Improved phosphorous remobilization and use efficiency in plants

Abstract

The disclosure encompasses genetically modified plants and methods for improving phosphorus redistribution and use efficiency. The genetically modified plants increase the expression of a nonspecific phospholipase C4 (NPC4) protein, enhancing phosphorus remobilization from old, senescing tissues to young, growing tissues and seeds. The genetic modification improves plant growth, increases seed and oil yield, and alters fatty acid composition. The genetically modified plants demonstrate enhanced phosphorus use efficiency under phosphorus-limited conditions, showing greater growth and yield improvements compared to wild-type plants. Specifically in camelina plants, the genetic modification increases seed yield, elevates oil content, and alters fatty acid composition of the oil under P-limited conditions.

Description

Polsinelli Docket No.: 077875-849424 Via EFS-web IMPROVED PHOSPHOROUS REMOBILIZATION AND USE EFFICIENCY IN PLANTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Patent Application No. 63 / 674,575, filed on July 23, 2024, the entire contents of which are incorporated by reference. INCORPORATION OF SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated herein by reference in its entirety. Said computer readable file was created on July 20, 2025, named 077875-849424 Sequence.xml and is 29,859 bytes in size. GOVERNMENTAL RIGHTS

[0003] This invention was made with government support under 2020-67013-30908 awarded by the USDA National Institute of Food and Agriculture. The government has certain rights in the invention. FIELD OF THE INVENTION

[0004] The present disclosure relates to genetic modifications in plants to enhance phosphorus redistribution, specifically to genetic modifications in camelina plants to improve plant growth, increase seed and oil yield, and alter fatty acid composition. BACKGROUND OF THE INVENTION

[0005] Phosphorus (P) is a macronutrient for plant growth and development. A significant portion of global cultivated land suffers from phosphorus deficiency, which severely limits crop productivity. The reliance on phosphorus fertilizers to mitigate this deficiency has led to increased agricultural costs, environmental pollution, and the accelerated depletion of finite phosphorus reserves. These challenges necessitate the development of innovative strategies to enhance phosphorus use efficiency in crops. Current solutions to address phosphorus deficiency in crops primarily focus on improving phosphorus acquisition efficiency (PAE) through modifications in root architecture, secretion 104866324.4Polsinelli Docket No.: 077875-849424 of organic acids, and enhancement of rhizosphere interactions. While these approaches have shown some success, they often fall short in addressing the internal phosphorus use efficiency (PUE), which involves the effective distribution and remobilization of acquired phosphorus within the plant. The limited progress in improving PUE has hindered efforts to enhance crop production without increasing phosphorus fertilizer applications.

[0006] Camelina, also known as false flax, is an oilseed crop valued for its high oil content and unique fatty acid profile, which is well-suited for nutritional, pharmacological, and industrial uses. Camelina is particularly attractive for cultivation on marginal lands due to its short life cycle and ability to grow in low-temperature environments. However, phosphorus deficiency significantly reduces camelina seed production.

[0007] Accordingly, there is a need for innovative solutions to enhance phosphorus use efficiency, reduce dependency on fertilizers, and improve crop yield under nutrient- limited conditions. SUMMARY OF THE INVENTION

[0008] In some aspects, provided herein is a method of improving phosphorus redistribution in a plant, the method comprising: introducing to the plant a recombinant DNA construct comprising a polynucleotide sequence encoding a nonspecific phospholipase C4 (NPC4) protein operably linked to a heterologous promoter, wherein one or more of the remobilization of phosphorus from old, senescing tissues to young, growing tissues and seeds is enhanced, plant growth and yield is enhanced, or an alteration in the fatty acid composition in the plant compared to a wild type plant.

[0009] In some aspects, the expression of one or more phosphorus transporter genes is enhanced in the old tissues. In some aspects, the plant is cultivated under phosphorus-limited conditions.

[0010] In some aspects, the growth and yield are enhanced in the plant under phosphorus-limited conditions when compared to phosphorus-sufficient conditions. In other aspects, the seed yield and oil production are increased in the plant under a phosphorus- limited condition when compared to a wild-type plant cultivated under similar conditions.

[0011] In some aspects, the fatty acid composition is altered in the plant when compared to a wild-type plant cultivated under similar conditions. In some aspects, altered fatty acid composition is selected from the group consisting of increased the linolenic acid (18:3) content, decreased linoleic acid (18:2) content, increased oleic acid (18:1) content, decreased stearic acid (18:0), palmitic acid (16:0), arachidic acid (20:0), and eicosadienoic 2 104866324.4Polsinelli Docket No.: 077875-849424 acid (20:2), increased 18:1 and gondoic acid (20:1), a decreased 20:2 fatty acid, and any combination thereof.

[0012] In some aspects, the phosphorus use efficiency (PUE) is enhanced in the plant under phosphorus-limited conditions.

[0013] In some aspects, the plant is selected from a camelina plant or a rice plant.

[0014] In some aspects, seed yield and oil content is enhanced when compared to a wild-type plant cultivated under the same conditions. In other aspects, the fatty acid composition is altered in the plant when compared to a wild-type camelina plant cultivated under the same conditions. In some aspects, altered fatty acid composition is selected from the group consisting of increased the linolenic acid (18:3) content, decreased linoleic acid (18:2) content, increased oleic acid (18:1) content, decreased stearic acid (18:0), palmitic acid (16:0), arachidic acid (20:0), and eicosadienoic acid (20:2), increased 18:1 and gondoic acid (20:1), a decreased 20:2 fatty acid, and any combination thereof.

[0015] In some aspects, the promoter is a constitutive promoter.

[0016] In some aspects, the NPC4 protein is selected from AtNPC4, OsNPC3, and OsNPC4. In some aspects, the polynucleotide encoding NPC4 encodes an amino acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1, 7, 9, 10, 11, 12, or 13. In some aspects, the polynucleotide encoding NPC4 comprises a nucleic acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2, 6, or 8.

[0017] In some aspects, the recombinant DNA construct comprises a CaMV promoter operably linked to a polynucleotide sequence encoding an AtNPC4 protein. In some aspects, the recombinant DNA construct comprises a nucleic acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 3.

[0018] In other aspects, provided herein is a method of improving yield and oil content or altering fatty acid composition in a plant, the method comprising: introducing to the plant a recombinant DNA construct comprising a polynucleotide sequence encoding an NPC4 protein, wherein the plant is a camelina plant or a rice plant.

[0019] In other aspects, provided herein is a genetically modified plant expressing a recombinant DNA construct comprising a polynucleotide sequence encoding a nonspecific phospholipase C4 (NPC4) protein operably linked to a heterologous promoter, wherein one or more of remobilization of phosphorus from old, senescing tissues to young, growing tissues, yield or oil content are enhanced or in the plant or the fatty acid composition is altered, and wherein the plant is a camelina plant or a rice plant. In some aspects, the promoter is a constitutive promoter. 104866324.4Polsinelli Docket No.: 077875-849424

[0020] In some aspects, the NPC4 protein is selected from AtNPC4, OsNPC3, and OSNPC4.

[0021] In some aspects, the heterologous promoter is a CaMV promoter.

[0022] In some aspects, growth, seed yield or oil production is enhanced, or fatty acid composition is altered in the plant when compared to a wild-type plant cultivated under similar conditions.

[0023] In some aspects, the polynucleotide encoding NPC4 encodes an amino acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1, 7, 9, 10, 11, 12, or 13.

[0024] In some aspects, the polynucleotide encoding NPC4 comprises a nucleic acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2, 6, or 8.

[0025] In other aspects, provided herein is a plant cell, seed, plant product thereof, of the genetically modified plant described herein.

[0026] In other aspects, provided herein is a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a NPC4 comprising a nucleic acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2, 6, or 8.

[0027] In other aspects, provided herein is a transgenic plant cell having in its genome the recombinant DNA construct described herein.

[0028] In other aspects, provided herein is a transgenic plant comprising the transgenic plant cell described herein.

[0029] In other aspects, provided herein is a crop product produced from the transgenic plant described herein.

[0030] In other aspects, provided herein is a transgenic progeny seed or propagatable plant part of the transgenic plant described herein.

[0031] In other aspects, provided herein is a kit for improving yield and oil content and altering fatty acid composition in a plant, the kit comprising one or more nucleic acid constructs for increasing the expression of a NPC protein in a plant, a plant comprising the one or more nucleic acid constructs for increasing expression of the NPC in the plant, or any combination thereof. BRIEF DESCRIPTION OF THE FIGURES

[0032] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain 104866324.4Polsinelli Docket No.: 077875-849424 embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0033] FIG. 1A shows expression of camelina CsNPC4s in response to Pi deficiencyand AtNPC4 OE in camelina. Phylogenetic tree of CsNPC4 genes with Arabidopsis NPC4 using (www.phylogeny.fr).

