 gene comprises a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a nucleic acid sequence of SEQ ID NO: 1 (SWEET39A) or SEQ ID NO: 2 (SWEET39B), wherein the nucleic acid sequence encodes a polypeptide that retains sucrose efflux transporter activity; (ii) the SWEET39 gene comprises the nucleic acid sequence of SEQ ID NO: 1 (SWEET39A) or SEQ ID NO: 2 (SWEET39B); (iii) the SWEET39 gene encodes a polypeptide comprising an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 3 (SWEET39A) or SEQ ID NO: 4 (SWEET39B), wherein the polypeptide retains sucrose efflux transporter activity; and / or (iv) the SWEET39 gene encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 (SWEET39A) or SEQ ID NO: 4 (SWEET39B).

[0221] In specific embodiments, the mutation that decreases the sucrose efflux transporter activity is located in one or two alleles of a Glycine max SWEET39A gene (e.g., Glyma.15G049200) and / or SWEET39B gene (e.g., Glyma.08G 183500) or a regulatory region thereof. For example, a mutation can be located in two copies of the SWEET39 gene or homolog thereof, or one copy of the SWEET39 gene and one copy of its homolog, in order to reduce the expression of each gene to result in an increased protein content. In specific embodiments, plants and plant parts are provided that comprise a mutation in a SWEET39 gene as well as a SWEET39 gene homolog, and exhibit an increased protein content.

[0222] In the plant or plant part provided herein comprising a mutation that decreases the sucrose efflux transporter activity, at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertion, substitution, or deletion can be located at least partially in a coding region of Glycine max SWEET39 gene. As used herein, where an insertion, a substitution,

[0223] 30

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[0225] 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. In some embodiments, the plant or plant part contains a deletion of one or more nucleotides of SEQ ID NO: 1 (Glycine max SWEET39 (Glyma.l5G049200)) and / or SEQ ID NO: 2 (Glycine max SWEET39B (Glyma.08G183500y) in the Glycine max SWEET39 gene or homolog thereof.

[0226] For example, the plant or plant part can contain (i) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8, or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene. The plant or plant part can contain (ii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene. The plant or plant part can contain (iii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene. The plant or plant part can contain (iv) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene. The plant or plant part can contain (v) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, The plant or plant part can contain (vi) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The plant or plant part can contain any combination of the mutations provided in the present disclosure, such as mutations (i)-(vi) provided hereinabove.

[0227] Plants or plant parts can have a mutation (e.g., insertion, substitution, deletion) in more than one SWEET39 genes or homolog thereof or their regulatory regions, or in more than one copy of a SWEET39 gene or homolog or their regulatory regions. For example, the plant or plant part can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The plant or plant part can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The plant or plant part can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8 or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid 31

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[0229] sequence of SEQ ID NO: 12 or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The plant or plant part can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8 or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13 or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The plant or plant part can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9 or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12 or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The plant or plant part can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9 or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13 or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The plant or plant part can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12 or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The plant or plant part can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene.

[0230] The mutation that decreases the sucrose efflux transporter activity in the plant or plant part disclosed herein can comprise an out-of-frame mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) SWEET39 gene or homolog thereof. Alternatively, the mutation in the plant or plant part can comprise an in-frame mutation, a nonsense mutation, or a missense mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) SWEET39 gene or homolog thereof.

[0231] A plant or plant part of the present disclosure can have a genetic mutation that decreases the sucrose efflux transporter activity in a gene that is a homolog, ortholog, or variant of a SWEET39 gene disclosed herein and expresses a functional SWEET39, or in a regulatory region of such homolog, ortholog, or variant of a SWEET39 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%,

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[0234] 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, plants or plant parts comprising polynucleotides that have sucrose efflux transporter activity and share at least 75% sequence identity to the sequences disclosed herein are encompassed by the present disclosure and can have a genetic mutation that decreases the sucrose efflux transporter activity.

[0235] 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 etal. (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 SWEET39 can be identified and used in the methods of the present disclosure. The variant sequences will retain the sucrose efflux transporter activity.

[0236] In certain instances, mutations in any SWEET39 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 detection method described herein. Such detection methods may comprise use of primers for detecting mutation in a SWEET39 gene. For example, a forward primer (e.g., SEQ ID NO: 14) and a reverse primer (e.g., SEQ ID NO: 15) can be used for detection of a mutation in the Glycine max SWEET39A gene (Glyma.15G049200) near the binding site of the Glyma.15G049200 guide RNA (e.g., SEQ ID NO: 7), e.g., a mutation generated by introducing the Glyma.15G049200 guide RNA (e.g., SEQ ID NO: 7) into the plant or plant part. A forward primer (e.g., SEQ ID NO: 14) and a reverse primer (e.g., SEQ ID NO: 15) can be used for detection of a mutation in the Glycine max SWEET39B gene (Glyma.08G 183500) near the binding site of the Glyma.08G 183500 guide RNA (e.g., SEQ ID NO: 7), e.g., a mutation generated by introducing the Glyma.08G183500 guide RNA (e.g., SEQ ID NO: 7) into the plant or plant part.

[0237] In certain instances, a kit comprising a set of primers can be used for detecting mutation of SWEET39 genes 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: 14) and a reverse primer (e.g., SEQ ID NO: 15) can be used for detection of mutation in a mutation in the Glycine max SWEET39A gene (Glyma.15G049200) in plants, plant parts, or plant products (e.g., seed composition, plant protein compositions) near the binding site of the Glyma.15G049200 guide RNA (e.g., SEQ ID NO: 7). A kit comprising a forward primer (e.g., SEQ ID NO: 14) and a reverse 33

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[0239] primer (e.g., SEQ ID NO: 15) can be used for detection of mutation in Glycine max SWEET39B gene (Glyma.08G 183500) in plants, plant parts, or plant products (e.g., seed composition, plant protein compositions) near the binding site of the Glyma.08G 183500 guide RNA (e.g., SEQ ID NO: 7).

[0240] 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.

[0241] 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 decreases the sucrose efflux transporter activity described herein.

[0242] One or mutations insertions, substitutions, or deletions located in at least one SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog in the genome of the plant or plant part can reduce the expression levels of the SWEET39 gene or homolog, reduce level or sucrose efflux transporter activity encoded by the SWEET39 gene or homolog, reduce sucrose efflux transporter activity, and / or increase protein content in plant or plant part (e.g., seeds) relative to a control plant or plant part, e.g., when grown under the same environmental condition, as further described in the present disclosure.

[0243] 2. Plants with one or more mutations in regulatory region of a SWEET39 gene The plants or plant parts described herein can comprise a mutation that decreases the sucrose efflux transporter 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] in a regulatory region of at least one (e.g., one, more than one but not all, or all) SWEET39 gene. The SWEET39 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) SWEET39 gene to confer to the plant or plant part an altered (e.g., reduced) transcription activity of the SWEET39 gene. 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).

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[0246] In some embodiments, the mutation is in a promoter region of at least one (e.g., one, more than one but not all, or all) SWEET39A gene (e.g., Glyma.15G049200) and / or SWEET39B gene (e.g., Glyma.08G 183500). 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 SWEET39 gene can alter the transcription initiation activity of the promoter. For example, the modified promoter can reduce transcription of the operably linked nucleic acid molecule (e.g., the SWEET39 gene), initiate transcription in a developmentally-regulated or temporally-regulated manner, initiate transcription in a cell-specific, cell-preferred, tissue-specific, or tissue-preferred manner, or initiate transcription in an inducible manner. A deletion, a substitution, or an insertion, e.g., introduction of a heterologous promoter sequence, a cis-acting factor, a motif or a partial sequence from any promoter, including those described elsewhere in the present disclosure, can be introduced into the promoter region of the SWEET39 gene to confer an altered (e.g., reduced) transcription initiation function according to the present disclosure. The insertion, substitution, or deletion 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. 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

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[0249] 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) SWEET39 gene.

[0250] 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) SWEET39A gene (e.g., Glyma.15G049200) and / or SWEET39B gene (e.g., Glyma.08G 183500). 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, cisand trans-regulation).

[0251] 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) SWEET39 gene at or near one or more transcriptional regulator (e.g., transcriptional enhancer) binding domains. Mutation at or near the transcriptional regulator binding site can alter (e.g., decrease) binding of a transcription factor (e.g., transcriptional enhancer) and alter (e.g., decrease) level or activity of the SWEET39 gene.

[0252] In some embodiments, the plant or plant part of the present disclosure comprises a deletion of one or more nucleotides at least partially in the promoter and / or 5’UTR of a Glycine max SWEET39A gene (e.g., Glyma.15G049200) and / or SWEET39B gene (e.g., Glyma.08G183500).

[0253] 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 SWEET39 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 reduce expression levels of the SWEET39 gene, e.g., by inhibiting transcription initiation activity of the SWEET39 gene promoter. In some embodiments, the mutation modifies or inserts transcription factor binding sites or enhancer

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[0256] elements that regulates SWEET39 gene expression into the regulatory region of the SWEET39 gene.

[0257] In some embodiments, the mutation inserts a part or whole of one or more negative regulatory elements of the SWEET39 gene into the genome of a plant cell or plant part. A “negative regulatory element” of a gene, as used herein, refers to a nucleic acid molecule that suppresses expression or activity of the SWEET39 gene, e.g., by suppressing transcription activity of the promoter. The negative regulatory sequence of the gene can be in a cis location or in a trans location. Negative regulatory elements of the one or more SWEET39 genes can also include upstream open reading frames (uORFs). In some instances, a negative regulatory element can be inserted in a region upstream of the SWEET39 gene in order to inhibit the expression and / or function of the gene.

[0258] 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 SWEET39 gene, or other regulatory region of a SWEET39 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.

[0259] 3. Plants with reduced sucrose efflux transporter activity

[0260] The plants, plant parts (e.g., seeds, leaves), or plant products (e.g., seed composition, plant protein composition) of the present disclosure can comprise reduced activity of SWEET39 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 reduced sucrose efflux transporter activity compared to a control (e.g., wild-type) population of plants or plant parts.

[0261] In particular, the sucrose efflux transporter activity in the plant, plant part, population of plants or plant parts, or plant product of the present disclosure can be reduced by about 10-100%, 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-99%, 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least

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[0264] 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to a control plant, plant part, population, or plant product.

[0265] Sucrose efflux transporter activity can be measured by standard methods for assaying sucrose transporters, such as protoplast-esculin assays and the Forster resonance energy transfer (FRET) sensor-based assays.

[0266] Sucrose efflux transporter activity can also be 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), or 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.

[0267] In some embodiments, sucrose efflux transporter activity can also assessed by measuring seed oil content by standard methods for measuring oil content in a plant sample, for example by NIR, GC-MS optionally with certain modifications (e.g., with or without initial lipid extraction, with or without isotope labeling of analytes), or NMR.

[0268] 4. Plants with reduced expression level of SWEET39 gene or SWEET39 protein The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure, e.g., comprising one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog can have reduced expression level of the SWEET39 gene(s) or homolog as compared to the expression level of the SWEET39 gene or homolog in a control plant, plant part, a population of plants or plant parts, or plant product, e.g., a plant, plant part, a population of plants or plant parts, or plant product without such mutation. 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 reduced expression level of SWEET39 gene(s) or SWEET39 compared to a control (e.g., wild-type) population of plants or plant parts.

[0269] In particular, the expression levels of SWEET39 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) of the present disclosure can be reduced 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, SO- 38

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[0271] 90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to a control plant, plant part, a population of plants or plant parts, or plant product. In specific embodiments, expression levels of SWEET39 gene(s) or homolog in the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure is reduced, but is not completely eliminated, i.e., reduced by more than 0% and less than 100% as compared to the expression level of the SWEET39 gene or homolog in a control plant, plant part, a population of plants or plant parts, or plant product. Expression levels of the SWEET39 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 SWEET39 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 SWEET39 encoded by the SWEET39 gene.

[0272] The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure, e.g., comprising one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog can have reduced expression of the SWEET39, e.g., the SWEET39 encoded by the SWEET39 gene or homolog (having the mutation in the gene or in its regulatory region), as compared to the expression level of the SWEET39 in a control plant, plant part, a population of plants or plant parts, or plant product, e.g., a plant, plant part, a population of plants or plant parts, or plant product without such mutation. In particular, the expression levels of a full length SWEET39 in the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure can be reduced as compared to a control plant, plant part, a population of plants or plant parts, or plant product. A “full-length” SWEET39, as used herein, refers to a SWEET39 comprising the complete amino acid sequence of a wild-type SWEET39, e.g., encoded by a native SWEET39 gene. A plant, plant part, a population of plants or plant parts, or plant product that contains a mutated SWEET39 gene can have reduced expression of full-length SWEET39 as compared to a control plant, plant part, a population of plants or plant parts, or plant product, e.g., a plant, plant part, a population of plants or plant parts, or plant product without such mutation, e.g., a plant, plant part, a population of plants or plant parts, or plant product comprising a native (e.g., wild-type) SWEET39 gene. In some embodiments, in the plant, plant part, a

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[0275] population of plants or plant parts, or plant product of the present disclosure [e.g., comprising one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog], expression of SWEET39, e.g., full length SWEET39, e.g., encoded by the SWEET39 gene is reduced 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to expression of SWEET39, e.g., full length SWEET39 in a control plant, plant part, a population of plants or plant parts, or plant product. In specific embodiments, expression of SWEET39, e.g., full length SWEET39 in the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure is completely eliminated; or alternatively, reduced, but is not completely eliminated, i.e., reduced by more than 0% and less than 100%, as compared to a control plant, plant part, a population of plants or plant parts, or plant product.