[0034] FIG. 1B shows expression of camelina CsNPC4s in response to Pi deficiency and AtNPC4 OE in camelina. CsNPC4 transcript levels under Pi-sufficient and deficient conditions in shoots and roots. Camelina seedlings under an ½ Hoagland medium with 500 μM or 0 μM Pi for 7 days were collected for RNA extraction and RT-qPCR analysis, and the expression level is relative to the value of respective NPC4s under 500 μM Pi. Values are mean ± SD (n = 3). Asterisks denote significant difference from 500 μM Pi (p<0.05) by Student’s t-test.

[0035] FIG. 1C shows expression of camelina CsNPC4s in response to Pideficiency and AtNPC4 OE in camelina. AtNPC4-OE construct.

[0036] FIG. 1D shows expression of camelina CsNPC4s in response to Pideficiency and AtNPC4 OE in camelina. PCR confirmation of AtNPC4 DNA in OE lines. PCR was performed using a forward primer in the 35S promoter and a reverse primer for the AtNPC4 sequence.

[0037] FIG. 1E shows expression of camelina CsNPC4s in response to Pi deficiency and AtNPC4 OE in camelina. Immunoblotting of AtNPC4-Strep in NPC4-OE and WT camelina. Total proteins from 7-day-old plants were immunodetected using an anti-Strep antibody.

[0038] FIG. 1F shows expression of camelina CsNPC4s in response to Pi deficiency and AtNPC4 OE in camelina. AtNPC4 expression level in OE lines. The NPC4 transcript level was normalized to UBQ10 as internal control. Values are mean ± SD (n = 4). Asterisks denote significant difference from WT (p<0.05) by Student’s t-test.

[0039] FIG. 2A shows effect of NPC4-OE on camelina seed and oil production. A representative image of flowering WT, OE6 and OE7 plants under 1000 μM Pi.

[0040] FIG. 2B shows effect of NPC4-OE on camelina seed and oil production. Branch number, seed yield, and oil content of WT and OE plants grown under 1000 μM 104866324.4Polsinelli Docket No.: 077875-849424 (P1000), 200 μM (P200), and 50 μM (P50) Pi. Values are means ± SD (n = 12) for branch number and seed yield and mean ± SD (n = 6) for seed oil content.

[0041] FIG. 2C shows effect of NPC4-OE on camelina seed and oil production. Oil yield was calculated from single plants (seed yield × oil content) under P1000, P200, and P50. Data are means ± SD (n = 12). Different letters denote significant differences at P < 0.05 among genotypes under the different conditions by one-way ANOVA.

[0042] FIG. 2D shows effect of NPC4-OE on camelina seed and oil production. Fatty acid composition of WT and OE lines under different Pi levels. Data = mean ± SD (n = 5). Asterisks denote significant difference at P < 0.05 compared with the WT, based on Student’s t-test.

[0043] FIG. 3A shows total P and free Pi contents in leaves and seeds in NPC4- OE and WT camelina under P sufficiency and deficiency. Total P and free Pi from leaves of three-week-old plants.

[0044] FIG. 3B shows total P and free Pi contents in leaves and seeds in NPC4-OE and WT camelina under P sufficiency and deficiency. Total P and free Pi from mature seeds of WT and OE plants at P1000, P200, and P50.

[0045] FIG. 3C shows total P and free Pi contents in leaves and seeds in NPC4-OEand WT camelina under P sufficiency and deficiency. Representative picture showing thesampling of senescing (SL), matured (ML), and young leaves (YL) collected from floweringplants.

[0046] FIG. 3D shows total P and free Pi contents in leaves and seeds in NPC4-OEand WT camelina under P sufficiency and deficiency. Total P and free Pi in different leavesunder P sufficiency in camelina. Values are mean ± SD (n = 5). Different letters showsignificant differences at P < 0.05 among different position leaves of three genotypes by one-way ANOVA.

[0047] FIG. 4A shows comparison of Pi remobilization and uptake between WT and NPC4-OE camelina. Total P in roots and different leaves of camelina plants under Pi deficiency for different days. Camelina seedlings grown on an ½ Hoagland medium with 100 μM Pi until leaf 6 appeared were transferred to the ½ Hoagland without Pi. The tissues of WT and OE lines were collected after 0 d, 6 d (leaf 7 of WT fully appeared), and 11 d (leaf 8 of WT fully appeared) of transfer. Total P and Pi distribution ratio were calculated by total P or Pi content in each tissue divided by total P or Pi in the whole plant, respectively.

[0048] FIG. 4B shows comparison of Pi remobilization and uptake between WT and NPC4-OE camelina. Total Pi in roots and different leaves of camelina plants under Pi 104866324.4Polsinelli Docket No.: 077875-849424 deficiency for different days. Camelina seedlings grown on an ½ Hoagland medium with 100 μM Pi until leaf 6 appeared were transferred to theHoagland without Pi. The tissues of WT and OE lines were collected after 0 d, 6 d (leaf 7 of WT fully appeared), and 11 d (leaf 8 of WT fully appeared) of transfer. Total P and Pi distribution ratio were calculated by total P or Pi content in each tissue divided by total P or Pi in the whole plant, respectively.

[0049] FIG. 4C shows comparison of Pi remobilization and uptake between WT and NPC4-OE camelina. Pi uptake in WT and OE lines in camelina. After growing on an ½ liquid Hoagland medium with 100 μM Pi for 10 d, the seedlings were transferred to an ½ Hoagland medium without Pi and grown for 14 d to delete plant P pools and then transferred to an ½ Hoagland medium with 100 μM Pi. Pi concentration of media was measured every 3 h. Value = mean ± (n = 5). Asterisks show significant differences at P <0.05 among genotypes under the same timepoint by Student’s t-test. DAT: day after transferring.

[0050] FIG. 5A shows expression of selected Pi transporter and response genes inWT and NPC4-OE camelina. Transcript levels of three CsNPCs in WT and NPC4-OE senescing leaf (SL), matured leaf (ML), and young leaf (YL) under Pi sufficient conditions.

[0051] FIG. 5B shows expression of selected Pi transporter and response genes in WT and NPC4-OE camelina. Effect of NPC4-OE on the expression of selected Pi transporter and response genes in different age leaves. Leaves of different ages were collected from WT and OE at the comparable flowering stage.

[0052] FIG. 5C shows expression of selected Pi transporter and response genes in WT and NPC4-OE camelina. Effect of NPC4-OE on the expression of selected Pi transporter and response genes in roots and leaves under Pi sufficient and deficient conditions. leaves and roots were collected from seedlings grown on an ½ Hoagland medium with 500 or 0 μM Pi for 7 days. CsACT2 (Csa19g026200.1) was used as an internal standard, and the relative level is relative to the value of the same gene in WT (red dashed line). Values are mean ± SD (n = 4). a, b, and c represent three copies of each gene in camelina.

[0053] FIG.6 shows lipid levels in sensing and young leaves as affected by NPC4- OE under Pi sufficiency and deficiency. Lipids were extracted from senescing and young leaf of WT and OE plants at a comparable early flowering stage under 1000 μM and 50 μM treatments. Values are mean ± SD (n = 5). Different letters show significant differences at P < 0.05 among genotypes under the different Pi treatments by one-way ANOVA. DAG, diacylglycerol; DGDG, digalactosyldiacylglycerol; MGDG, 7 104866324.4Polsinelli Docket No.: 077875-849424 monogalactosyldiacylglycerol; P, phosphorus; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG,phosphatidylglycerol; PS, phosphatidylserine; PL, sum of all phospholipids analyzed; GL, sum of galactolipids MGDG and DGDG.

[0054] FIG. 7A-7C shows effect of NPC4-OE on camelina growth and development.

[0055] FIG. 8A-8C shows expression of selected Pi transporter and response genes in WT and NPC4-OE7 camelina.

[0056] FIG. 9A-9B shows levels of lipids in different position leaves under Pi sufficiency and deficiency in WT and NPC4-OE7 camelina.

[0057] FIG. 10 shows levels of lipid molecular species on different position leaves under Pi sufficient and deficient conditions.

[0058] FIG. 11A is a photograph displaying the morphology of wildtype (WT), NPC4 Overexpression (OE), and NPC4 CRISPR-knockout (CR) mutant rice plants grown under for 20 days.

[0059] FIG. 11B is a bar graph quantifying the length of roots observed in WT, NPC4-OE, or NPC4-CR rice plants grown in phosphate-sufficient and starvation conditions for 20 days. Values are means ± SD of five replicates. Different letters indicate statistical differences at P < 0.05 among genotypes under the same condition, as determined by one- way ANOVA.