[0276] Expression of a SWEET39, such as a full length SWEET39, 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 SWEET39 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 SWEET39, e.g., the full-length SWEET39.

[0277] 5. Plants with loss-of-function or reduced function of SWEET39

[0278] The plant, plant part (e.g., seeds, leaves), or plant product (e.g., seed composition, plant protein composition) of the present disclosure, e.g., comprising one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog can have loss-of-function or reduced function in the SWEET39, e.g., loss of sucrose efflux transporter activity or reduced sucrose efflux transporter activity, as compared to the SWEET39 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 loss-of-function or reduced function of the SWEET39 compared to a control (e.g., wild-type) population of plants or plant parts. 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, or a plant, plant part, a population of plants or plant parts, or plant product having wild-type sucrose efflux transporter activity. The 40

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[0280] SWEET39 with loss-of-function or reduced function can comprise a mutation compared to a wildtype SWEET39 that causes loss or reduction of SWEET39 function. In some embodiments, the function or sucrose efflux transporter activity encoded by the SWEET39 gene or homolog having a mutation (e.g., one or more insertions, substitutions, or deletions) in the gene or its regulatory region is reduced 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to function or activity of a control SWEET39 encoded by a control SWEET39 gene or homolog without such mutation. In specific embodiments, the SWEET39 function in the plant, plant part, a population of plants or plant parts, or plant product of the present disclosure is completely eliminated; or alternatively, reduced, but is not completely eliminated, i.e., reduced by more than 0% and less than 100%, as compared to a control plant, plant part, a population of plants or plant parts, or plant product.

[0281] SWEET39 function can be measured by standard enzymatic assay (e.g., chemiluminescence assay, fluorescence assay) for detecting products (including intermediates) generated by conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) and water, sucrose efflux transporter activity can also be measured by chemiluminescence or fluorescence measurement of ATP generated by combined actions of SWEET39 and pyruvate kinase.

[0282] SWEET39 function can also be 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), or 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.

[0283] 6. Plants with increased protein content

[0284] 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 that decreases sucrose efflux transporter activity, e.g., comprising one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or 41

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[0286] homolog, can have increased protein content as compared to a control plant, plant part, or plant product, e.g., without such mutation. 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 content as compared to a control population.

[0287] 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 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 may express a native (e.g., wild-type) SWEET39 gene endogenously or transgenically, and / or may have a wild-type sucrose efflux transporter activity. A plant, plant part, a population of plants or plant parts, or plant product of the present disclosure may 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.

[0288] 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, protein content, as expressed by % dry weight (% dry basis), 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%, 1.0-1.8%, 1.3-1.8%, 1.3-2.0%, 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%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, or 42

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[0290] 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 protein content. In specific embodiments, the plant, plant part, or population of the plants or plant parts has protein content that is greater by about 0.8%, about 1.0%, about 1.4% about 2.0%, about 2.7%, about 7.2%, about 11%, about 11.7%, at least about 0.8%, at least about 1.0%, at least about 1.4% at least about 2.0%, at least about 2.7%, at least about 7.2%, at least about 11%, at least about 11.7%, at least about 0.8-11.7%, at least about 0.8-11.0%, at least about 0.8-1.4%, at least about 1.4-11.7%, at least about 1.4-11%, at least about 7-11%, or at least about 11-12% dry weight relative to a control plant, plant part, or population.

[0291] 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 SWEET39, 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(l):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 specific embodiments, a control soybean cultivar has about 41.5%, about 44%, or about 46% seed protein content. In contrast, the soybean seeds or population of soybean seeds provided herein can have seed protein content (e.g., average seed protein content) of at least 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% or more by dry weight. In specific embodiments, the soybean seeds or population of soybean seeds provided herein can have seed protein content of about 42-52.5% dry weight, about 42-43% dry weight, about 42-53% dry weight, at least about 42.3% dry weight, at least about 43.5% dry weight, at least about 50% dry weight, at least about 51% dry weight, at least about 52% dry weight, at least about 52.5% dry weight, or at least about 53% dry weight, whereas control soybean seeds have protein content of about 41.5% dry weight. In specific embodiments, the soybean seeds or population of soybean seeds provided herein can have seed protein content of about 45.5-55% dry weight, about 45.5-48.5% dry weight, about 48.5-51% dry weight, about 51-55% dry weight, at least about 45.8% dry weight, at least about 48.5% dry weight, at least about 51.1% dry weight, or at least about 54.8% dry weight, whereas control soybean seeds have protein content of about 44-46% dry weight.

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[0294] Mutations in two or more SWEET39 genes can have synergistic effects in increase in protein content. For example, seeds or population of seeds having a mutation in one SWEET39 gene can have seed protein content of about 42-43% dry weight, about 42.3% dry weight, about 42.5% dry weight, about 42.9% dry weight, about 43.5% dry weight, and seeds or population of seeds having mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can have seed protein content of about 52-54% dry weight, about 52.5% dry weight, or about 53.2% dry weight, whereas control seeds have protein content of about 41.5% dry weight, seeds or population of seeds having a mutation in one SWEET39 gene can have seed protein content of about 48.5% dry weight, and seeds or population of seeds having mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can have seed protein content of about 51-55% dry weight, about 51.1% dry weight, or about 54.8% dry weight, whereas control seeds have protein content of about 44-46% dry weight. Protein content in a plant, plant part, plant product, or a population of plants or plant parts can be measured by standard methods for measuring total and specific amino acids in a plant sample, for example by high performance liquid chromatography (HPLC), spectrophotometer, mass spectrometry (MS), and combination thereof. 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), or nuclear magnetic resonance spectrometry (NMR). In specific embodiments, protein content is 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.

[0295] In specific embodiments, the plant, plant part, or a population of plants or plant parts of the present disclosure has 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 seed weight, seed size, seed count, or yield. In some embodiments, total seed count can be increased by about 1-10%, 2-10%, 3-10%, 4-10%, 5-10%, 6-10%, 7-10%, 8-10%, 2-9%, 3-9%, 4-9%, 5-9%, 6-9%, 7-9%, 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%, or more than 100% (e.g., by about 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or more than 100%), e.g., by about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 75.%, 8%, 85%, 9%, 9.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, or at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%,

[0296] 44

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[0298] 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in the plants or plant parts of the present disclosure as compared to a control plant or plant part. In specific embodiments, a control soybean cultivar has about 63 total average seed count per plant. In contrast, the plant, plant part, or population of plants or plant parts of the present disclosure can have total seed count of about 65-85 seeds per plant, e.g., about 65-70, 70-75, 75-80, 80-85, about 67, about 68, about 69, about 77, about 80, about 81, about 82, about 83, about 84, about 85, at least about 67, at least about 68, at least about 69, at least about 77, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, or at least about 85 average seeds per plant. Mutations in two or more SWEET39 genes can have synergistic effects in increase in total seed count. For example, seeds or population of seeds having a mutation in one SWEET39 gene can have average total seed count of about 67-77, about 67.4, about 68.3, about 70.1, or about 76.9 and seeds or population of seeds having mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can have average total seed count of about 80-84, about 80.1, or about 83.7, whereas control seeds have average total seed count of about 62.9.

[0299] In some embodiments, a reduction in seed weight (indicative of seed size) or yield in the plant, plant part, or population of plants or plant parts of the present disclosure, having increased protein content or increased seed count, 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 seed weight or yield as compared to a control plant, plant part, or population of plants or plant parts. In some embodiments, seed weight per 100 soybean seed is no more decreased by about 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, or 20 g per 100 seeds, e.g., no more decreased by about 1-20 g, 1-15 g, 1-11 g, 1-4 g, 4-11 g, or 9-11 g per 100 seeds in the plant, plant part, or population of plants or plant parts of the present disclosure as compared to a control plant, plant part, or plant population. In specific embodiments, a control soybean cultivar has seed weight of about 16.6 g per 100 seeds. In contrast, the soybean seeds provided herein can have seed weight of about 6-16 g, about 6-7 g, about 13-16 g, about 6.3 g, about 6.7 g, about 13.1 g, Ibout 13.8 g, about 14.7 g, or about 15.4 g.

[0300] Mutations in two or more SWEET39 genes can have synergistic effects in increase in seed weight. For example, seeds or population of seeds having a mutation in one SWEET39 gene can have seed weight of about 13-15.5 g, about 13.1 g, about 13.8 g, about 14.7 g, or about 15.4 g per 100 seeds, and seeds or population of seeds having mutations in two or more SWEET39 genes 45

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[0302] (e.g., a SWEET39A gene and a SWEET39B gene) can have seed weight of about 6-7 g, about 6.3 g, or about 6.7 g per 100 seeds, whereas control seeds have seed weight of about 16.6 g per 100 seeds.

[0303] Seed weight or 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.

[0304] In specific embodiments, provided herein are seeds and a population of seeds with decreased sucrose efflux transporter activity provided herein, having increased protein content as compared to control seeds or a population of seeds.

[0305] 7. Plants with altered oil content or sugar content, or seed size

[0306] 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 that decreases sucrose efflux transporter activity, e.g., comprising one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog, can have decreased oil content, decreased sucrose content, altered seed composition, or decreased seed size as compared to a control plant, plant part, or plant product, e.g., without such mutation. 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 decreased oil content, decreased sucrose content, decreased seed composition, and / or decreased seed size as compared to a control population. The plant or plant part having decreased oil content, decreased sucrose content, altered seed composition, or decreased seed size in the plant, plant part, or population of plants or plant parts can also have increased protein content. Content of a composition (e.g., protein content, sucrose content, oil content, e.g., X% dry basis) in a population of plants or plant parts as used herein may refer to average content (e.g., average protein content) of X% dry basis in the population, at least a portion of the soybean seeds in the population having the content (e.g., protein content) of X% dry basis, or the majority of the soybean seeds in the population having the content (e.g., protein content) of X% dry basis. In some embodiments, seed size is indicated from seed weight. The plant, plant part, or population of plants or plant parts provided herein can have decreased seed size, as indicated by decreased seed weight described in the present disclosure.

[0307] 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 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 may express a native (e.g., wild-type) SWEET39 gene endogenously or transgenically, and / or may have a wild-type sucrose efflux transporter activity. A plant, plant part, a 46

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[0309] population of plants or plant parts, or plant product of the present disclosure may have increased protein content, decreased oil content, decreased sucrose content, decreased seed composition, and / or decreased seed size 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.

[0310] In some embodiments, total oil content can be decreased by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50%, 1-40%, 1-30%, 1-20% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the plant, plant part, or population of the plants or plant parts of the present disclosure as compared to a control plant, plant part, or population of plants or plant parts. In some embodiments, oil content, as expressed by % dry weight (% dry basis), in the plant, plant part, or a population of plant or plant parts provided herein is less 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% dry weight (e.g., by about 0.25-0.5%, 0.5-0.75%, 0.75-1.0%, 1.0-1.5%, 1.0-1.8%, 1.3-1.8%, 1.3-2.0%, 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% dry weight), by about 0.25%, 0.5%, 0.75%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10% dry weight, 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% dry weight, or more oil content. In specific embodiments, the plant, plant part, or population of the plants or plant parts has oil content that is decreased by at least about 0.7%, about 1.1%, about 1.3%, about 1.9%, about 5.7%, about 8.0%, about 8.4%, about 8.9%, at least about 0.7%, at least about 1.1%, at least about 1.3%, at least about 1.9%, at least about 5.7%, at least about 8.0%, at least about 8.4%, at least about 8.9%, about 0.7-8.9%, about 0.7-1.3%, about 1.3-1.8%, about 1.8-8.0%, about 8.0-8.9%, about 0.7-8.4%, or about 1.1-8.4% dry weight relative to a control plant, plant part, or population.

[0311] In specific embodiments, provided herein are seeds or a population of seeds having seed oil content lower than that in control seeds or a control population of seeds (e.g., control seeds or population having a native SWEET39, reference seeds or population, commodity seeds or

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[0314] population). The seeds can be legume seeds, e.g., pea seeds or soybean seeds. Seed oil content of typical soybean cultivars ranges approximately 8-28% in dry weight (Clemente & Cahoon 2009, Plant Physiol. 151:1030-1040). In specific embodiments, a control soybean cultivar has about 18.5-20% seed oil content. In contrast, the soybean seeds or population of soybean seeds provided herein can have seed oil content (e.g., average seed oil content) of about % or lessl 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 19.5% or less by dry weight. In specific embodiments, the soybean seeds or population of soybean seeds provided herein can have seed oil content of about 11-19.2% dry weight, about 19-19.5% dry weight, about 11-11.5% dry weight, about 11% dry weight, about 11.5% dry weight, about 18.6% dry weight, about 18.8% dry weight, about 19.1% dry weight, or about 19.2% dry weight, whereas control soybean seeds have oil content of about 19.9% dry weight. In specific embodiments, the soybean seeds or population of soybean seeds provided herein can have seed oil content of about 11-17% dry weight, about 11-14% dry weight, about 14-17% dry weight, about 11.3% dry weight, about 14.0% dry weight, or about 16.7% dry weight, whereas control soybean seeds have oil content of about 18.5-20% dry weight.