[0060] FIG. 11C is a bar graph quantifying the number of root tips observed in WT, NPC4-OE, or NPC4-CR rice plants grown in phosphate-sufficient and starvation conditions for 20 days. Values are means ± SD of five replicates. Different letters indicate statistical differences at P < 0.05 among genotypes under the same condition, as determined by one- way ANOVA.

[0061] FIG. 12A is a table displaying different lipid classes (first row) or PA species (second row) immobilized on filters.

[0062] FIG. 12B is a photographic image displaying a representative western blot for PHR1 produced in E. coli.

[0063] FIG. 12C is a photographic image displaying lipid levels on filter paper blotted with an empty vector (EV) according to the table shown in FIG. 12A.

[0064] FIG. 12D is a photographic image displaying lipid levels on filter paper blotted with PHR1 according to the table shown in FIG.12A. 104866324.4Polsinelli Docket No.: 077875-849424 DETAILED DESCRIPTION

[0065] The present disclosure encompasses genetically modified plants and methods of using these plants to improve phosphorus redistribution and use efficiency. The present disclosure provides genetically modified plants that increase the expression of a nonspecific phospholipase (NPC) protein. The inventors discovered that this genetic modification enhances phosphorus remobilization from old, senescing tissues to young, growing tissues and seeds, thereby improving plant growth, increasing seed and oil yield, and improving oil production.

[0066] The inventors discovered that overexpression of NPC also increases the expression of phosphorus transporter genes in the old tissues of the genetically modified plants, further facilitating efficient phosphorus redistribution. More specifically, the inventors discovered that overexpressing nonspecific phospholipase C4 (NPC4) in camelina improves growth and increases seed and oil yield under phosphorus-limited conditions compared to phosphorus-sufficient conditions, and alters fatty acid composition of the produced oil. The modified camelina plants show increased seed yield and oil production, as well as enhanced phosphorus use efficiency (PUE) and altered fatty acid composition, under nutrient-limited conditions. The genetic modification strategy described herein can reduce dependency on phosphorus fertilizers and improve crop yield, thereby addressing the global challenge of phosphorus deficiency in agriculture. I. Genetically modified plants

[0067] One aspect of the present disclosure encompasses a genetically modified plant comprising improved yield and oil content compared to an unmodified plant. The genetically modified plant comprises one or more genetic modifications that increase expression of a nonspecific phospholipase C (NPC) protein in the plant when compared to a plant without the genetic modification. (a) Plants

[0068] The present disclosure provides a genetically modified plant comprising a genetic modification that increases expression of an NPC protein in the plant when compared to a wild type plant. The genetically modified plant exhibits improved yield and oil content, and altered fatty acid composition of the oil produced.

[0069] As used herein, a “plant” refers to any of various photosynthetic, eukaryotic multi-cellular organisms of the kingdom Plantae, characteristically producing embryos, 9 104866324.4Polsinelli Docket No.: 077875-849424 containing chloroplasts, having cellulose cell walls and lacking locomotion. As used herein, a “plant” includes any plant or part of a plant at any stage of development, including seeds, suspension cultures, plant cells, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, microspores, and progeny thereof. Also included are cuttings, and cell or tissue cultures. As used in conjunction with the present disclosure, plant tissue includes, without limitation, whole plants, plant cells, plant organs, e.g., leaves, stems, roots, meristems, plant seeds, protoplasts, callus, cell cultures, and any groups of plant cells organized into structural and / or functional units.

[0070] Non-limiting examples of suitable plants may include, for example, species of the Family Gramineae, including Sorghum bicolor and Zea mays; species of the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, and Triticum.

[0071] In some aspects, plants can include, for example, those from corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., 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), duckweed (Lemna), soybean (Glycine max), 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 nucijra), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia spp.), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers. 104866324.4Polsinelli Docket No.: 077875-849424

[0072] Non-limiting examples of suitable crop plants and model plants may include, for example, Arabidopsis, corn, rice, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum, wheat, tobacco, and lemna.

[0073] Non-limiting examples of suitable leguminous plants may include, for example, guar, locust bean, fenugreek, soybean (Glycine), garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, peanuts (Arachis sp.), crown vetch (Vicia sp.), hairy vetch, adzuki bean, lupine (Lupinus sp.), trifolium, common bean (Phaseolus sp.), field bean (Pisum sp.), clover (Melilotus sp.), Lotus, trefoil, lens, and false indigo.

[0074] In some aspects, the plant is an oil-producing plant. Oil-producing plants are a diverse group of plants that synthesize and store oils in their seeds, fruits, or other tissues. These plants play an important role in various industries, including food, cosmetics, pharmaceuticals, and biofuels. The oils produced by these plants are generally classified into two broad categories: essential oils and fixed oils. Essential oils are volatile and aromatic, often used in fragrances, flavorings, and therapeutic products. Fixed oils, on the other hand, are non-volatile and typically extracted for use in cooking, manufacturing, and as raw materials for biofuel production.

[0075] In some aspects, the plant is an oil-producing crop plant. Oil-producing crop plants are specifically cultivated for their ability to yield significant quantities of oil. Non- limiting examples of oil-producing crop plants include oil palm, soybean, sunflower, canola (rapeseed), and olive. These crops are fundamental to the agricultural economy and global trade, providing oils that serve multiple purposes. For instance, soybean and canola oils are predominantly used in cooking and food processing due to their nutritional profiles. Sunflower and olive oils are prized for their culinary qualities and health benefits. Additionally, crops like oil palm produce oils that are versatile in both food production and industrial applications, including biofuel. These oils can be further processed into biodiesel, offering a renewable energy source that contributes to sustainability efforts. The variety and utility of the oils derived from these plants highlight their significance in meeting both nutritional needs and industrial demands.

[0076] In some aspects, the plant is camelina. Camelina (Camelina sativa) seeds are high in oil content, with a unique fatty acid profile well-suited for nutritional, pharmacological, and industrial uses. Camelina oil is highly unsaturated with 30–40% of health. Moreover, its short life cycle and ability to grow in low temperature and on marginal 11 104866324.4Polsinelli Docket No.: 077875-849424 lands further increased the interest to expand camelina production for biofuels and renewable industrial applications. Camelina was reported to produce moderate yields on poor soils. Phosphorus (P) is a major limiting macronutrient for crop production, and about 70% of global cultivated land is deficient in the available inorganic phosphate. It has been previously identified that P limitation greatly decreases camelina seed production and alters oil fatty acid composition. Thus, improving camelina resilience to P limitation can greatly benefit the effort to expand camelina growth and seed oil production.

[0077] In some aspects, a genetically modified camelina plant of the instant disclosure comprises a genetic modification that increases expression of an NPC protein in the camelina plant when compared to a wild type plant. Expression of the NPC protein remobilizes (redistributes) phosphorus from old, senescing tissues to young, growing tissues in the genetically modified camelina plant thereby improving yield and oil content and altering the fatty acid composition in the genetically modified camelina plant.

[0078] In other aspects, the plant is oryza. In some aspects, the plant is an oryza species selected from the group consisting of Oryza sativa, Oryza glaberrima, Oryza rufipogon, Oryza barthii, Oryza longistaminata, Oryza punctata, Oryza officinalis, Oryza nivara, Oryza australiensis, Oryza glumaepatula, Oryza granulate, Oryza brachyantha, Oryza minuta, Oryza latifolia, Oryza coarctata, Oryza ridleyi, and Oryza eichingeri. In some aspects, the plant is Oryza sativa. In some aspects, a genetically modified oryza plant of the instant disclosure comprises a genetic modification that increases expression of an NPC protein in the oryza plant when compared to a wild type plant. Expression of the NPC protein remobilizes (redistributes) phosphorus from old, senescing tissues to young, growing tissues in the genetically modified oryza plant thereby improving yield and oil content and altering the fatty acid composition in the genetically modified oryza plant.

[0079] In some aspects, the NPC protein is an NPC4 protein. The NPC4 may be selected from a AtNPC4, OsNPC3, and OSNPC4. In some aspects, the genetic modification comprises an expression construct comprising a promoter operably linked to a polynucleotide encoding a nonspecific phospholipase C4 (NPC4) protein. In some aspects, the expression construct comprises a constitutively expressed promoter operably linked to the polynucleotide encoding the NPC4 protein. In some aspects, the polynucleotide encoding the NPC4 protein encodes an AtNPC4, OsNPC3, or OSNPC4 protein. In some aspects, the expression construct comprises a CaMV promoter operably linked to a polynucleotide encoding an AtNPC4 protein. The expression construct can be as further described in Sections I(b) and II herein below. 104866324.4Polsinelli Docket No.: 077875-849424 (b) Phospholipase C

[0080] Genetically modified plants of the instant disclosure comprise a genetic modification that results in increased expression of a phospholipase C (PLC) protein when compared to plants that do not comprise the genetic modification. PLC enzymes are a diverse family of proteins that play roles in cellular signaling and lipid metabolism. These enzymes catalyze the hydrolysis of phospholipids, specifically cleaving the phosphodiester bond just before the phosphate group. In eukaryotic cells, the primary substrate for most PLCs is phosphatidylinositol 4,5-bisphosphate (PIP2), which is hydrolyzed to produce two important second messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These second messengers trigger various cellular responses, including calcium release from intracellular stores and activation of protein kinase C.