[0315] Mutations in two or more SWEET39 genes can have synergistic effects in increase in oil content. For example, seeds or population of seeds having a mutation in one SWEET39 gene can have seed oil content of about 18-19.5% dry weight, about 18.6% dry weight, about 18.8% dry weight, about 19.1% dry weight, or about 19.2% dry weight, and seeds or population of seeds having mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can have seed oil content of about 11-12% dry weight, about 11% dry weight, or about 11.5% dry weight, whereas control seeds have oil content of about 20% dry weight, seeds or population of seeds having a mutation in one SWEET39 gene can have seed oil content of about 17% dry weight or about 16.7% dry weight, and seeds or population of seeds having mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can have seed oil content of about 11-14% dry weight, about 11.3% dry weight, or about 14% dry weight, whereas control seeds have oil content of about 18.5-20% dry weight.

[0316] In some embodiments, total sucrose content can be decreased by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50%, 1-40%, 1-30%, 1-20% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the plant, plant part, or population of the plants or plant parts of the present disclosure as compared to a control plant, plant part, or population of 48

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[0318] plants or plant parts. In some embodiments, sucrose content, as expressed by % dry weight (% dry basis), in the plant, plant part, or a population of plant or plant parts provided herein is less 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%, 1.0-1.8%, 1.3-1.8%, 1.3-2.0%, 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%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 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 sucrose content. In specific embodiments, the plant, plant part, or population of the plants or plant parts has sucrose content that is decreased by at least about 0.4%, about 0.5%, about 0.6%, about 3.8%, about 4.5%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 3.8%, at least about 4.5%, about 0.4-4.5%, about 0.4-0.6%, about 0.6-3.8%, about 3.8-4.5%, about 0.4-3.8%, or about 0.6-4.5% dry weight relative to a control plant, plant part, or population.

[0319] In specific embodiments, provided herein are seeds or a population of seeds having seed sucrose content lower than that in control seeds or a control population of seeds (e.g., control seeds or population having a native SWEET39, reference seeds or population, commodity seeds or population). The seeds can be legume seeds, e.g., pea seeds or soybean seeds. In specific embodiments, a control soybean cultivar has about 7% seed sucrose content. In contrast, the soybean seeds or population of soybean seeds provided herein can have seed sucrose content (e.g., average seed sucrose content) of less than 7%, about 6.6% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, or about 2% or less by dry weight. In specific embodiments, the soybean seeds or population of soybean seeds provided herein can have seed sucrose content of about 2.5-7.0% dry weight, about 2.5-3.2% dry weight, about 3.2-6.4 g dry weight, about 6.4-7.0% dry weight, about 2.5% dry weight, about 3.2% dry weight, about 6.4% dry weight, about 6.5% dry weight, about 6.6% dry weight, or about 7.0% dry weight, whereas control soybean seeds have sucrose content of about 7.0% dry weight.

[0320] Mutations in two or more SWEET39 genes can have synergistic effects in increase in sucrose content. For example, seeds or population of seeds having a mutation in one SWEET39 gene can have seed sucrose content of about 6-7% dry weight, about 6.4% dry weight, about 6.5% dry weight, about 6.6% dry weight, or about 7.0% dry weight, and seeds or population of seeds having mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B

[0321] 49

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[0323] gene) can have seed sucrose content of about 2-3.5% dry weight, about 2.5% dry weight, or about 3.2% dry weight, whereas control seeds have sucrose content of about 7.0% dry weight.

[0324] In specific embodiments, provided herein are seeds or a population of seeds having seed oil content, seed composition, and / or seed size (e.g., average seed oil content, average seed composition, and / or average seed size) that are different (e.g., decreased) relative to control seeds or a control population of seeds (e.g., control seeds or population having a native SWEET39, reference seeds or population, commodity seeds or population). The seeds or population of seeds with decreased oil content, decreased sucrose content, altered seed composition, or decreased seed size can also have increased protein content. The seeds can be legume seeds, e.g., pea seeds or soybean seeds. Seed composition (e.g., protein content, oil content, sucrose content) can be measured by standard methods for measuring oil content, sucrose content, or seed composition in a plant sample, for example by NIR, GC-MS optionally with certain modifications (e.g., with or without initial lipid extraction, with or without isotope labeling of analytes), or NMR. The seed composition (e.g., protein content, oil content, sucrose content) may be expressed as % dry basis (% DB).

[0325] In specific embodiments, provided herein are plants, plant parts (e.g., seeds), or a population of plants or plant parts (e.g., seeds) having increased protein content in combination with decreased oil content, decreased sucrose content, altered seed composition, and / or decreased seed size as compared to control plants, plant parts (e.g., seeds), or a population of plants or plant parts (e.g., seeds). For example, plants, plant parts (e.g., seeds), or a population of plants or plant parts (e.g., seeds) can have increased protein content that is greater by about 0.8%, about 1.0%, about 1.4% about 2.0%, about 2.7%, about 7.2%, about 11%, about 11.7%, or at least about 0.8-11.7%, at least about 0.8-2.7%, at least about 7-12%, or at least about 11-12% dry weight, e.g., having protein content of about 42-52.5% dry weight, about 42-44% dry weight, about 50-53% dry weight, at least about 42.3% dry weight, at least about 43.5% dry weight, at least about 50% dry weight, at least about 51% dry weight, at least about 52% dry weight, at least about 52.5% dry weight, or at least about 53% dry weight, whereas control soybean seeds have protein content of about 41.5% dry weight; or having protein content of about 45.5-55% dry weight, about 45.5-48.5% dry weight, about 48.5-51% dry weight, about 51-55% dry weight, at least about 45.8% dry weight, at least about 48.5% dry weight, at least about 51.1% dry weight, or at least about 54.8% dry weight, whereas control soybean seeds have protein content of about 44-46% dry weight, in combination with one or more of the following characteristics:

[0326] (i) decreased oil content that is decreased by at least about 0.7%, about 1.1%, about 1.3%, about 1.9%, about 5.7%, about 8.0%, about 8.4%, about 8.9%, at least about 0.7%, at least about 50

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[0328] 1.1%, at least about 1.3%, at least about 1.9%, at least about 5.7%, at least about 8.0%, at least about 8.4%, at least about 8.9%, about 0.7-8.9%, about 0.7-1.3%, about 1.3-1.8%, about 1.8-8.0%, about 8.0-8.9%, about 0.7-8.4%, or about 1.1-8.4% dry weight relative to a control plant, plant part, or population, e.g., having seed oil content of about 11-19.2% dry weight, about 19-19.5% dry weight, about 11-11.5% dry weight, about 11% dry weight, about 11.5% dry weight, about 18.6% dry weight, about 18.8% dry weight, about 19.1% dry weight, or about 19.2% dry weight, whereas control soybean seeds have oil content of about 19.9% dry weight; or having seed oil content of about 11-17% dry weight, about 11-14% dry weight, about 14-17% dry weight, about 11.3% dry weight, about 14.0% dry weight, or about 16.7% dry weight, whereas control soybean seeds have oil content of about 18.5-20% dry weight; (ii) decreased sucrose content that is decreased by at least about 0.4%, about 0.5%, about 0.6%, about 3.8%, about 4.5%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 3.8%, at least about 4.5%, about 0.4-4.5%, about 0.4-0.6%, about 0.6-3.8%, about 3.8-4.5%, about 0.4-3.8%, or about 0.6-4.5% dry weight relative to a control plant, plant part, or population, e.g., having seed sucrose content (e.g., average seed sucrose content) of less than 7% dry weight, 2.5-7.0% dry weight, about 2.5-3.2% dry weight, about 3.2-6.4 g dry weight, about 6.4-7.0% dry weight, about 2.5% dry weight, about 3.2% dry weight, about 6.4% dry weight, about 6.5% dry weight, or about 6.6% dry weight, whereas control soybean seeds have sucrose content of about 7.0% dry weight;

[0329] (iii) increased total seed count of about 65-85 seeds per plant, e.g., about 65-70, 70-75, 75-80, 80-85, about 67, about 68, about 69, about 77, about 80, about 81, about 82, about 83, about 84, about 85, at least about 67, at least about 68, at least about 69, at least about 77, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, or at least about 85 seeds per plant, whereas a control soybean plant has total seed count of about 63 seeds per plant; and (iv) decreased seed size as measured by seed weight that is decreased by about 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, or 11 g per 100 seeds, e.g., about 1-11 g, 1-4 g, 4-9 g, or 9-11 g per 100 seeds, e.g., having seed weight of about 6-16 g, about 6-7 g, about 13-16 g, about 6.3 g, about 6.7 g, about 13.1 g, Ibout 13.8 g, about 14.7 g, or about 15.4 g per 100 seeds whereas a control soybean cultivar has seed weight of about 16.6 g per 100 seeds.

[0330] In specific embodiments, (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis in the plant or plant part relative to a control plant or plant part.

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[0333] B. Plant parts and plant products

[0334] The present disclosure provides plant parts and plant products obtained from the plant of the present disclosure. 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 feed, food, or 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.

[0335] 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)). “White flake” or “white flake protein” as used herein 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.

[0336] 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, anti-caking agents, or any other ingredient to make the protein fit for the

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[0339] 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 (in) 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.

[0340] “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%

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[0343] 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.

[0344] In specific embodiments, the plant protein compositions provided herein are obtained from a soybean plant (Glycine max) that contains a mutation that decreases sucrose efflux transporter activity, e.g., one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 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 decreases sucrose efflux transporter activity, e.g., one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog. The plant parts, population of plant parts,

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[0351] and plant products of the present disclosure can have reduced sucrose efflux transporter activity, reduced expression level of the SWEET39 gene or homolog, reduced expression level of the SWEET39 [e.g., the full-length SWEET39], loss of function or reduced function or sucrose efflux transporter activity, increased protein content as compared to a control plant part, population, or plant product, e.g., without the mutation, comprising a native (e.g., wild-type) SWEET39 gene or SWEET39, or comprising wild-type sucrose efflux transporter activity.

[0352] C. Soybean compositions with increased protein content and decreased oil and / or sucrose content

[0353] Soybean compositions with increased protein content in combination with decreased oil and / or sucrose content are provided. “Soybean compositions” or “soybean products” as used herein refer to any compositions or products produced from soybeans, including seed compositions, oil products (oil compositions), sugar products, fiber products, protein products (protein compositions, 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 feed, food, or beverage products. Soybean compositions and soybean products are plant products provided herein, and can be intended for animal or human consumption as described in the present disclosure in the context of plant products.

[0354] Provided herein is a soybean composition comprising protein content of 50% dry basis or more in combination with oil content of 15% dry basis or less and / or sucrose content of 5% dry basis or less. For example, a soybean composition provided herein can contain protein content of 50%, 51%, 52%, or 53% dry basis or more, and oil content of 15%, 14%, 13%, 12%, 11.5%, or ll% dry basis or less. A soybean composition provided herein can contain protein content of 50%, 51%, 52%, 53% dry basis or more, and sucrose content of 5%, 4%, 3.5%, 3%, 2.5%, or 2% dry basis or less. A soybean composition provided herein can contain protein content of 50%, 51%, 52%, 53% dry basis or more, oil content of 15%, 14%, 13%, 12%, 11.5%, or ll% dry basis orless, and sucrose content of 5%, 4%, 3.5%, 3%, 2.5%, or 2% dry basis or less.

[0355] The soybean composition provided herein can comprise a SWEET39 gene, homolog thereof, or regulatory region thereof, comprising a mutation, or a polynucleotide fragment comprising the mutation. The mutation can be any mutation of a SWEET39 gene, homolog thereof, or regulatory region thereof provided herein, including a loss-of-function mutation. For example, the soybean composition provided herein can contain (i) a polynucleotide comprising a nucleic acid sequence of 55

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[0357] SEQ ID NO: 8, or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene. The soybean composition provided herein can contain (ii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene. The soybean composition provided herein can contain (iii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene. The soybean composition provided herein can contain (iv) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene. The soybean composition provided herein can contain (v) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The soybean composition provided herein can contain (vi) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene.

[0358] The soybean composition can contain any combination of the mutations provided in the present disclosure, such as mutations (i)-(vi) provided hereinabove, or more than one mutated SWEET39 gene or homolog or regulatory regions, or fragments thereof. For example, the soybean composition can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The soybean composition can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The soybean composition can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8 or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12 or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The soybean composition can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8 or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13 or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The soybean composition can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9 or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of 56

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[0360] SEQ ID NO: 12 or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The soybean composition can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9 or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13 or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The soybean composition can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12 or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene. The soybean composition can contain a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene.

[0361] The soybean composition can contain a fragment of any of the polynucleotides described herein, wherein the fragment at least partially comprises the mutation described herein.

[0362] IV. Increasing Protein Content in Plants

[0363] Methods are provided herein for altering (e.g., increasing) protein content in a plant or plant part. In some aspects, the methods comprise reducing sucrose efflux transporter activity in the plant or plant part, by, e.g., reducing levels or sucrose efflux transporter activity. Levels or activity of SWEET39 in a plant or plant part can be reduced by any methods known in the art for reducing protein activity or reducing gene expression, including the methods provided herein.

[0364] In some aspects, the methods comprise introducing one or more genetic mutations that alters (e.g., decreases) sucrose efflux transporter activity into a plant or plant part. The method can further comprise introducing the genetic mutation that alters (e.g., decreases) sucrose efflux transporter 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., decrease) SWEET39 level or activity, alter (e.g., decrease) expression levels of at least one SWEET39 gene encoding SWEET39, alter (e.g., decrease) SWEET39 levels or activity, 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 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) SWEET39 gene endogenously or transgenically. A control plant of the present disclosure may be grown under the same environmental conditions (e.g., same or similar

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[0367] temperature, humidity, air quality, soil quality, water quality, and / or pH conditions) as a plant to which the one or more mutations are 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 that decreases sucrose efflux transporter activity, altered (e.g., decreased) expression levels of at least one SWEET39 gene or homolog thereof, altered (e.g., decreased) SWEET39 levels or activity, altered (e.g., increased) protein content, altered (e.g., decreased) oil and / or sucrose content, altered seed composition, and / or altered (e.g., decreased) seed size as compared to a control plant or plant part, when the plant or plant part of the present disclosure is grown under the same environmental conditions as the control plant or plant part.

[0368] A. Altering expression or function of SWEET39 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 one or more genetic mutations that alters (e.g., decreases) sucrose efflux transporter activity into a plant or plant part. The method can further comprise introducing the genetic mutation that alters (e.g., decreases) sucrose efflux transporter activity into a plant cell, and regenerating a plant or plant part from the plant cell (e.g., transformed plant cell). The genetic mutation that is introduced into the plant or plant part according to the methods provided herein can comprise one or more insertions, substitutions, or deletions into the genome of the plant or plant part. The genetic mutation that alters (e.g., decreases) the sucrose efflux transporter activity can be introduced into at least one native SWEET39 gene or homolog thereof; a regulatory region of the native SWEET39 gene or homolog thereof; in a coding region, a noncoding 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 SWEET39 gene located at a genomic site or in a plant in a non-naturally occurring matter is a “native” SWEET39 gene if its nucleic acid sequence can be found in a plant existing in nature.

[0369] 1. Introducing mutation to SWEET39 sene, or its homolog, ortholog, or variant In some aspects, the methods provided herein comprise introducing one or more genetic mutations that decreases the sucrose efflux transporter 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 58

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[0371] 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 SWEET39 gene or homolog thereof and / or in a regulatory region of said at least one native SWEET39 gene or homolog 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 SWEET39 gene, each encoding a SWEET39. In particular, the plant or plant part to which the one or more mutations are introduced according to the methods can comprise at least 2 genes encoding a SWEET39, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes that have at least 80% (e.g., less than 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 93%, 82%, 81%, or 80%; or 80-85%, 80-90%, 80-99%, 85-90%, 85-95%, 85-99%, 90-95%, 90-99%, or 95-99%) sequence identity to one another, and retain sucrose efflux transporter (e.g., SWEET) activity. 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 SWEET39 gene or homolog; into a regulatory region of one SWEET39 gene or homolog; into more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all SWEET39 genes or homologs; into regulatory regions of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10), but not all SWEET39 genes or homologs; into all SWEET39 genes or homologs; and / or into regulatory regions of all SWEET39 genes or homologs in the plant or plant part.

[0372] 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 SWEET39 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.

[0373] In some embodiments, the one or more mutations are introduced at least partially into a SWEET39 gene or its regulatory region, and (i) the SWEET39 gene comprises a nucleic acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a nucleic acid sequence of SEQ ID NO: 1 or 2, wherein the nucleic acid sequence encodes a polypeptide that retains sucrose efflux transporter activity; (ii) the SWEET39 gene comprises the nucleic acid sequence of SEQ ID NO: 1 or 2; (iii) the SWEET39 gene encodes a polypeptide comprising an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence of SEQ ID NO: 3 or 4, wherein the polypeptide retains sucrose efflux

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[0376] transporter activity; and / or (iv) the SWEET39 gene encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or 4.

[0377] The methods provided herein to introduce a mutation that decreases the sucrose efflux transporter activity can include introducing at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) insertion, substitution, or deletion at least partially into in a coding region of Glycine max SWEET39 gene or homolog thereof in the plant or plant part. For instance, where an insertion, a substitution, or a deletion is at least partially in an exon, the whole part of the insertion, the substitution, or the deletion can be within the exon, or can span across the exon and a region (e.g., an intron, a regulatory region) upstream or downstream of the exon.

[0378] In some specific embodiments according to the methods provided herein, the mutation that decreases the sucrose efflux transporter activity is introduced in one or two alleles of a Glycine max SWEET39A gene (e.g., Glyma.l5G049200) and / or SWEET39B gene (e.g., Glyma.08G183500) or a regulatory region thereof. For example, a mutation can be introduced in two copies of the SWEET39 gene or homolog thereof, or one copy of the SWEET39 gene and one copy of its homolog, in order to reduce the expression of each gene to result in an increased protein content. In specific embodiments, the methods introduce a mutation in a SWEET39 gene as well as a SWEET39 gene homolog, and exhibit an increased protein content. In some embodiments, the methods introduce a deletion of one or more nucleotides of SEQ ID NO: 1 (Glycine max SWEET39A (Glyma.l5G049200)) and / or SEQ ID NO: 2 (Glycine max SWEET39B (Glyma.08G183500y) in the Glycine max SWEET39 gene or homolog thereof.

[0379] For example, in some embodiments, (i) the one or more mutations comprise a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39 gene, or said plant or plant part comprises SEQ ID NO: 8 when said deletion is introduced. In embodiments, (ii) the one or more mutations comprise a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part comprises SEQ ID NO: 9 when said deletion is introduced. In embodiments, (iii) the one or more mutations comprise a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part comprises SEQ ID NO: 10 when said deletion is introduced. In embodiments, (iv) the one or more mutations comprise a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part comprises SEQ ID NO: 11 when said deletion is introduced. In embodiments, (v) the one or more mutations comprise a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NO: 9 when said deletion is introduced. In embodiments, (vi) the one or more mutations 60

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[0381] comprise a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39 gene, or said plant or plant part comprises SEQ ID NO: 10 when said deletion is introduced. The one or more mutations can comprise any combination of mutations provided herein, e.g., any combination of (i)-(vi) provided hereinabove.

[0382] In some embodiments, the one or more mutations comprise or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 10 and 13 when said deletions are introduced. In some embodiments, the one or more mutations comprise a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 11 and 12 when said deletions are introduced. In some embodiments, the one or more mutations comprise a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 8 and 12 when said deletions are introduced. In some embodiments, the one or more mutations comprise a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 8 and 13 when said deletions are introduced. In some embodiments, the one or more mutations comprise a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 9 and 12 when said deletions are introduced. In some embodiments, the one or more mutations comprise a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 9 and 13 when said deletions are introduced. In some embodiments, the one or more mutations comprise a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 10 and 12 when said deletions are introduced. In some embodiments, the one or more mutations comprise a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 11 and 13 when said deletions are introduced.

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[0385] 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) SWEET39 gene or homolog thereof. Alternatively, the mutation introduced into the plant or plant part according to the methods can comprise an in-frame mutation, a nonsense mutation, or missense mutation of one or both alleles of at least one (e.g., one, more than one but not all, or all) SWEET39 gene or homolog thereof.

[0386] A genetic mutation that decreases the sucrose efflux transporter activity can be introduced into a gene that is a homolog, ortholog, or variant of a SWEET39 gene disclosed herein and expresses a SWEET39 with SWEET39 function, or in a regulatory region of such homolog, ortholog, or variant of a SWEET39 gene, according to the methods provided herein.

[0387] Variant sequences (e.g., homologs, orthologs) can be isolated by PCR. In this manner, variant sequences encoding SWEET39 can be identified and used in the methods of the present disclosure. The variant sequences will retain the sucrose efflux transporter activity.

[0388] In certain instances, mutations introduced into any SWEET39 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 SWEET39 gene. For example, a forward primer (e.g., SEQ ID NO: 14) and a reverse primer (e.g., SEQ ID NO: 15) can be used for detection of a mutation in the Glycine max SWEET39A gene (e.g., Glyma.15G049200) near the binding site of the Glyma.15G049200 guide RNA (e.g., SEQ ID NO: 7), e.g., a mutation generated by introducing the Glyma.15G049200 guide RNA (e.g., SEQ ID NO: 7) into the plant or plant part. A forward primer (e.g., SEQ ID NO: 14) and a reverse primer (e.g., SEQ ID NO: 15) can be used for detection of a mutation in the Glycine max SWEET39B gene (Glyma.08G 183500) near the binding site of the Glyma.08G 183500 guide RNA (e.g., SEQ ID NO: 7), e.g., a mutation generated by introducing the Glyma.08G183500 guide RNA (e.g., SEQ ID NO: 7) into the plant or plant part.

[0389] 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.

[0390] Introducing one or mutations insertions, substitutions, or deletions into at least one SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog in the genome of the plant or plant part can reduce the expression levels of the SWEET39 gene or homolog, reduce level or sucrose efflux transporter activity encoded by the SWEET39 gene or 62

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[0392] homolog, reduce sucrose efflux transporter activity, and / or increase protein content in the plant, plant part, or a population of plants or plant parts relative to a control plant or plant part, e.g., when grown under the same environmental condition, as further described in the present disclosure.

[0393] 2. Introducing regulatory modi fications

[0394] The methods described herein can comprise introducing a mutation that decreases the sucrose efflux transporter 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 into a regulatory region of at least one (e.g., one, more than one but not all, or all) SWEET39A gene (e.g., Glyma.15G049200) and / or SWEET39B gene (e.g., Glyma.08G 183500). A “regulatory region” of a gene can include a promoter region, 5’UTR, a genomic site where a RNA polymerase, a transcription factor, or other transcription modulators bind and interact to control mRNA synthesis of the gene, such as a binding site (e.g., enhancer sequence) for transcription modulator proteins (e.g., transcription factors), and other genomic regions that contribute to regulation of transcription of the gene. A regulatory region of the gene can be located in the 5’ untranslated region of the gene.

[0395] For example, one or more insertions, substitutions, and / or deletions can be introduced into a promoter region, a transcription modulator protein (e.g., transcription factor) binding site, or other regulatory regions of at least one (e.g., one, more than one but not all, or all) SWEET39 gene to confer to the plant or plant part an altered (e.g., reduced) transcription activity of the SWEET39 gene.

[0396] In some embodiments, the methods provided herein include introducing a mutation into a promoter region of at least one (e.g., one, more than one but not all, or all) SWEET39A gene (e.g., Glyma.15G049200) and / or SWEET39B gene (e.g., Glyma.08G 183500). The one or more insertions, substitutions, and / or deletions in the promoter region of the SWEET39 gene can alter the transcription initiation activity of the promoter. For example, the modified promoter can reduce transcription of the operably linked nucleic acid molecule (e.g., the SWEET39 gene), initiate transcription in a developmentally-regulated or temporally-regulated manner, initiate transcription in a cell-specific, cell-preferred, tissue-specific, or tissue-preferred manner, or initiate transcription in an inducible manner. A deletion, a substitution, or an insertion, e.g., introduction of a heterologous promoter sequence, a cis-acting factor, a motif or a partial sequence from any promoter, including those described elsewhere in the present disclosure, can be introduced into the promoter region of the SWEET39 gene to confer an altered (e.g., reduced) transcription initiation function according to the present disclosure.

[0397] The promoter sequence of one or more SWEET39 genes can be inactivated 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,

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[0400] 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 sequence of one or more of SWEET39 genes can be inactivated 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 SWEET39 genes can also be inactivated 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.

[0401] In some instances, the promoter sequence of one or more SWEET39 genes is inactivated by correction of the promoter sequence. A promoter sequence may be corrected by deletion, modification, and / or correction of one or more polymorphisms or mutations that would otherwise enhance the activity of the promoter sequence. In particular, the promoter sequence of one or more SWEET39 genes can be inactivated 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.

[0402] In some instances, the promoter sequence of one or more SWEET39 genes is inactivated 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.

[0403] In some instances, the promoter sequence of one or more SWEET39 genes is inactivated by addition, insertion, and / or engineering of cis-acting factors that interact with and modify the promoter sequence.

[0404] In some embodiments, a mutation is introduced to locate at least partially in 5’UTR of one or more (e.g., one, more than one but not all, or all) SWEET39A gene (e.g., Glyma.15G049200) and / or SWEET39B gene (e.g., Glyma.08G183500\ wherein the 5’UTR regulates translation of the main coding sequence (reinitiation of translation, cis- and trans-regulation).

[0405] In some embodiments, the method provided herein introduces mutation comprising a deletion of one or more nucleotides at least partially in the promoter and / or 5’UTR of a

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[0408] SWEET39A gene (e.g., Glyma.l5G049200) and / or SWEET39B gene (e.g., Glyma.08G 183500). Function and / or expression of the one or more SWEET39 genes can also be decreased or inhibited by modulation (e.g., increase or decrease) of expression of one or more transcription factor genes. For example, modulation of expression of the one or more transcription factor genes can inactivate or inhibit transcription initiation activity of the promoter of the one or more of SWEET39 genes and / or inhibit expression of the one or more SWEET39 genes.

[0409] Function and / or expression of the one or more SWEET39 genes can also be decreased 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 decrease function and / or expression of SWEET39 genes. Alternatively, modification and / or engineering of existing transcription factor binding sites or enhancer elements can decrease function and / or expression of SWEET39 genes.