[0081] In mammals, there are 13 known PLC isozymes, categorized into six classes unique features and is regulated by different factors, such as G-protein-coupled receptors, receptor tyrosine kinases, calcium ions, and small GTPases. These enzymes are involved in numerous physiological processes, including cell growth, differentiation, metabolism, and immune responses.

[0082] Plants also possess PLC enzymes, which play important roles in stress responses, growth, and development. Unlike mammalian PLCs, plant PLCs are generally divided into two main groups: phosphatidylinositol-specific PLCs (PI-PLCs) and nonspecific PLCs (NPCs). PI-PLCs in plants are structurally similar to mammalian PLCs and primarily hydrolyze PIP2. They are involved in various signaling pathways, including responses to abiotic and biotic stresses, hormone signaling, and developmental processes.

[0083] In some aspects, the genetically modified plants of the instant disclosure express an NPC protein. NPC proteins in plants form a distinct group that differs from PI- PLCs in their substrate specificity and structure. NPCs can hydrolyze various phospholipids, including phosphatidylcholine, which is more abundant in plant membranes than PIP2. The Arabidopsis thaliana genome encodes six NPC genes (NPC1-NPC6), each with distinct expression patterns and functions.

[0084] Among the NPCs, NPC4 has been shown to play significant roles in plant phosphorus metabolism and stress responses. NPC4 is primarily expressed in roots and is induced under phosphate starvation conditions. It contributes to phosphate homeostasis by hydrolyzing membrane phospholipids, releasing phosphate for other cellular processes. 13 104866324.4Polsinelli Docket No.: 077875-849424

[0085] NPC4 has been shown to affect phosphorus redistribution in plants. During phosphate deficiency, NPC4 activity increases, leading to the breakdown of phospholipids in cell membranes. This process releases phosphate, which can then be remobilized to other parts of the plant where it is needed most, such as growing tissues or reproductive organs. The released phosphate can also be used to maintain essential cellular functions under low-phosphate conditions.

[0086] The mechanism of action for NPC4 involves its localization to the plasma membrane, where it can access its phospholipid substrates. Upon activation, NPC4 hydrolyzes phospholipids, particularly phosphatidylcholine, producing DAG and a phosphate-containing head group. This activity not only releases phosphate but also alters membrane composition, which can affect membrane properties and potentially influence various cellular processes, including signaling and transport.

[0087] Recent studies have also implicated NPC4 in plant responses to various stresses, including salt stress and pathogen infection. For example, NPC4 has been shown to contribute to the production of lipid signaling molecules that play roles in plant defense responses. Additionally, the DAG produced by NPC4 activity can serve as a precursor for other signaling lipids or be further metabolized to provide energy during stress conditions.

[0088] In some aspects, genetically modified plants of the instant disclosure comprise increased expression of an NPC4 protein. In some aspects, the NPC4 protein is an A. thaliana NPC4 protein. In some aspects, the NPC4 protein comprises an amino acid sequence comprising about 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 with the amino acid sequence of SEQ ID NO: 1. In some aspects, the NPC4 protein comprises an amino acid sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0089] In some aspects, the NPC4 protein is encoded by a nucleic acid sequence comprising a nucleotide sequence comprising about 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 with the nucleic acid sequence of SEQ ID NO: 2. In some aspects, the NPC4 protein is encoded by a nucleic acid sequence comprising a nucleotide sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 2. 104866324.4Polsinelli Docket No.: 077875-849424

[0090] In other aspects, genetically modified plants of the instant disclosure comprise increased expression of an NPC3 protein. In some aspects, the NPC3 protein is an O. sativa NPC3 protein. In some aspects, the NPC3 protein comprises an amino acid sequence comprising about 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 with the amino acid sequence of SEQ ID NO: 7. In some aspects, the NPC3 protein comprises an amino acid sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7.

[0091] some aspects, the NPC3 protein is encoded by a nucleic acid sequence comprising a nucleotide sequence comprising about 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 with the nucleic acid sequence of SEQ ID NO: 6. In some aspects, the NPC3 protein is encoded by a nucleic acid sequence comprising a nucleotide sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 6.

[0092] In other aspects, genetically modified plants of the instant disclosure comprise increased expression of an NPC4 protein. In some aspects, the NPC4 protein is an O. sativa NPC4 protein. In some aspects, the NPC4 protein comprises an amino acid sequence comprising about 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 with the amino acid sequence of SEQ ID NO: 9. In some aspects, the NPC4 protein comprises an amino acid sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9.

[0093] some aspects, the NPC4 protein is encoded by a nucleic acid sequence comprising a nucleotide sequence comprising about 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 with the nucleic acid sequence of SEQ ID NO: 8. In some aspects, the NPC4 protein is encoded by a nucleic acid sequence comprising a nucleotide sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 8. 104866324.4Polsinelli Docket No.: 077875-849424

[0094] In other aspects, genetically modified plants of the instant disclosure comprise increased expression of an NPC4 protein. In some aspects, the NPC4 protein is a C. sativa NPC4 protein. In some aspects, the NPC4 protein comprises an amino acid sequence comprising about 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 with any one of the amino acid sequences set forth in SEQ ID NOs: 10- 13. In some aspects, the NPC4 protein comprises an amino acid sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with any one of the amino acid sequences set forth in SEQ ID NOs: 10- 13.

[0095] In some aspects, the genetically modified plant expresses a polynucleotide encoding a NPC4. In such aspects, the NPC4 encodes an amino acid sequence with about 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 sequence of SEQ ID NO: 1, 7, 9, 10, 11, 12, or 13. In specific aspect, the NPC4 encodes an amino acid sequence with 100% sequence identity to the sequence of SEQ ID NO: 1, 7, 9, 10, 11, 12, or 13. The genetically modified plant, in one aspect, the polynucleotide encodes a NPC4 comprising a nucleic acid sequence with about 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 sequence of SEQ ID NO: 2, 6, or 8. Alternatively, the polynucleotide encodes a NPC4 comprising a nucleic acid sequence with 100% sequence identity to the sequence of SEQ ID NO: 2, 6, or 8.

[0096] The disclosure further provides a plant cell, seed, plant product thereof, of the genetically modified plant. (c) Increased expression of NPC

[0097] Genetically modified plants of the instant disclosure comprise one or more genetic modifications that increase expression of an NPC protein in the plant when compared to a plant that does not comprise the genetic modification. Non-limiting examples of genetic modifications that increase expression of a protein such as an NPC of the instant disclosure include expression constructs that express the NPC protein, programmable nucleic acid modification systems programmed to increase expression of the NPC protein or any combination thereof. 104866324.4Polsinelli Docket No.: 077875-849424

[0098] In some aspects, a genetic modification of the instant disclosure comprises an expression construct comprising a promoter operably linked to a polynucleotide encoding an NPC protein. In some aspects, the expression construct increases the expression of an NPC4 protein in the plant. Exemplary expression constructs are described in Section II herein below.

[0099] In some aspects, a genetic modification of the instant disclosure comprises a programmable nucleic acid modification system programmed to increase expression of the NPC protein. As used herein, a “programmable nucleic acid modification system” is a system capable of targeting a nucleic acid sequence in a polynucleotide and modifying the nucleic acid sequence of the polynucleotide or modifying the expression of the polynucleotide. Accordingly, a programmable nucleic acid modification system can be used to alter a polynucleotide or to alter a protein encoded by the nucleic acid sequence. A programmable nucleic acid modification system can also be used to modify the expression of a nucleic acid sequence encoded by the polynucleotide or modify the expression of a polypeptide encoded by the polynucleotide. In the context of a genetic modification of a polynucleotide encoding an NPC protein, the programmable nucleic acid modification system can specifically target a nucleic acid sequence in a polynucleotide encoding the NPC protein and increases expression of the NPC protein. The programmable nucleic acid modification system can comprise an interfering nucleic acid molecule such as RNAi or a nucleic acid editing system.

[0100] Nucleic acid editing systems generally comprise a programmable nucleic acid binding domain and a nuclease domain to generate a programmable nucleic acid nuclease system or a transcription factor domain to create a programmable transcription regulation system. A programmable nucleic acid binding domain of an editing system is capable of targeting a nucleic acid sequence in a polynucleotide, a nuclease is capable of facilitating modification of the nucleic acid sequence of the polynucleotide, and a transcription factor is capable of regulating transcription and thus expression of the polynucleotide.