[0410] Function and / or expression of the one or more SWEET39 genes can also be decreased or inhibited by insertion of one or more negative regulatory elements of the gene. For example, to inhibit the expression and / or function of the SWEET39 gene, a part or whole of one or more negative regulatory elements of the SWEET39 gene can be inserted in the genome of a plant cell or plant part. The negative regulatory sequence of the gene can be in a cis location. Alternatively, the negative regulatory sequence of the gene may be in a trans location. Negative regulatory elements of the one or more SWEET39 genes can also include upstream open reading frames (uORFs). In some instances, a negative regulatory sequence can be inserted in a region upstream of the SWEET39 gene in order to inhibit the expression and / or function of the gene.

[0411] 3. RNA interference

[0412] Function or sucrose efflux transporter activity in a plant or plant part can be altered by inhibiting or silencing the expression of the SWEET39 gene. Methods of the present disclosure can inhibit expression of the SWEET39 gene in a plant or plant part by RNA interference (RNAi). RNA interference is a biological process in which double-stranded RNA (dsRNA) molecules are involved in sequence-specific suppression of gene expression through translation or transcriptional repression. RNAi can be conducted using two types of small RNA molecules - microRNA (miRNA) and small interfering RNA (siRNA). RNAs are the direct products of genes, and these small RNAs can direct enzyme complexes to degrade messenger RNA (mRNA) molecules and thus decrease their activity by preventing translation, via post-transcriptional gene silencing. Moreover, transcription can be inhibited via the pre-transcriptional silencing mechanism of RNA interference, through which an enzyme complex catalyzes DNA methylation at genomic positions complementary to complexed siRNA or miRNA.

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[0415] Provided herein are methods for suppressing the expression of a SWEET39 gene by using siRNA and / or miRNA molecules that are directed to the SWEET39 gene or its mRNA transcript. In particular, methods of the present disclosure can inhibit or silence the SWEET39 gene in the genome of cells or parts of a plant by RNA interference, using siRNA and / or miRNA molecules that are directed to the SWEET39 gene.

[0416] siRNA and / or miRNA molecules for use in the present methods can be complementary to about 1-23, 2-23, 3-23, 4-23, 5-23, 6-23, 7-23, 8-23, 9-23, or 10-23 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) nucleotides of the SWEET39 gene, or the corresponding RNA transcripts.

[0417] In some embodiments, the siRNA and / or miRNA molecules can be complementary to a nucleotide region that comprises 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 NO: 1, 2, 5, and 6. For example, the siRNA and / or miRNA molecules can be complementary to a nucleotide region that comprises the nucleic acid sequence of any one of SEQ ID NO: 1, 2, 5, and 6.

[0418] 4. Reducing sucrose efflux transporter activity

[0419] The methods of the present disclosure (e.g., introducing mutations into a SWEET39 gene or its regulatory region; RNAi; modification of transcriptional regulation of the SWEET39 gene; insertion of a regulatory element) can reduce activity of SWEET39 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 plant, plant part, a population of plants or plant parts, or plant product. In particular, methods provided herein can reduce the sucrose efflux transporter 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-99%, 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to a control plant, plant part, a population of plants or plant parts, or plant product.

[0420] Sucrose efflux transporter activity can be measured by standard methods for assaying sucrose transporters, such as protoplast-esculin assays and the Forster resonance energy transfer (FRET) sensor-based assays.

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[0423] Sucrose efflux transporter activity can also be 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), or 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.

[0424] In some embodiments, sucrose efflux transporter activity can also assessed by measuring seed oil content by standard methods for measuring oil content in a plant sample, for example by NIR, GC-MS optionally with certain modifications (e.g., with or without initial lipid extraction, with or without isotope labeling of analytes), or NMR.

[0425] 5. Reducing expression level of SWEET39 gene or SWEET39 protein

[0426] The methods provided herein (e.g., introducing mutations into a SWEET39 gene or its regulatory region; RNAi; modification of transcriptional regulation of the SWEET39 gene; insertion of a regulatory element) can reduce the expression levels of SWEET39 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to the expression level of the SWEET39 gene or homolog in a control plant, plant part, a population of plants or plant parts, or plant product. In specific embodiments, the methods provided herein can reduce expression levels of a Glycine max SWEET39 gene. Expression levels of the SWEET39 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).

[0427] Expression levels of the SWEET39 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 SWEET39 encoded by the SWEET39 gene.

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[0430] The methods of the present disclosure (e.g., introducing mutations into a SWEET39 gene or its regulatory region; RNAi; modification of transcriptional regulation of the SWEET39 gene; insertion of a regulatory element) can reduce expression levels of the SWEET39, e.g., the SWEET39 encoded by the SWEET39 gene or homolog (having the mutation in the gene or in its regulatory region) 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 SWEET39 in a control plant, plant part, a population of plants or plant parts, or plant product, e.g., a plant, plant part, a population of plants or plant parts, or plant product without such mutation. In particular, the methods provided herein can reduce the expression levels of a full length SWEET39 having the complete amino acid sequence of a wild-type SWEET39, e.g., encoded by a native SWEET39 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. The methods provided herein can introduce a mutation into at least one SWEET39 gene or its regulatory regions in the plant or plant part, which can reduce expression of full-length SWEET39 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, e.g., product without such mutation, e.g., comprising a native (e.g., wild-type) SWEET39 gene. In particular, the methods provided herein, e.g., introducing one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog, can reduce expression levels of SWEET39, e.g., full length SWEET39, e.g., encoded by the SWEET39 gene 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, as compared to expression of SWEET39 in a control plant, plant part, a population of plants or plant parts, or plant product. In specific embodiments, the methods completely eliminates expression of the SWEET39; in other specific embodiments, the method decreases, but does not completely eliminate, the expression levels of SWEET39 in the plant, plant part, a population of plants or plant parts, or plant product provided herein, i.e., decrease the SWEET39 expression levels by more than 0% and less than 100% as compared to a control plant, plant part, a population of plants or plant parts, or plant product. Expression of a SWEET39, such as a full length

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[0433] SWEET39, in a plant, plant part, or plant product can be determined by one or more standard methods of determining protein levels. For example, expression of a SWEET39 can be determined by western blot analysis, ELISA, or dot blot analysis of a protein sample obtained from a plant, plant part, or plant product using an antibody directed to the SWEET39, e.g., the full-length SWEET39.

[0434] 6. Reducing or eliminating activity of SWEET39

[0435] The methods of the present disclosure (e.g., introducing mutations into a SWEET39 gene or its regulatory region; RNAi; modification of transcriptional regulation of the SWEET39 gene; insertion of a regulatory element) can reduce or eliminate (e.g., reduce to zero) function in the SWEET39, e.g., reduce or eliminate sucrose efflux transporter activity, as compared to the SWEET39 in a control plant, plant part, a population of plants or plant parts, 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, or a plant, plant part, a population of plants or plant parts, or plant product having wild-type sucrose efflux transporter activity. The methods disclosed herein can produce a SWEET39 with loss-of-function or reduced function having a mutation compared to a wild-type SWEET39 that causes loss or reduction of SWEET39 function. In some embodiments, the methods provided herein can reduce the function of the SWEET39 encoded by the SWEET39 gene or homolog to which a mutation (e.g., one or more insertions, substitutions, or deletions) has been introduced in the gene or its regulatory region 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% as compared to a control SWEET39 encoded by a control SWEET39 gene or homolog without such mutation. In some embodiments, the methods provided herein can reduce the sucrose efflux transporter activity in the plant, plant part, a population of plants or plant parts, or plant product to which the mutation (e.g., one or more insertions, substitutions, or deletions) has been introduced 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%, or 100% (e.g., by about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or 100%), e.g., by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 69

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[0437] 100% as compared to a control plant, plant part, or plant product, e.g., a plant, plant part, or plant product without such mutation. In specific embodiments, the method completely eliminates the function or sucrose efflux transporter activity; in other specific embodiments, the method decreases, but does not completely eliminate, the function or activity of SWEET39 in the plant, plant part, a population of plants or plant parts, or plant product provided herein, i.e., decrease the SWEET39 function or activity by more than 0% and less than 100% as compared to a control plant, plant part, a population of plants or plant parts, or plant product.

[0438] SWEET39 function can be measured by standard enzymatic assay (e.g., chemiluminescence assay, fluorescence assay) for detecting products (including intermediates) generated by conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) and water. SWEET39 function can also be measured by chemiluminescence or fluorescence measurement of ATP generated by combined actions of SWEET39 and pyruvate kinase.

[0439] SWEET39 function can also be 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), or 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.

[0440] B. Introducing mutations into the genome of plant cells

[0441] Introducing one or more mutations into the plant genome, e.g., into at least one SWEET39 gene or its regulatory region, and modulating the level or activity of SWEET39 in a plant or plant part may be achieved in any method of creating a change in a nucleic acid of a plant. For example, one or more mutations can be introduced into the plant genome, e.g., into at least one SWEET39 gene (e.g., Glycine maxBSl or BS2) or its regulatory region through the use of precise genomeediting 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 SWEET39 gene 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)

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[0444] 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 .4 rqr m / c -mediated DNA insertion (Gao et al. 2016 Nat Biotechnol doi:10.1038 / nbt.3547); Cre-lox site-specific recombination (Dale et al. 1995 Plant 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 (Yau 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.

[0445] 1. Editing reagent

[0446] Inserting, substituting, or deleting one or more nucleotides at a precise location of interest in at least one SWEET39 gene and / or a regulatory region of the SWEET39 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.

[0447] As used herein, the terms “nuclease” or “endonuclease” refers to naturally-occurring or engineered enzymes, which cleave a phosphodiester bond within a polynucleotide chain. Nucleases that can be used in precise genome-editing technologies to modulate the expression of the native sequence (e.g., at least one SWEET39 gene and / or a regulatory region of the SWEET39 gene) include, but are not limited to, meganucleases designed against the plant genomic sequence of interest (D’Halluin et al (2013) Plant Biotechnol 711: 933-941); Cas9 endonuclease; Casl2a (Cpfl) endonuclease; ortholog of Cas 12a endonuclease; Cmsl endonuclease; transcription activator-like effector nucleases (TALENs); zinc finger nucleases (ZFNs); and a deactivated CRISPR nuclease (e.g., a deactivated Cas9, Casl2a, or Cmsl endonuclease) fused to a

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[0450] 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 Casl2a nuclease, herein used interchangeably with a Cpfl nuclease, e.g., a McCpfl nuclease. In some embodiments, the CRISPR nuclease is a Casl2a nuclease ortholog, e.g., Lb5Casl2a, CMaCasl2a, BsCasl2a, BoCasl2a, MICas 12a, Mb2Casl2a, TsCasl2a, and MAD7 endonucleases.

[0451] 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. J. 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 SWEET39 gene and / or a regulatory region of the SWEET39 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 SWEET39 gene, thereby modulating the transcription of the SWEET39 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 / ncommsl3274), 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 72

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[0453] 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 Casl2a ortholog nucleases and crRNAs (CRISPR RNAs) for forming gRNAs that are coactive with the Casl2a nucleases. A “TALEN” nuclease is an endonuclease comprising a DNA-binding domain comprising a plurality of TAL domain repeats fused to a nuclease domain or an active portion thereof from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, and yeast HO endonuclease. A “zinc finger nuclease” or “ZFN” refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, and yeast HO endonuclease.

[0454] The editing system, editing reagents, or construct described herein can comprise one or more guide RNAs (gRNAs). “Guide RNA” as used herein refers to a RNA molecule that function as guides for RNA- or DNA-targeting enzymes, e.g., nucleases. To introduce one or more mutations into at least one SWEET39 gene and / or the promoter region of the SWEET39 gene, antisense constructions, complementary to at least a portion of the sequence of the SWEET39 gene messenger RNA (mRNA), SWEET39 gene, or regulatory region of the SWEET39 gene can be constructed. Antisense nucleotides are designed to hybridize with the corresponding mRNA or genomic nucleic acid sequence. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA or genomic sequence. In this manner, antisense constructions having at least 75%, optimally 80%, more optimally 85%, 90%, 95% or greater sequence identity to the corresponding sequences to be edited may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene.

[0455] Accordingly, a gene editing system, editing reagents, or a construct of the present disclosure can contain a guide RNA (gRNA) cassette, comprising one or more gRNAs or encoding one or more gRNAs, to drive mutations at the locus of at least one SWEET39 gene or the regulatory region of the SWEET39 gene. The one or more gRNAs can be designed to specifically target a regulatory region (e.g., promoter, 5’UTR) of a SWEET39 gene, or exons or introns of a SWEET39 gene.

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[0458] 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 NO: 1, 2, 5, and 6. The gRNA can be specific to the nucleic acid sequence of any one of SEQ ID NO: 1, 2, 5, and 6 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 SWEET39 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 SWEET39 gene or a regulatory region of the SWEET39 gene, and cause non-homologous end joining or homology-directed repair to introduce a mutation at the cleavage site. In specific embodiments, at least one of the one or more gRNAs targets a SWEET39 gene Glyma.15G049200 guide RNA and 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: 7; or (ii) the nucleic acid sequence of SEQ ID NO: 7. In specific embodiments, at least one of the one or more gRNAs targets a SWEET39 gene homolog Glyma.08G 183500 guide RNA and 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: 7; or (ii) the nucleic acid sequence of SEQ ID NO: 7.