[0101] Programmable nucleic acid binding domains rely for specificity on the delivery of exogenous protein(s), and / or a guide RNA (gRNA), deadRNA (dRNA), or single guide RNA (sgRNA) having a sequence which binds specifically to a gene sequence of interest. Non-limiting examples of programmable nucleic acid binding domains that can be used in a programmable nucleic acid editing system include, without limit, a programmable nucleic acid domain of RNA-guided clustered regularly interspersed short palindromic 17 104866324.4Polsinelli Docket No.: 077875-849424 repeats (CRISPR) / CRISPR- associated (Cas) (CRISPR / Cas) nuclease systems, a CRISPR / Cpf1 nuclease system, a zinc finger nuclease (ZFN), a transcription activator-like effector (TALE), a meganuclease, and a ribozyme. Other suitable programmable nucleic acid binding domains will be recognized by individuals skilled in the art.

[0102] When the programmable nucleic acid modification system comprises more than one component, such as a binding protein, a nuclease, a transcription factor or a guide nucleic acid, the multi-component modification system can be modular, in that the different components can optionally be distributed among two or more nucleic acid constructs as described herein. The system components can be delivered by a plasmid or viral vector or as a synthetic oligonucleotide. II. Nucleic acid constructs

[0103] A further aspect of the present disclosure provides one or more nucleic acid constructs for introducing a genetic modification described in Section I herein above. The nucleic acid constructs can be DNA or RNA, linear or circular, single-stranded or double-stranded, or any combination thereof. The nucleic acid constructs can be codon- optimized for efficient translation into protein, and possibly for transcription into an RNA donor polynucleotide transcript in the cell of interest. Codon optimization programs are available as freeware or from commercial sources. The expression construct as disclosed herein may be a recombinant DNA construct comprising a polynucleotide sequence a nonspecific phospholipase C4 (NPC4) protein. The expression construct in a specific aspect may be a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a NPC4. The NPC4 may be selected from a AtNPC4, OsNPC3, and OSNPC4.

[0104] Expression constructs generally comprise DNA coding sequences operably linked to at least one promoter control sequence for expression in a cell of interest. Promoter control sequences can control expression of the transposase, the programmable targeting nuclease, the donor polynucleotide, or combinations thereof in bacterial (e.g., E. coli) cells or eukaryotic (e.g., yeast, insect, mammalian, or plant) cells. Suitable bacterial promoters include, without limit, T7 promoters, lac operon promoters, trp promoters, tac promoters (which are hybrids of trp and lac promoters), variations of any of the foregoing, and combinations of any of the foregoing. Non-limiting examples of suitable eukaryotic promoters include constitutive, regulated, or cell- or tissue-specific promoters. As explained above, methylation of the MeSWEET10a gene can be targeted in leaves by 104866324.4Polsinelli Docket No.: 077875-849424 specifically expressing the system in leaves using a leaf-specific promoter, allowing for fine- tuning pathogen resistance and normal plant growth and development.

[0105] Suitable eukaryotic constitutive promoter control sequences include, but are not limited to, pU10, cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor (ED1)-alpha promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, fragments thereof, or combinations of any of the foregoing. Examples of suitable eukaryotic regulated promoter control sequences include, without limit, those regulated by heat shock, metals, steroids, antibiotics, or alcohol. Non- limiting examples of tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, promoter, SYN1 promoter, and WASP promoter.

[0106] Promoters can also be plant-specific promoters, or promoters that can be used in plants. A wide variety of plant promoters are known to those of ordinary skill in the art, as are other regulatory elements that can be used alone or in combination with promoters.

[0107] Plant promoters can be divided into two types, namely, constitutive promoters and non-constitutive promoters. Constitutive promoters are classified as providing for a range of constitutive expression. Thus, some are weak constitutive promoters, and others are strong constitutive promoters. Non-constitutive promoters include tissue-preferred promoters, tissue-specific promoters, cell-type specific promoters, and inducible promoters. Suitable plant-specific constitutive promoter control sequences include, but are not limited to, CaMV35S promoter, CaMV 19S, GOS2, Arabidopsis At6669 promoter, Rice cyclophilin, Maize H3 histone, Synthetic Super MAS, an opine promoter, a plant ubiquitin (Ubi) promoter, an actin 1 (Act-1) promoter, pEMU, Cestrum yellow leaf curling virus promoter (CYMLV promoter), and an alcohol dehydrogenase 1 (Adh-1) promoter. Other constitutive promoters include those in U.S. Pat. Nos.5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

[0108] Regulated plant promoters respond to various forms of environmental stresses, or other stimuli, including, for example, mechanical shock, heat, cold, flooding, 19 104866324.4Polsinelli Docket No.: 077875-849424 drought, salt, anoxia, pathogens such as bacteria, fungi, and viruses, and nutritional deprivation, including deprivation during times of flowering and / or fruiting, and other forms of plant stress. For example, the promoter can be a promoter which is induced by one or more, but not limited to one of the following: abiotic stresses such as wounding, cold, desiccation, ultraviolet-B, heat shock or other heat stress, drought stress or water stress. The promoter can further be one induced by biotic stresses including pathogen stress, such as stress induced by a virus or fungi, stresses induced as part of the plant defense pathway or by other environmental signals, such as light, carbon dioxide, hormones or other signaling molecules such as auxin, hydrogen peroxide and salicylic acid, sugars and gibberellin or abscisic acid and ethylene. Suitable regulated plant promoter control sequences include, but are not limited to, salt-inducible promoters such as RD29A; drought- inducible promoters such as maize rab17 gene promoter, maize rab28 gene promoter, and maize Ivr2 gene promoter; heat-inducible promoters such as heat tomato hsp80-promoter from tomato.

[0109] Tissue-specific plant promoters can include, but are not limited to, fiber-specific, green tissue-specific, root-specific, stem-specific, flower-specific, callus- specific, pollen-specific, egg-specific, promoters specific to male or female reproductive tissues, and seed coat-specific. Suitable tissue-specific plant promoter control sequences include, but are not limited to, leaf-specific promoters , seed-preferred promoters [e.g., from seed-specific genes , Brazil Nut albumin, legumin , Glutelin (rice) , Zein, napA, Wheat SPA , sunflower oleosin , endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 , wheat a, b, and g gliadins , Barley ltrl promoter, barley B1, C, D hordein, Barley DOF, Biz2 , Synthetic promoter , rice prolamin NRP33, rice-globulin Glb-1 , rice alpha-globulin REB / OHP-1 , rice ADP-glucose PP , maize ESR gene family , sorgum gamma-kafirin , embryo-specific promoters [e.g., rice OSH1 , KNOX , rice oleosin , and flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA), LAT52 , apetala-3].

[0110] Nucleic acids encoding a genetic modification that can increase expression of an NPC protein in plants can be present in a construct. Suitable constructs include plasmid constructs, viral constructs, and self-replicating RNA (Yoshioka et al., Cell Stem Cell, 2013, 13:246-254). For instance, the nucleic acid encoding one or more components of an engineered DNA methylation system and / or transcription activation system can be present in a plasmid construct.

[0111] Non-limiting examples of suitable plasmid constructs include pUC, pBR322, pET, pBluescript, and variants thereof. Alternatively, the nucleic acid encoding 104866324.4Polsinelli Docket No.: 077875-849424 one or more components of an engineered DNA methylation system and / or transcription activation system can be part of a viral vector (e.g., lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, and so forth).

[0112] The plasmid or viral vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable reporter sequences (e.g., antibiotic resistance genes), origins of replication, T-DNA border sequences, and the like. The plasmid or viral vector can further comprise RNA processing elements such as glycine tRNAs, or Csy4 recognition sites. Such RNA processing elements can, for instance, intersperse polynucleotide sequences encoding multiple gRNAs under the control of a single promoter to produce the multiple gRNAs from a transcript encoding the multiple gRNAs. When a cys4 recognition cite is used, a vector can further comprise sequences for expression of Csy4 RNAse to process the gRNA transcript. Additional information about vectors and use thereof can be found in “Current Protocols in Molecular Biology”, Ausubel et al., John Wiley & Sons, New York, 2003, or “Molecular Cloning: A Laboratory Manual”, Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0113] The plasmid or viral vector can also comprise a transit peptide for targeting of a protein product, particularly to a chloroplast, leucoplast or other plastid organelle or vacuole or an extracellular location. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of other such isolated chloroplast proteins include, but are not limited to those associated with the small subunit (SSU) of ribulose- 1,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS) and transit peptides described in U.S. Pat. No. 7,193,133, herein incorporated by reference. It has been demonstrated in vivo and in vitro that non-chloroplast proteins can be targeted to the chloroplast by use of protein fusions with a heterologous CTP and that the CTP is sufficient to target a protein to the chloroplast. Incorporation of a suitable chloroplast transit peptide, such as, the Arabidopsis thaliana EPSPS CTP , and the Petunia hybrida EPSPS CTP (CTP4) has been show to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants. The production of glyphosate tolerant plants by expression of a fusion protein comprising an amino-terminal CTP with a glyphosate resistant EPSPS enzyme is well known by those skilled in the art. 104866324.4Polsinelli Docket No.: 077875-849424

[0114] In some aspects, nucleic acid constructs for increasing expression of an NPC protein in a plant can be an expression construct comprising a promoter operably linked to a polynucleotide encoding the NPC protein. In some aspects, an expression construct of the instant disclosure comprises a 35S cauliflower mosaic virus promoter operably linked to a nucleic acid sequence encoding a NPC protein. In some aspects, an expression construct of the instant disclosure comprises a 35S cauliflower mosaic virus promoter operably linked to a nucleic acid sequence encoding a NPC4 protein. The NPC4 protein can be a AtNPC4, OsNPC3, or OSNPC4.

[0115] A recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a NPC4, wherein the NPC4 comprises a nucleic acid sequence with about 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 sequence of SEQ ID NO: 2, 6, or 8. Alternatively, provided herein is a recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a NPC4, wherein the NPC4 comprises a nucleic acid sequence with 100% sequence identity to the sequence of SEQ ID NO: 2, 6, or 8.

[0116] In some aspects, an expression construct of the instant disclosure comprises a 35S cauliflower mosaic virus promoter operably linked to a nucleic acid sequence encoding an AtNPC4 protein. In some aspects, the expression construct comprises a nucleic acid sequence comprising about 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 with the nucleic acid sequence of SEQ ID NO: 3. In some aspects, the expression construct comprises a nucleic acid sequence comprising about 75% or more, at least about 85% or more, at least about 95% or more, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 3.

[0117] The disclosure further encompasses a transgenic plant cell having in its genome the recombinant DNA construct. A transgenic plant comprising the transgenic plant cell. A crop product produced from the transgenic plant, a transgenic progeny seed or propagatable plant part of the transgenic plant are further provided. III. Methods

[0118] A further aspect of the present disclosure encompasses methods of improving phosphorus redistribution in plants, improving yield and oil production in plants, and methods of altering fatty acid composition of oil produced in plants. The methods 22 104866324.4Polsinelli Docket No.: 077875-849424 comprise genetically modifying a plant to overexpress a nonspecific phospholipase C (NPC) protein when compared to wild type plants. Increasing expression of the NPC protein remobilizes (redistributes) phosphorus from old, senescing tissues to young, growing tissues and seeds in the genetically modified plant thereby enhancing plant growth and yield and altering fatty acid composition in the genetically modified plant. The method comprises introducing to the plant a recombinant DNA construct comprising a polynucleotide sequence encoding a nonspecific phospholipase C4 (NPC4) protein operably linked to a heterologous promoter. The NPC protein and the plant can be as described in Section I herein above. In some aspects, the NPC protein is an NPC4 protein. The NPC4 may be selected from a AtNPC4, OsNPC3, and OSNPC4. In some aspects, the plant is camelina. In other aspects, the plant is oryza. In some aspects, a method of the instant disclosure comprises genetically modifying a camelina plant or a oryza plant to overexpress a nonspecific phospholipase C4 (NPC4) protein when compared to wild type plants.

[0119] In some aspects, overexpressing the NPC4 protein increases expression of phosphorus transporter genes in the old tissues of the genetically modified plant. The plant can be cultivated under phosphorus-limited conditions.

[0120] In some aspects, the genetically modified plant demonstrates enhanced phosphorus use efficiency (PUE) under phosphorus-limited conditions. In some aspects, the genetically modified plant exhibits greater growth and yield improvements under phosphorus-limited conditions compared to phosphorus-sufficient conditions. In some aspects, the genetically modified plant shows increased seed yield and oil production under phosphorus-limited conditions when compared to a wild-type plant cultivated under the same conditions. Altered fatty acid composition can comprise increased linolenic acid (18:3) content, decreased linoleic acid (18:2) content, increased oleic acid (18:1) content, decreased stearic acid (18:0), palmitic acid (16:0), arachidic acid (20:0), and eicosadienoic acid (20:2), increased 18:1 and gondoic acid (20:1), a decreased 20:2 fatty acid, and any combination thereof, when compared to a wild-type plant cultivated under the same conditions.

[0121] Another aspect of the instant disclosure encompasses a method of improving yield and oil content and altering fatty acid composition in a Camelina sp. plant or a Oryza sp. plant. The method comprises genetically modifying the camelina plant or the oryza plant to overexpress an NPC4 protein. 104866324.4Polsinelli Docket No.: 077875-849424 (d) Introduction into the cell

[0122] The method comprises introducing a nucleic acid construct into a cell of interest. As explained above, an engineered protein can be encoded on more than one nucleic acid sequence. Accordingly, a method of the instant disclosure can comprise introducing more than one nucleic acid construct into the cell.

[0123] The one or more nucleic acid constructs described above can be introduced into the cell by a variety of means. Suitable delivery means include microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposomes and other lipids, dendrimer transfection, heat shock transfection, nucleofection transfection, gene gun delivery, dip transformation, supercharged proteins, cell-penetrating peptides, viral vectors, magnetofection, lipofection, impalefection, optical transfection, Agrobacterium tumefaciens mediated foreign gene transformation, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions. The choice of means of introducing the system into a cell can and will vary depending on the cell, or the system or nucleic acid nucleic acid constructs encoding the system, among other variables. (e) Culturing a cell

[0124] The method can further comprise culturing a cell under conditions suitable for expressing the engineered protein. Methods of culturing cells are known in the art. In some aspects, the cell is from an animal, fungi, oomycete or prokaryote. In some aspects, the cell is a plant cell, plant, or plant part. When the cell is in tissue ex vivo, or in vivo within a plant or within a plant part, the plant part and / or plant may also be maintained under appropriate conditions for insertion of the donor polynucleotide. In general, the plant, plant part, or plant cell is maintained under conditions appropriate for cell growth and / or maintenance. Those of skill in the art appreciate that methods for culturing plant cells are known in the art and may and will vary depending on the cell type. Routine optimization may be used, in all cases, to determine the best techniques for a particular cell type. IV. Kits

[0125] A further aspect of the present disclosure provides kits comprising one or more genetically modified plants comprising improved yield and oil content and altered fatty acid composition, one or more nucleic acid constructs for increasing the expression of a NPC protein in a plant, a plant comprising the one or more nucleic acid constructs for 24 104866324.4Polsinelli Docket No.: 077875-849424 increasing expression of the NPC in the plant, or any combination thereof. The genetically modified plant can be as described in Section I. The one or more nucleic acid constructs can be as described in Section II. A plant comprising the one or more nucleic acid constructs can be as described in Section I herein above.

[0126] The kits can further comprise transfection reagents, cell growth media, selection media, in vitro transcription reagents, nucleic acid purification reagents, protein purification reagents, buffers, and the like. The kits provided herein generally include instructions for carrying out the methods detailed below. Instructions included in the kits may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” may include the address of an internet site that provides the instructions. SEQUENCES25 104866324.4Polsinelli Docket No.: 077875-849424104866324.4Polsinelli Docket No.: 077875-849424104866324.4Polsinelli Docket No.: 077875-84942428 104866324.4Polsinelli Docket No.: 077875-849424104866324.4Polsinelli Docket No.: 077875-849424104866324.4Polsinelli Docket No.: 077875-849424104866324.4Polsinelli Docket No.: 077875-849424DEFINITIONS

[0127] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0128] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are 104866324.4Polsinelli Docket No.: 077875-849424 intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0129] A “genetically modified” cell refers to a cell in which the nuclear, organellar or extrachromosomal nucleic acid sequences of a cell has been modified, i.e., the cell contains at least one nucleic acid sequence that has been engineered to contain an insertion of at least one nucleotide, a deletion of at least one nucleotide, and / or a substitution of at least one nucleotide.

[0130] The terms “genome modification” and “genome editing” refer to processes by which a specific nucleic acid sequence in a genome is changed such that the nucleic acid sequence is modified. The nucleic acid sequence may be modified to comprise an insertion of at least one nucleotide, a deletion of at least one nucleotide, and / or a substitution of at least one nucleotide. The modified nucleic acid sequence is inactivated such that no product is made. Alternatively, the nucleic acid sequence may be modified such that an altered product is made.