[0459] 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 NO: 1, 2, 5, and 6. The two or more gRNA can be specific to the nucleic acid sequence of any one of SEQ ID NO: 1, 2, 5, and 6 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 max SWEET39A gene (e.g., Glyma.15G049200) or SWEET39B gene (e.g., Glyma.08G 183500) 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. Introducing two or more gRNAs targeting different genes can produce mutations of two or more genes being targeted (e.g., double mutant plants, multiple mutant plants). For example, in specific embodiments, one or more gRNAs targeting a SWEET39A gene Glyma.15G049200 (e.g., comprising the nucleic acid sequence of SEQ ID NO: 7) and one or more gRNAs targeting a SWEET39B gene Glyma.08G 183500 (e.g.,

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[0462] comprising the nucleic acid sequence of SEQ ID NO: 7) are introduced to the plants or plant parts, e.g., for introducing mutations in Glyma.15G049200 and / or Glyma.08G 183500.

[0463] In some instances, a gRNA may 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. The targeting region (i.e. spacer) of a gRNA that binds to the region of at least one SWEET39 gene or a regulatory region of the SWEET39 gene for use in the method described herein above 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 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 NO: 1, 2, 5, and 6. 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 NO: 1, 2, 5, and 6. 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% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the nucleic acid sequence of any one of SEQ ID NO: 1, 2, 5, and 6 or a nucleic acid sequence of any one of SEQ ID NO: 1, 2, 5, and 6.

[0464] The gRNA or a combination of two or more gRNAs provided herein can introduce a deletion of one or more 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 SWEET39 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., of a Glycine max SWEET39 gene and introduce into the plant, plant part, or plant cell:

[0465] In some embodiments, a gene editing efficiency of the one or more gRNAs is greater than 0.5% (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

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[0468] least one SWEET39 gene and its regulatory region. In some embodiments, the methods introduce mutations into all alleles each comprising a SWEET39 gene and its regulatory region.

[0469] 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., APOB EC 1), 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.

[0470] The gene editing system (e.g., CRISPR-Casl2a system), editing reagents, or a construct of the present disclosure can comprise at least one CRISPRRNA (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.

[0471] The methods described herein, comprising introducing into such plant a non-naturally occurring heterologous CRISPR-Casl2a genomic editing system of a type as variously described herein, can cause the editing reagents to introduce mutations in at least one SWEET39 gene or a regulatory region of the SWEET39 gene and alter the level or activity of the SWEET39 gene or SWEET39. 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.

[0472] 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 SWEET39 genes or a regulatory region of the SWEET39 gene.

[0473] 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 76

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[0475] 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.

[0476] 2. Promoter

[0477] 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 etal. (1985) Nature 313:810-812); rice actin (McElroy etal. (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 etal. (1984) EMBO J. 3:2223-2230); ALS promoter (U.S. Patent No. 5,659,026), and the like.

[0478] Alternatively, promoters for use in the methods of the present disclosure can be tissuepreferred promoters. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J.

[0479] 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 etal. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp etal. (1996) Plant Physiol. 112(2):525-535; Canevascini etal. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. 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 etal. (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 et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor etal. (1993) Plant J. 3:509-18; Orozco etal. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0480] 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

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[0483] particular developmental stage. Such promoters have been described in the art, e.g., US Patent No.

[0484] 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.

[0485] 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.

[0486] Alternatively, promoters for use in the methods of the present disclosure can be cellpreferred 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 al. (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.

[0487] 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 cv.s-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 etal. (2009) Plant Physiol 150: 535-546; Rushton etal. (2002) Plant Cell 14: 749-762). G'.s-regulatory elements may also be used to alter promoter expression profiles, as described in Venter (2007) Trends Plant Sci 12: 118-124.

[0488] 3. Transfer DNA

[0489] 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 78

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[0491] 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).

[0492] 4. Regulatory signal

[0493] 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.

[0494] 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.

[0495] 5. Reporter senes / selectable marker senes

[0496] 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, etal., (1990) EMBO J. 9:2517-2522; Kain, etal., (1995) Bio Techniques 19:650-655 and Chiu, et al., (1996) Current Biology 6:325-330, herein incorporated by reference in their entirety.

[0497] 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, etal., (1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron, et al., (1985) Plant Mol. Biol. 5:103-108 andZhijian, etal., (1995) Plant Science 108:219-227); streptomycin (Jones, etal., (1987) 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.

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[0500] 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.

[0501] 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.

[0502] 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).

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[0505] Selectable marker genes can further include genes encoding resistance to: chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; and phosphinothricin.

[0506] 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, etal., (1987) Nucleic Acids Res. 15(19):8115 and Luehrsen, etal., (1992) Methods EnzymoL 216:397-414), red fluorescent protein (DsRFP, RFP, etc), beta-galactosidase, and the maize genes encoding for anthocyanin production (Ludwig, etal., (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.

[0507] 6. Terminator

[0508] 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;

[0509] Sanfacon etal. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272;

[0510] Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res . 17:7891-7903; and Joshi etal. (1987) Nucleic Acids Res. 15:9627-9639.

[0511] 7. Vector

[0512] 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 SWEET39 gene or a regulatory region of the SWEET39 gene.

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[0515] 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 SWEET39 gene or the regulatory region of the SWEET39 gene.

[0516] 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 SWEET39 gene or the regulatory region of the SWEET39 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).

[0517] 8. Cells

[0518] 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 SWEET39 gene and / or a regulatory region of the SWEET39 gene. The cell can be a plant cell, a bacterial cell, and a fungal cell. The cell can be a bacterium, e.g., an Agrobacterium lumefaciens. containing the gRNA targeting at least one SWEET39 gene and / or a regulatory region of the SWEET39 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.

[0519] C. Increasing protein content in plants

[0520] The methods of the present disclosure, by introducing a mutation that decreases sucrose efflux transporter activity, e.g., comprising one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog in 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 plant, plant part, population of plants or plant parts, or plant product, e.g., without such mutation.

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[0523] A control plant or plant part can be a plant or plant part to which a mutation provided herein has not been introduced, e.g., by methods of the present disclosure. Thus, a control plant, plant part, a population of plants or plant parts, or plant product may express a native (e.g., wild-type) SWEET39 gene endogenously or transgenically, and / or may have a wild-type sucrose efflux transporter activity. The methods provided herein can increase protein content in plant, plant part, a population of plants or plant parts, or plant product 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.

[0524] 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 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%, 1.0-1.8%, 1.3-1.8%, 1.3-2.0%, 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%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 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 protein content. In specific embodiments, the methods can increase protein content by about 0.8%, about 1.0%, about 1.4% about 2.0%, about 2.7%, about 7.2%, about 11%, about 11.7%, at least about 0.8%, at least about 1.0%, at least about 1.4% at least about 2.0%, at least about 2.7%, at 83

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[0526] least about 7.2%, at least about 11%, at least about 11.7%, at least about 0.8-11.7%, at least about 0.8-11.0%, at least about 0.8-1.4%, at least about 1.4-11.7%, at least about 1.4-11%, at least about 7-11%, or at least about 11-12% dry weight dry weight relative to a control plant, plant part, or population.

[0527] In specific embodiments, the methods increase protein content in soybean seeds or a population of soybean seeds compared to a control soybean seeds or population of soybean seeds (e.g., control seed population having native SWEET39, 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 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(l):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 specific embodiments, the methods can increase seed protein content to about 42-52.5% dry weight, about 42-43% dry weight, about 42-53% dry weight, at least about 42.3% dry weight, at least about 43.5% dry weight, at least about 50% dry weight, at least about 51% dry weight, at least about 52% dry weight, at least about 52.5% dry weight, or at least about 53% dry weight, whereas control soybean seeds have protein content of about 41.5% dry weight. In specific embodiments, the methods can increase seed protein content to about 45.5-55% dry weight, about 45.5-48.5% dry weight, about 48.5-51% dry weight, about 51-55% dry weight, at least about 45.8% dry weight, at least about 48.5% dry weight, at least about 51.1% dry weight, or at least about 54.8% dry weight, whereas control soybean seeds have protein content of about 44-46% dry weight.

[0528] Introducing mutations in two or more SWEET39 genes in plants or plant parts can have synergistic effects in increase in protein content. For example, introducing a mutation in one SWEET39 gene in a plant or plant part can increase seed protein content to about 42-43% dry weight, about 42.3% dry weight, about 42.5% dry weight, about 42.9% dry weight, about 43.5% dry weight, and introducing mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) in a plant or plant part can have seed protein content to about 52-54% dry 84

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[0530] weight, about 52.5% dry weight, or about 53.2% dry weight, whereas control seeds have protein content of about 41.5% dry weight. Introducing a mutation in one SWEET39 gene in a plant or plant part can increase seed protein content to about 48.5% dry weight, and introducing mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) in a plant or plant part can increase seed protein content to about 51-55% dry weight, about 51.1% dry weight, or about 54.8% dry weight, whereas control seeds have protein content of about 44-46% dry weight.

[0531] 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), or nuclear magnetic resonance spectrometry (NMR). In specific embodiments, protein content is 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.

[0532] In specific embodiments, the methods provided herein can increase protein content in a plant, plant part, population of plants or plant parts, or plant product, as compared to a control plant, plant part, population, or plant product, without a significant decrease in seed weight, seed size, seed count, or yield.

[0533] For example, the methods can increase total seed count (e.g., average total seed count per plant) by about 1-10%, 2-10%, 3-10%, 4-10%, 5-10%, 6-10%, 7-10%, 8-10%, 2-9%, 3-9%, 4-9%, 5-9%, 6-9%, 7-9%, 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%, or more than 100% (e.g., by about 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or more than 100%), e.g., by about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 75.%, 8%, 85%, 9%, 9.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, or at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in the plants or plant parts of the present disclosure as compared to a control plant or plant part. In specific embodiments, a control soybean cultivar has about 63 average total seed count. In contrast, the method can increase average total seed count to about 65-85 seeds per plant, e.g., about 65-70, 70-75, 75-80, 80-85, about 67, about 68, about 69, about 77, about 80, about 81, about 82, about 83, about 84, about 85, at least about 67, at least about 68, at least about 69, at least about 77, at least about 80, at least about 81, at least about 82, at least about 85

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[0535] 83, at least about 84, or at least about 85 seeds per plant. Introducing mutations in two or more SWEET39 genes can have synergistic effects in increase in total seed count. For example, introducing a mutation in one SWEET39 gene in a plant or plant part can increase seed average total seed count to about 67-77, about 67.4, about 68.3, about 70.1, or about 76.9, and introducing mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) in a plant or plant part can increase average total seed count to about 80-84, about 80.1, or about 83.7, whereas control seeds have average total seed count of about 62.9.

[0536] In some embodiments, the methods reduce in seed weight, seed size, or 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. In some embodiments, seed weight per 100 soybean seed is no more decreased by about 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, or 20 g per 100 seeds, e.g., no more decreased by about 1-20 g, 1-15 g, 1-11 g, 1-4 g, 4-11 g, or 9-11 g per 100 seeds by the methods of the present disclosure as compared to a control plant, plant part, or plant population. In specific embodiments, a control soybean cultivar has seed weight of about 16.6 g per 100 seeds. In contrast, the methods can reduce seed weight to about 6-16 g, about 6-7 g, about 13-16 g, about 6.3 g, about 6.7 g, about 13.1 g, Ibout 13.8 g, about 14.7 g, or about 15.4 g.

[0537] Introducing mutations in two or more SWEET39 genes can have synergistic effects in increase in seed weight. For example, introducing a mutation in one SWEET39 gene can reduce seed weight to about 13-15.5 g, about 13.1 g, about 13.8 g, about 14.7 g, or about 15.4 g per 100 seeds, and introducing mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can reduce seed weight to about 6-7 g, about 6.3 g, or about 6.7 g per 100 seeds, whereas control seeds have seed weight of about 16.6 g per 100 seeds.

[0538] Seed weight or 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.

[0539] In some embodiments the methods can decrease oil content, decrease sucrose content, and / or alter seed composition in the plant or plant part. The decrease in oil content or sucrose content, and / or change in seed composition can be in addition to the increase in protein content.

[0540] For example, the methods can decrease total oil content by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 1-50%, 10-50%,

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[0543] 20-50%, 30-50%, 40-50%, 1-40%, 1-30%, 1-20% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the plant, plant part, or population of the plants or plant parts of the present disclosure as compared to a control plant, plant part, or population of plants or plant parts. In some embodiments, the methods can decrease oil content, as expressed by % dry weight (% dry basis), in the plant, plant part, or a population of plant or plant parts by 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% dry weight, or more than 10% dry weight (e.g., by about 0.25-0.5%, 0.5-0.75%, 0.75-1.0%, 1.0-1.5%, 1.0-1.8%, 1.3-1.8%, 1.3-2.0%, 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% dry weight), by about 0.25%, 0.5%, 0.75%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10% dry weight, 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% dry weight, or more oil content. In specific embodiments, the methods decrease oil content that by at least about 0.7%, about 1.1%, about 1.3%, about 1.9%, about 5.7%, about 8.0%, about 8.4%, about 8.9%, at least about 0.7%, at least about 1.1%, at least about 1.3%, at least about 1.9%, at least about 5.7%, at least about 8.0%, at least about 8.4%, at least about 8.9%, about 0.7-8.9%, about 0.7-1.3%, about 1.3-1.8%, about 1.8-8.0%, about 8.0-8.9%, about 0.7-8.4%, or about 1.1-8.4% dry weight in the plant or plant part relative to a control plant, plant part, or population.