[0131] The terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms may encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and / or phosphate moieties. In general, an analog of a particular nucleotide has the same base-pairing specificity, i.e., an analog of A will base-pair with T. The nucleotides of a nucleic acid or polynucleotide may be linked by phosphodiester, phosphothioate, phosphoramidite, phosphorodiamidate bonds, or combinations thereof.

[0132] The term "nucleotide" refers to deoxyribonucleotides or ribonucleotides. The nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or nucleotide analogs. A nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety. A nucleotide analog may be a naturally occurring nucleotide (e.g., inosine) or a non-naturally occurring nucleotide. Non-limiting examples of modifications on the sugar or base moieties of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7-deaza purines). Nucleotide analogs also include dideoxy nucleotides, 104866324.4Polsinelli Docket No.: 077875-849424 2’-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.

[0133] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.

[0134] As used herein, the terms "target site", "target sequence", or “nucleic acid locus” refer to a nucleic acid sequence that defines a portion of a nucleic acid sequence to be modified or edited and to which a homologous recombination composition is engineered to target.

[0135] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences may also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) may be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm may be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res.14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP may be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non- redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs may be found on the GenBank website. 104866324.4Polsinelli Docket No.: 077875-849424 With respect to sequences described herein, the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value therebetween. Typically the percent identities between sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity.

[0136] As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense. EXEMPLARY EMBODIMENTS 1. A method of improving phosphorus redistribution in plants, the method comprising genetically modifying a plant to increase expression of a nonspecific phospholipase C4 (NPC4) protein when compared to wild type plants, wherein increasing expression of the NPC4 protein remobilizes (redistributes) phosphorus from old, senescing tissues to young, growing tissues and seeds in the genetically modified plant thereby enhancing plant growth and yield, and altering fatty acid composition in the genetically modified plant. 2. The method of embodiment 1, wherein overexpressing the NPC4 protein increases expression of phosphorus transporter genes in the old tissues of the genetically modified plant. 3. The method of embodiment 1 or embodiment 2, wherein the genetically modified plant is cultivated under phosphorus-limited conditions. 4. The method of embodiment 3, wherein the genetically modified plant exhibits greater growth and yield improvements under phosphorus-limited conditions when compared to phosphorus-sufficient conditions. 5. The method of embodiment 4, wherein the genetically modified plant shows increased seed yield and oil production under phosphorus-limited conditions when compared to a wild-type plant cultivated under the same conditions. 104866324.4Polsinelli Docket No.: 077875-849424 6. The method of embodiment 4, wherein the genetically modified plant comprises altered fatty acid composition when compared to a wild-type plant cultivated under the same conditions. 7. The method of embodiment 6, wherein altered fatty acid composition comprises increased the linolenic acid (18:3) content, decreased linoleic acid (18:2) content, increased oleic acid (18:1) content, decreased stearic acid (18:0), palmitic acid (16:0), arachidic acid (20:0), and eicosadienoic acid (20:2), increased 18:1 and gondoic acid (20:1), a decreased 20:2 fatty acid, and any combination thereof, when compared to a wild-type plant cultivated under the same conditions. 8. The method of embodiment 4, wherein the genetically modified plant demonstrates enhanced phosphorus use efficiency (PUE) under phosphorus-limited conditions. 9. The method of any of the preceding embodiments, wherein the genetically modified plant is a genetically modified camelina plant. 10. The method of embodiment 9, wherein enhancing plant growth and increasing yield in the genetically modified camelina plant comprises increased seed yield and elevated oil content when compared to a wild-type camelina plant cultivated under the same conditions. 11. The method of embodiment 9, wherein the genetically modified camelina plant comprises altered fatty acid composition when compared to a wild-type camelina plant cultivated under the same conditions. 12. The method of embodiment 11, wherein altered fatty acid composition comprises increased the linolenic acid (18:3) content, decreased linoleic acid (18:2) content, increased oleic acid (18:1) content, decreased stearic acid (18:0), palmitic acid (16:0), arachidic acid (20:0), and eicosadienoic acid (20:2), increased 18:1 and gondoic acid (20:1), a decreased 20:2 fatty acid, and any combination thereof, when compared to a wild-type plant cultivated under the same conditions. 13. The method of any one of the preceding embodiments, wherein the expression construct comprises a constitutively expressed promoter operably linked to the polynucleotide encoding the NPC4 protein. 104866324.4Polsinelli Docket No.: 077875-849424 14. The method of embodiment 13, wherein the polynucleotide encoding the NPC4 protein encodes an AtNPC4 protein. 15. The method of embodiment 13, wherein the expression construct comprises a CaMV promoter operably linked to a polynucleotide encoding an AtNPC4 protein. 16. A method of improving yield and oil content and altering fatty acid composition in a camelina sp. plant, the method comprising genetically modifying the camelina plant to overexpress an NPC4 protein. 17. A genetically modified camelina plant comprising improved yield and oil content and altered fatty acid composition, the genetically modified Camelina plant comprising an expression construct comprising a promoter operably linked to a polynucleotide encoding a nonspecific phospholipase C4 (NPC4) protein, wherein expression of the NPC4 protein remobilizes (redistributes) phosphorus from old, senescing tissues to young, growing tissues in the genetically modified camelina plant thereby improving yield and oil content and altering the fatty acid composition in the genetically modified camelina plant. 18. The genetically modified Camelina plant of embodiment 17, wherein the expression construct comprises a constitutively expressed promoter operably linked to the polynucleotide encoding the NPC4 protein. 19. The genetically modified Camelina plant of embodiment 18, wherein the polynucleotide encoding the NPC4 protein encodes an AtNPC4 protein. 20. The genetically modified Camelina plant of embodiment 18, wherein the expression construct comprises a CaMV promoter operably linked to a polynucleotide encoding an AtNPC4 protein. 21. The genetically modified Camelina plant of embodiment 15, wherein the genetically modified Camelina plant comprises enhanced growth, increased seed yield and elevated oil production, and altered fatty acid composition when compared to a wild-type camelina plant cultivated under the same conditions. 22. A kit for improving yield and oil content and altering fatty acid composition in a plant, the kit comprising one or more nucleic acid constructs for increasing the expression of a 104866324.4Polsinelli Docket No.: 077875-849424 NPC protein in a plant, a plant comprising the one or more nucleic acid constructs for increasing expression of the NPC in the plant, or any combination thereof. EXAMPLES

[0137] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the present disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0138] The publications discussed throughout are provided solely for their disclosure before the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0139] The following examples are included to demonstrate the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosure. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes could be made in the disclosure and still obtain a like or similar result without departing from the spirit and scope of the disclosure, therefore all matter set forth is to be interpreted as illustrative and not in a limiting sense. Example 1. NPC4 expression and manipulation in camelina

[0140] Membrane phospholipids in plants contain approximately 1 / 3 of total cellular P. In response to P shortage, the membrane lipids in plants undergo remodeling, with a decrease in phospholipids and an increase in non-P-containing lipids (Li et al., 2006; Moellering and Benning, 2010; Okazaki et al., 2013). Specific phospholipid-hydrolyzingan important role in lipid remodeling. Genetic impairments of the lipid remodeling compromise plant growth in plant response to P-deficiency (Cruz-Ramírez et al., 2006; Li et al., 2006; Gaude et al., 2008; Yang et al., 2021, 2023). For example, the disruption of NPC4 function led to impaired root hair and root elongation under Pi deficiency in Arabidopsis (Chandrika et al., 2013; Su et al., 2018; Yang et al., 2021). Recent studies in rapeseed showed that increased expression of NPC4 promoted plant growth and seed production, whereas the loss of NPC4 had the opposite effect particularly under P-limited growth 104866324.4Polsinelli Docket No.: 077875-849424 conditions (Yang et al., 2023). NPC4 in Arabidopsis was found to prefer hydrolyzing phosphosphingolipids to phosphoglycerolipids, affecting primarily the change of sphingolipids (Yang et al., 2021). By comparison, NPC4-knockout and overexpression (OE) in rapeseed alter the level of phosphosphingolipids and phosphoglycerolipids, indicating that NPC4 hydrolyzes both phosphosphingolipids and phosphoglycerolipids in rapeseed (Yang et al., 2023). The NPC4-medated hydrolysis of phospholipids is expected to release Pi from phospholipids for other critical cellular needs for plant growth under P deprivation (Tjellström et al., 2008). However, the mechanism by which NPC4 promotes plant growth under Pi deficiency remains largely unknown.