[0544] In specific embodiments, the methods decrease oil content in seeds or a population of seeds relative to control seeds or a control population of seeds (e.g., control seeds or population having a native SWEET39, reference seeds or population, commodity seeds or population). The seeds can be legume seeds, e.g., pea seeds or soybean seeds. Seed oil content of typical soybean cultivars ranges approximately 8-28% in dry weight (Clemente & Cahoon 2009, Plant Physiol. 151:1030-1040). In specific embodiments, a control soybean cultivar has about 18.5-20% seed oil content. In contrast, the methods can decrease seed oil content (e.g., average seed oil content) of about % or lessl 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 19.5% or less by dry weight. In specific embodiments, the methods can decrease seed oil content of about 11-19.2% dry weight, about 19-19.5% dry weight, about 11-11.5% dry weight, about 11% dry weight, about 11.5% dry weight, about 18.6% dry weight, about 18.8% dry weight, about 19.1% dry weight, or about 19.2% dry weight, whereas control soybean seeds have oil content of about 19.9% dry weight. In specific 87

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[0546] embodiments, the methods can decrease seed oil content of about 11-17% dry weight, about 11-14% dry weight, about 14-17% dry weight, about 11.3% dry weight, about 14.0% dry weight, or about 16.7% dry weight, whereas control soybean seeds have oil content of about 18.5-20% dry weight.

[0547] Introducing mutations in two or more SWEET39 genes can have synergistic effects in increase in oil content. For example, introducing a mutation in one SWEET39 gene can reduce seed oil content to about 18-19.5% dry weight, about 18.6% dry weight, about 18.8% dry weight, about 19.1% dry weight, or about 19.2% dry weight, and introducing mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can reduce seed oil content to about 11-12% dry weight, about 11% dry weight, or about 11.5% dry weight, whereas control seeds have oil content of about 20% dry weight. Introducing a mutation in one SWEET39 gene can reduce seed oil content to about 17% dry weight or about 16.7% dry weight, and introducing mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can reduce seed oil content to about 11-14% dry weight, about 11.3% dry weight, or about 14% dry weight, whereas control seeds have oil content of about 18.5-20% dry weight.

[0548] The methods can decrease total sucrose content by about 1-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50%, 1-40%, 1-30%, 1-20% (e.g., by about 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%), e.g., by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the plant, plant part, or population of the plants or plant parts of the present disclosure as compared to a control plant, plant part, or population of plants or plant parts. In some embodiments, the methods can reduce sucrose content, as expressed by % dry weight (% dry basis), in the plant, plant part, or a population of plant or plant parts by 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%, 1.0-1.8%, 1.3-1.8%, 1.3-2.0%, 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%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 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 sucrose content. In specific embodiments, the methods decreases sucrose content in a plant or plant part by at least about 0.4%, about 0.5%, about 0.6%, about 3.8%, about 4.5%, at least about 0.4%, at least 88

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[0550] about 0.5%, at least about 0.6%, at least about 3.8%, at least about 4.5%, about 0.4-4.5%, about 0.4-0.6%, about 0.6-3.8%, about 3.8-4.5%, about 0.4-3.8%, or about 0.6-4.5% dry weight relative to a control plant, plant part, or population.

[0551] In specific embodiments, the methods decrease sucrose content in seeds or a population of seeds relative to that in control seeds or a control population of seeds (e.g., control seeds or population having a native SWEET39, reference seeds or population, commodity seeds or population). The seeds can be legume seeds, e.g., pea seeds or soybean seeds. In specific embodiments, a control soybean cultivar has about 7% seed sucrose content. In contrast, the methods can reduce sucrose content (e.g., average seed sucrose content) to less than 7%, about 6.6% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, or about 2% or less by dry weight in the plant or plant part. In specific embodiments, the methods can reduce seed sucrose content to about 2.5-7.0% dry weight, about 2.5-3.2% dry weight, about 3.2-6.4 g dry weight, about 6.4-7.0% dry weight, about 2.5% dry weight, about 3.2% dry weight, about 6.4% dry weight, about 6.5% dry weight, about 6.6% dry weight, or about 7.0% dry weight in a plant or plant part, whereas control soybean seeds have sucrose content of about 7.0% dry weight.

[0552] Introducing mutations in two or more SWEET39 genes can have synergistic effects in increase in sucrose content. For example, introducing a mutation in one SWEET39 gene can reduce seed sucrose content of about 6-7% dry weight, about 6.4% dry weight, about 6.5% dry weight, about 6.6% dry weight, or about 7.0% dry weight, and introducing mutations in two or more SWEET39 genes (e.g., a SWEET39A gene and a SWEET39B gene) can reduce seed sucrose content to about 2-3.5% dry weight, about 2.5% dry weight, or about 3.2% dry weight, whereas control seeds have sucrose content of about 7.0% dry weight.

[0553] In specific embodiments, the methods can alter seed oil content, seed composition, and / or seed size (e.g., average seed oil content, average seed composition, and / or average seed size) relative to control seeds or a control population of seeds (e.g., control seeds or population having a native SWEET39, reference seeds or population, commodity seeds or population). The methods can decrease oil content, decrease sucrose content, alter seed composition, or decrease seed size as well as increase protein content. The seeds can be legume seeds, e.g., pea seeds or soybean seeds. Seed composition (e.g., protein content, oil content, sucrose content) can be measured by standard methods for measuring oil content, sucrose content, or seed composition in a plant sample, for example by NIR, GC-MS optionally with certain modifications (e.g., with or without initial lipid extraction, with or without isotope labeling of analytes), or NMR. The seed composition (e.g., protein content, oil content, sucrose content) may be expressed as % dry basis (% DB).

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[0556] In specific embodiments, the methods provided herein increase protein content in combination with decrease oil content, decreased sucrose content, altered seed composition, and / or decrease seed size in plants, plant parts (e.g., seeds), or a population of plants or plant parts (e.g., seeds). For example, the methods can increase protein content by about 0.8%, about 1.0%, about 1.4% about 2.0%, about 2.7%, about 7.2%, about 11%, about 11.7%, or at least about 0.8-11.7%, at least about 0.8-2.7%, at least about 7-12%, or at least about 11-12% dry weight, e.g., to about 42-52.5% dry weight, about 42-44% dry weight, about 50-53% dry weight, at least about 42.3% dry weight, at least about 43.5% dry weight, at least about 50% dry weight, at least about 51% dry weight, at least about 52% dry weight, at least about 52.5% dry weight, or at least about 53% dry weight, whereas control soybean seeds have protein content of about 41.5% dry weight; or to about 45.5-55% dry weight, about 45.5-48.5% dry weight, about 48.5-51% dry weight, about 51-55% dry weight, at least about 45.8% dry weight, at least about 48.5% dry weight, at least about 51.1% dry weight, or at least about 54.8% dry weight, whereas control soybean seeds have protein content of about 44-46% dry weight, in combination with introducing one or more of the following characteristics to the plant or plant part:

[0557] (i) decreased oil content that is decreased by at least about 0.7%, about 1.1%, about 1.3%, about 1.9%, about 5.7%, about 8.0%, about 8.4%, about 8.9%, at least about 0.7%, at least about 1.1%, at least about 1.3%, at least about 1.9%, at least about 5.7%, at least about 8.0%, at least about 8.4%, at least about 8.9%, about 0.7-8.9%, about 0.7-1.3%, about 1.3-1.8%, about 1.8-8.0%, about 8.0-8.9%, about 0.7-8.4%, or about 1.1-8.4% dry weight relative to a control plant, plant part, or population, e.g., having seed oil content of about 11-19.2% dry weight, about 19-19.5% dry weight, about 11-11.5% dry weight, about 11% dry weight, about 11.5% dry weight, about 18.6% dry weight, about 18.8% dry weight, about 19.1% dry weight, or about 19.2% dry weight, whereas control soybean seeds have oil content of about 19.9% dry weight; or having seed oil content of about 11-17% dry weight, about 11-14% dry weight, about 14-17% dry weight, about 11.3% dry weight, about 14.0% dry weight, or about 16.7% dry weight, whereas control soybean seeds have oil content of about 18.5-20% dry weight; (ii) decreased sucrose content that is decreased by at least about 0.4%, about 0.5%, about 0.6%, about 3.8%, about 4.5%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 3.8%, at least about 4.5%, about 0.4-4.5%, about 0.4-0.6%, about 0.6-3.8%, about 3.8-4.5%, about 0.4-3.8%, or about 0.6-4.5% dry weight relative to a control plant, plant part, or population, e.g., having seed sucrose content (e.g., average seed sucrose content) of less than 7% dry weight, 2.5-7.0% dry weight, about 2.5-3.2% dry weight, about 3.2-6.4 g dry weight, about 6.4-7.0% dry weight, about 2.5% dry weight, about 3.2% dry

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[0560] weight, about 6.4% dry weight, about 6.5% dry weight, or about 6.6% dry weight, whereas control soybean seeds have sucrose content of about 7.0% dry weight;

[0561] (iii) increased total seed count of about 65-85 seeds per plant, e.g., about 65-70, 70-75, 75-80, 80-85, about 67, about 68, about 69, about 77, about 80, about 81, about 82, about 83, about 84, about 85, at least about 67, at least about 68, at least about 69, at least about 77, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, or at least about 85 seeds per plant, whereas a control soybean plant has total seed count of about 63 seeds per plant; and (iv) decreased seed size as measured by seed weight that is decreased by about 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, or 11 g per 100 seeds, e.g., about 1-11 g, 1-4 g, 4-9 g, or 9-11 g per 100 seeds, e.g., having seed weight of about 6-16 g, about 6-7 g, about 13-16 g, about 6.3 g, about 6.7 g, about 13.1 g, Ibout 13.8 g, about 14.7 g, or about 15.4 g per 100 seeds whereas a control soybean cultivar has seed weight of about 16.6 g per 100 seeds.

[0562] In specific embodiments, the methods (i) increase protein content by at least 5% dry basis and (ii) decrease the oil content is decreased by at least 5% dry basis and / or decrease the sucrose content by at least 3% dry basis relative to a control plant or plant part.

[0563] In specific embodiments, the methods provided herein can decrease sucrose efflux transporter activity in a population of seeds and increase seed protein content as compared to control population.

[0564] D. Plants, plant parts, population, and plant products produced by present methods

[0565] 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 reduced sucrose efflux transporter 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 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 91

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[0567] 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 feed, food, or 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.

[0568] 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)). 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 (in) 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).

[0569] In specific embodiments, the plant protein compositions provided herein are obtained from a soybean (Glycine max') plant or plant part produced according to the methods of the present disclosure, e.g., a soybean plant or plant part to which a mutation that decreases sucrose efflux transporter activity, e.g., one or more insertions, substitutions, or deletions is introduced into at least one native SWEET39 gene or homolog or into a regulatory region of such SWEET39 gene or homolog.

[0570] 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 92

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[0572] 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.

[0573] 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.

[0574] The plants, plant parts, and plant products, including plant protein compositions and plantbased food / beverage products produced according to the methods of the present disclosure can contain a mutation that decreases sucrose efflux transporter activity, e.g., one or more insertions, substitutions, or deletions in at least one native SWEET39 gene or homolog or in a regulatory region of such SWEET39 gene or homolog. The plants, plant parts, and plant products produced according to the methods of the present disclosure can have reduced sucrose efflux transporter activity, reduced expression level of the SWEET39 gene or homolog, reduced expression level of the SWEET39 (e.g., the full-length SWEET39) encoded by the SWEET39 gene, loss of function or reduced function or sucrose efflux transporter activity encoded by the SWEET39 gene, and / or increased protein content compared to a control plant part or plant product, e.g., without the mutation, comprising a native (e.g., wild-type) SWEET39 gene or SWEET39, or comprising wildtype sucrose efflux transporter activity.

[0575] E. Transformation of plants

[0576] 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 SWEET39 gene and / or a regulatory region of the SWEET39 gene. 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.

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[0579] 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 SWEET39 gene and / or a regulatory region of the SWEET39 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) 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 SWEET39 gene and / or a regulatory region of the SWEET39 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) 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.

[0580] Any mutation or any polynucleotide of interest (e.g., editing reagents, e.g., a nuclease and a guide RNA) 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 etal. (1986) Proc. Natl. Acad. Sci. USA ?> S6GlU6Q6. Agrohaclerium-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 etal. (1988) Biotechnology 6:923-926); and Lecl 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 / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh etal. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Dattac / o / . (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) roc. 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 etal. (1988) Plant Physiol . 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Patent No.

[0581] 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae)

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[0584] De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, 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 (\99 ) 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.

[0585] 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-an biolistic-mediated transformation remain the two predominantly employed approaches. However, transformation may be performed by infection, transfection, microinjection, electroporation, microprojection, biolistics or particle bombardment, electroporation, silica / carbon fibers, ultrasound mediated, PEG mediated, calcium phosphate co-precipitation, polycation DMSO technique, DEAE dextran procedure, viral infection, Agrobacterium and viral mediated (Caulimoriviruses, Geminiviruses, RNA plant viruses), liposome mediated and the like. 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.

[0586] 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 95

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[0588] 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.

[0589] Alteration of the SWEET39 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).

[0590] Alteration of the SWEET39 level or activity may be achieved by inserting a transposable element into at least one SWEET39 gene and / or a regulatory region of the SWEET39 gene.

[0591] 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.

[0592] 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 (Cigna radiata), cowpea (Cigna 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.). 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. In specific embodiments, a plant or plant part to be transformed according to the methods of the present disclosure is Pisum sativum or a part of Pisum sativum. 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, corn (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

[0593] 96

[0594] 4908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)

[0595] (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), quinoa (Chenopodium quinoa). chicory (Cichorium inlybus). lettuce (Lactuca sativa), safflower (Carthamus tinctorius), wheat (Triticum aestivum , soybean (Glycine max), tobacco (Nicotiana spp., e.g., Nicotiana labacum, 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...

Claims

Atorney Docket No.: B88552 1710WO (00456)What is claimed is:

1. A plant or plant part comprising decreased sucrose efflux transporter activity compared to a control plant or plant part, wherein said plant or plant part comprises one or more genetic mutations that decreases Sugars Will Eventually be Exported Transporter (SWEET) activity.

2. The plant or plant part of claim 1, wherein the one or more mutations comprise one or more insertions, substitutions, or deletions in at least one SWEET39 gene or homolog thereof or regulatory region thereof in said plant or plant part, wherein:an expression level of said at least one SWEET39 gene or homolog thereof is reduced compared to a corresponding native SWEET39 gene or homolog thereof without said mutation; and / orlevel or activity of SWEET39 encoded by said at least one SWEET39 gene or homolog thereof is reduced compared to SWEET39 encoded by a corresponding native SWEET39 gene or homolog thereof without said mutation.

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 claim 2 or 3, wherein the one or more mutations comprise one or more insertions, substitutions, or deletions in two or more SWEET39 genes or homologs thereof or regulatory regions thereof in said plant or plant part, wherein:an expression level of said two or more SWEET39 genes or homologs thereof is reduced compared to a corresponding native SWEET39 gene or homolog thereof without said mutation; and / orlevel or activity of SWEET39 encoded by said two or more SWEET39 genes or homologs thereof is reduced compared to SWEET39 encoded by a corresponding native SWEET39 gene or homolog thereof without said mutation.

5. The plant or plant part of any one of claims 2-4, wherein the at least one SWEET39 gene is a SWEET39A gene or a SWEET39B gene.

6. The plant or plant part of any one of claims 2-5, wherein the one or more mutations are located at least partially in a SWEET39 gene or homolog thereof:1124908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)(i) comprising a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1 or 2, wherein said nucleic acid sequence encodes a polypeptide that retains sucrose efflux transporter activity;(ii) comprising the nucleic acid sequence of SEQ ID NO: 1 or 2;(iii) encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 3 or 4, wherein said polypeptide retains sucrose efflux transporter activity;(iv) encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or 4; and / or in a regulatory region of said SWEET39 gene or homolog thereof.

7. The plant or plant part of claim 6, comprising a deletion of one or more nucleotides of SEQ ID NO: 1 in the Glycine max SWEET39A gene and / or SEQ ID NO: 2 in the Glycine max SWEET39B gene.

8. The plant or plant part of claim 7, comprising:(i) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8, or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene;(ii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene;(iii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene;(iv) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene;(v) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene; and / or(vi) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene.

9. The plant or plant part of claim 8, comprising:(i) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or(ii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or a 1134908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene.

10. The plant or plant part of any one of claims 2-9, wherein said one or more mutations comprise an out-of-frame mutation of the at least one SWEET39 gene or homolog thereof.

11. The plant or plant part of any one of claims 2-10, wherein said mutation comprises a nonsense mutation of the at least one SWEET39 gene or homolog thereof.

12. The plant or plant part according to any one of claims 1-11, wherein said plant or plant part comprises 2 or more genes encoding SWEET39.

13. The plant or plant part according to claim 12, wherein said 2 or more genes have at least 80% sequence identity to one another and retain sucrose efflux transporter activity.

14. The plant or plant part of any one of claims 1-13, comprising decreased oil content, decreased sucrose content, and / or decreased seed size compared to a control plant or plant part.

15. The plant or plant part of claim 14, wherein (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis compared to a control plant or plant part.

16. The plant or plant part of any one of claims 1-15, wherein said plant or plant part is a legume.

17. The plant or plant part of claim 16, 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 (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.).

18. The plant or plant part of any one of claims 1-15, wherein said plant or plant part is selected from the group consisting of corn (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),1144908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)foxtail millet (Setaria italica), finger millet Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aesliviim). tobacco (Nicotiana labacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsulum), sweet potato (Ipomoea balalus), 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 integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

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

20. A population of plants or plant parts comprising the plant or plant part of any one of claims 1-19, wherein the population comprises decreased sucrose efflux transporter activity, increased protein content, decreased oil content, decreased sucrose content, and / or decreased seed size compared to a control population.

21. The population of plants or plant parts of claim 20, wherein said plant or plant part is a seed, and said population is a population of seeds.

22. The population of plants or plant parts of claim 20 or 21, wherein (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis compared to a control population.

23. A method of increasing protein content in a plant or plant part, said method comprising reducing level or activity of a Sugars Will Eventually be Exported Transporter (SWEET) in said plant or plant part.

24. The method of claim 23, comprising introducing one or more genetic mutations that decrease SWEET activity into said plant or plant part.

25. The method of claim 24, further comprising introducing the one or more genetic mutations into a plant cell, and regenerating said plant or plant part from said plant cell.1154908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)26. The method of claim 24 or 25, wherein the one or more mutations comprise one or more insertions, substitutions, or deletions in at least one SWEET39 gene or homolog thereof or in a regulatory region thereof in said plant or plant part, wherein:an expression level of said at least one SWEET39 gene or homolog thereof is reduced compared to a corresponding native SWEET39 gene or homolog thereof without said mutation; and / orlevel or activity of SWEET39 encoded by said at least one SWEET39 gene or homolog thereof is reduced compared to SWEET39 encoded by a corresponding native SWEET39 gene or homolog thereof without said mutation.

27. The method of claim 26, wherein the one or more mutations comprise one or more insertions, substitutions, or deletions in two or more SWEET39 genes or homologs thereof or regulatory regions thereof in said plant or plant part, wherein:an expression level of said two or more SWEET39 genes or homologs thereof is reduced compared to a corresponding native SWEET39 gene or homolog thereof without said mutation; and / orlevel or activity of SWEET39 encoded by said two or more SWEET39 genes or homologs thereof is reduced compared to SWEET39 encoded by a corresponding native SWEET39 gene or homolog thereof without said mutation.

28. The method of claim 26 or 27, wherein the at least one SWEET39 gene is a SWEET39A gene or a SWEET39B gene.

29. The method of any one of 24-28, wherein the one or more mutations are introduced at least partially into a SWEET39 gene or homolog thereof:(i) comprising a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NO: 1 or 2, wherein said nucleic acid sequence encodes a polypeptide that retains sucrose efflux transporter activity;(ii) comprising the nucleic acid sequence of SEQ ID NO: 1 or 2;(iii) encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 3 or 4, wherein said polypeptide retains sucrose efflux transporter activity;(iv) encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or 4; and / or in a regulatory region of said SWEET39 gene or homolog thereof.1164908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)30. The method of any one of claims 24-29, wherein the one or more mutations comprise a deletion of one or more nucleotides of SEQ ID NO: 1 in the Glycine max SWEET39A gene and / or SEQ ID NO: 2 in the Glycine max SWEET39B gene.

31. The method of claim 30, wherein:(i) the one or more mutations comprise a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39 gene, or said plant or plant part comprises SEQ ID NO: 8 when said deletion is introduced;(ii) the one or more mutations comprise a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part comprises SEQ ID NO: 9 when said deletion is introduced;(iii) the one or more mutations comprise a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part comprises SEQ ID NO: 10 when said deletion is introduced;(iv) the one or more mutations comprise a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or said plant or plant part comprises SEQ ID NO: 11 when said deletion is introduced;(v) the one or more mutations comprise a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NO: 9 when said deletion is introduced;(vi) the one or more mutations comprise a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39 gene, or said plant or plant part comprises SEQ ID NO: 10 when said deletion is introduced;(vii) the one or more mutations comprise or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 10 and 13 when said deletions are introduced, and / or(viii) the one or more mutations comprise a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene and a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, or said plant or plant part comprises SEQ ID NOs: 11 and 12 when said deletions are introduced.1174908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)32. The method of any one of claims 26-31, wherein introducing the one or more mutations comprise introducing an out-of-frame mutation into said at least one SWEET39 gene or homolog thereof.

33. The method of any one of claims 24-32, further comprising introducing editing reagents or a nucleic acid construct encoding said editing reagents into said plant, plant part, or plant cell.

34. The method of claim 33, wherein said editing reagents comprise at least one nuclease, wherein the nuclease cleaves a target site in said at least one SWEET39 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.

35. The method of claim 34, wherein the at least one nuclease comprises a CRISPR nuclease.

36. The method of claim 35, 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.

37. The method of any one of claims 33-36, wherein the editing reagents comprise one or more guide RNAs (gRNAs).

38. The method of claim 37, wherein the one or more gRNAs comprise a nucleic acid sequence complementary to a region of a genomic DNA sequence encoding the SWEET39 or regulating transcription or translation of the SWEET39 in said plant or plant part.

39. The method of claim 37 or 38, 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: 7; or(ii) the nucleic acid sequence of SEQ ID NO: 7.

40. The method of any one of claims 23-39, wherein said plant or plant part is a legume.

41. The method of claim 40, 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),1184908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)fava bean (Vicia faba), pea (Pisum sativum), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentils (Lens culinaris, Lens esculenla), 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.).

42. The method of any one of claims 23-39, wherein said plant or plant part is selected from the group consisting of com (Zea mays), Brassica species, Brassica napus, Brassica rapa, Brassica juncea, rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet, pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Per sea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

43. A plant or plant part produced by the method of any one of claims 23-42, wherein said plant or plant part comprises reduced sucrose efflux transporter activity, increased protein content, decreased oil content, decreased sucrose content, and / or decreased seed size compared to a control plant or plant part.

44. The plant or plant part of claim 43, wherein (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis compared to a control plant or plant part.

45. The plant or plant part of claim 43 or 44, wherein said plant or plant part is a seed.

46. A population of plants or plant parts produced by the method of any one of claims 20-42, wherein the population comprises decreased sucrose efflux transporter activity, increased protein content, decreased oil content, decreased sucrose content, and / or decreased seed size compared to a control population.1194908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)47. The population of plants or plant parts of claim 46, wherein said population is a population of seeds.

48. The population of plants or plant parts of claim 46 or 47, wherein (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis compared to a control population.

49. A plant composition produced from the plant, plant part, or population plants or plant parts of any one of claims 1-22 and 43-47, wherein the plant composition is a seed composition, a protein composition, and / or an oil composition.

50. A plant product comprising the plant, plant part, or population of plants or plant parts of any one of claims 1-22 and 43-47 or the plant composition of claim 49, wherein the plant product is a feed, food, or beverage product.

51. The plant composition or plant product of claim 49 or 50, wherein (i) the protein content is increased by at least 5% dry basis and (ii) the oil content is decreased by at least 5% dry basis and / or the sucrose content is decreased by at least 3% dry basis compared to a control plant composition or plant product.

52. A soybean composition comprising protein content of 50% dry basis or more and oil content of 15% dry basis or less, wherein the soybean composition is a seed composition, a protein composition, an oil composition, or a feed, food, or beverage product produced from soybean plants or seeds.

53. The soybean composition of claim 52, comprising sucrose content of 5% dry basis or less.

54. The soybean composition of claim 52 or 53, comprising a SWEET39 gene, homolog thereof, regulatory region thereof, or fragment thereof comprising a mutation.

55. The soybean composition of claim 54, comprising:(i) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 8, or a deletion of nucleotides 509-519 of SEQ ID NO: 1 in the Glycine max SWEET39A gene;(ii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, or a deletion of nucleotides 513-517 of SEQ ID NO: 1 in the Glycine max SWEET39A gene;1204908-2861-7864vlAtorney Docket No.: B88552 1710WO (00456)(iii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene;(iv) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene;(v) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene;(vi) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene;(vii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, or a deletion of nucleotides 502-520 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, and a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 13, or a deletion of nucleotides 905-909 of SEQ ID NO: 2 in the Glycine max SWEET39B gene, and / or(viii) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 11, or a deletion of nucleotides 512-522 of SEQ ID NO: 1 in the Glycine max SWEET39A gene, or a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 12, or a deletion of nucleotides 883-914 of SEQ ID NO: 2 in the Glycine max SWEET39B gene,or a fragment of any thereof comprising the mutation.

56. A nucleic acid molecule comprising a nucleic acid sequence of a mutated SWEET39 gene or coding sequence thereof, wherein said nucleic acid sequence comprises any one of SEQ ID NOs: 1, 2, 5, and 6 comprising one or more insertions, substitutions, or deletions therein.

57. The nucleic acid molecule of claim 56, wherein the nucleic acid sequence of the mutated SWEET39 gene or coding sequence comprises any one of SEQ ID NOs: 8-1313.

58. 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 56 or 57.

59. A cell comprising the nucleic acid molecule of claim 56 or 57, or the DNA construct of claim 58.

60. The cell of claim 59, wherein the cell is a plant cell or a bacteria cell.1214908-2861-7864vl

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