[0141] The plant P use efficiency consists of two distinctive, yet interrelated components: P acquisition efficiency (PAE) and internal P use efficiency (PUE) (Raghothama, 1999). PAE can be improved by changing root growth and architecture (e.g., increasing proliferation of lateral roots and root hairs), secreting cellular components (e.g., organic acids and acid phosphatases) into the soil, and / or enhancing rhizosphere interactions to improve P bioavailability (Schachtman et al., 1998; Hurley et al., 2010). PUE (the amount of biomass produced per unit P) measures how efficiently the acquired P is used to produce biomass or seeds, which involves the distribution and remobilization of, and cellular response to acquired P in plants. Great advances have been made in understanding PAE, but the progress in PUE and using it to improve plant production without increasing Pi applications has been limited.

[0142] In this and following examples, the effects of NPC4 on plant growth and seed oil production in camelina, an emerging industrial oilseed crop with potential for production in marginal lands that are often deficient in Pi are investigated. The results showed that OE of NPC4 increased camelina seed and oil production under Pi-limited conditions. Moreover, the study reveals that NPC4 increases P remobilization from old to young tissues to promote P utilization for plant growth and seed production.

[0143] Camelina is a hexaploid species and a close relative of Arabidopsis (Kagale et al., 2014). Its genome contains 3 NPC4s, NPC4a, NPC4b, and NPC4c (FIG. 1A). To test whether these genes are induced by Pi deficiency, their transcript levels were quantified in young camelina seedlings under Pi sufficient and deficient conditions. Among the three NPCs, NPC4b is induced most highly under Pi deficiency (FIG.1B). The transcript level of NPC4b increased about 5- and 3-fold in leaves and roots, respectively, whereas that of NPC4a and NPC4c was almost doubled compared to that under sufficient Pi (FIG. 104866324.4Polsinelli Docket No.: 077875-849424 1B). The basal level of NPC4s in roots was almost 10-fold higher than that in leaves (FIG. 1B).

[0144] For greenhouse experiments, seeds of Suneson and overexpression lines OE6, and OE7 were sown in a cubic pot (cm3) filled with soil of 1:1(v / v) vermiculite and perlite. Plants were grown in the greenhouse under the following conditions: 20-21°C, 50% humidity, 16-hr light / 8-hr dark cycle, supplemental light threshold of 566 pmol / m2 / s (supplemental lights turned off when outside solar radiation was 566 pmol / m2 / s or above), 100% shading with shade cloth when solar radiation at 1415 pmol / m2 / s or above, and 50% shading at 1132 pmol / m2 / s or above (Li et al., 2015). Seedlings were watered using half- strength Hoagland nutrition solution (Hoagland and Arnon, 1950) with varying P concentrations (25, 50, and 500 μM) during the first 3 weeks, then with 50, 200, and 1000 μM Pi afterwards, respectively. Trays were dipped in the nutrition solution for 10 min as one fertilization treatment. Seedlings were fertilized once a week during the first three weeks, and every three days for the rest of time until the end of the experiments. Plant materials were collected and analyzed at 21 days for vegetative growth parameters. The fresh weight of shoots (leaf and stem) was measured, and then the samples were dried in an oven set at 105°C for 30 minutes, and at 80°C for 48 hours. The biomass was measured as dry weight. When plants grew completely matured, plant height and branch number were measured and counted.

[0145] To facilitate functional studies of NPC4, camelina lines expressing a 35S::AtNPC4 construct were generated (FIG.1C). The Arabidopsis gene encoding NPC4 was used because it was shown to improve plant growth under Pi deficiency (Yang et al., 2021; 2023). To overexpress NPC4, the full length coding DNA sequence of NPC4 was amplified by PCR using Col-0 Arabidopsis leaf cDNA as a template (Peters, et al., 2010). The construct was cloned into a modified p35S-FAST vector before the Strep coding sequence at SacI and PacI sites under the control of a 35S cauliflower mosaic virus promoter. The construct was introduced into the Agrobacterium tumefaciens strain C58C1 after verifying the sequence of the construct. Camelina sativa (cv. Suneson) plants were transformed by Agrobacterium tumefaciens containing 35S-FAST::AtNPC4 using a floral dip technique (Lu and Kang, 2008). The transgenic plants were screened by kanamycin resistance selection and confirmed by PCR using a forward primer (GCP169) targeting the 35S promoter and a reverse primer GCP218 specific to the NPC4 gene. Several independent homozygous overexpression lines were obtained in T4 generation. The transgenic plants were verified by PCR amplification of the inserted DNA from the genomic 40 104866324.4Polsinelli Docket No.: 077875-849424 DNA using a forward primer targeting the 35S promoter region and a reverse primer in the introduced NPC4 (FIG.1D).

[0146] In addition, proteins from the transgenic plants were immunoblotted with anti-Strep antibody. Overexpression of 35S::AtNPC4-Strep was verified by immunoblot analysis using anti-Strep. Briefly, total protein was extracted from leaves of 3-week old plants using the extraction buffer (50mM Tris pH7.4, 50 μM NaCl, 5% glycerol) and added with protease inhibitor (10 μl to 1 ml total volume). Ten μg protein of supernatant protein was separated by 10% SDS-PAGE gel after centrifuge at 12,000 RPM for 5 min. The protein samples were transferred onto a polyvinylidene difluoride membrane. After blocking into 5% Nonfat milk in TBST for 30 min, the membrane was blotted against with anti-Strep antibody (5000:1, Sigma-Aldrich: SAB2702227) conjugated with horseradish peroxidase for 1 h, then rinsed 3 times × 5 min with TBST buffer. The membrane was incubated with LumiGLO substrate, and the signal was detected with iBright500 (Invitrogen).

[0147] Immunoblotting showed the production of the NPC4-Strep protein (FIG.1E). Transgenic lines 6 and 7 displayed more than 6-fold higher level of NPC4 expression than that of WT and were used for further study (FIG.1F). Example 2. NPC4-OE enhanced camelina seed production in response to P levels.

[0148] To determine whether NPC4-OE alters camelina growth, data was collected on different growth and development stages of camelina plants grown under different Pi levels, 1000 μM, 200 μM, and 50 μM, designated as P1000, P200, and P50, respectively. P1000 provides sufficient Pi, whereas P200 and P50 are deficient for camelina growth (Li et al., 2023). Camelina plant growth was impeded under the P200 and P50 conditions (FIGs. 7A-7B). Compared with plants grown at P sufficient P1000 conditions, the leaf area decreased by approximately 40% and 35% at P200, and 65% and 70% at P50, respectively, in 3-week-old plants (FIGs.7A-7B). NPC4-OE and WT plants were similar in these traits under the same Pi level (FIGs.7A-7B). Pi deficiency at either P200 or P50 delayed flowering for almost 2 days compared to the Pi-sufficient P1000 treatment, with NPC4-OE flowering about 2 days earlier regardless of Pi levels (FIG.2A; FIGs.7A-7C).

[0149] NPC4-OE plants tended to have more branches at P1000, P200, and P50, and the number of branches was about 20% greater in NPC4-OE lines than WT at P200 (FIGs. 2A, 2B upper panel). The seed yield per plant was higher in NPC4-OE lines compared to WT under the same P level (FIG.2B middle panel). With the three P levels 41 104866324.4Polsinelli Docket No.: 077875-849424 tested, NPC4-OE plants displayed the greatest increase in seed yield (40%) at P200, whereas the increase in seed yield was about 20% at P50 and 10% at P1000 over WT. Those data indicate that increased NPC4 expression enhances camelina growth and seed production, with the greatest increase in the moderate Pi deficient condition tested. Example 3. NPC4-OE increases seed oil content and alters fatty acid composition.

[0150] Camelina is a seed oil crop, and its seed oil yield and fatty acid composition are the primary determinants of its value. The oil content and fatty acid composition of camelina seeds harvested from plants grown under different Pi concentrations were analyzed. For each sample, five camelina seeds were put in labeled glass tubes with Teflon-lined screw caps, and then 2 mL of methanol with 1.5% H2SO4 and 0.01% butylated hydroxytoluene (BHT) was added using a glass pipette. Twenty-five microliters of 16.2 μmol / ml heptadecanoic acid (C17:0) were added as the internal standard for each sample. The samples were incubated at 90°C for 2 hours for oil extraction and transmethylation. After cooling down, 1 ml of autoclaved ddH2O and 1 ml of hexane were added using a glass pipette. After vortexing, the samples were centrifuged at 900 rpm for 5 minutes. The supernatant was transferred into a labeled vial and stored in a box at -20°C until ready for gas chromatography (GC) analysis. Fatty acid methyl esters (FAMEs) were loaded onto a GC equipped with a SUPELCOWAX-10 (0.25 mm × 30 m) column using helium as a carrier gas at 20 ml / min and underwent flame ionization. The column temperature was maintained at 170°C for 1 min and gradually increased to 210°C by 3°C per minute. FAMEs from seed lipids were identified by comparing their retention times to those of standards. Six biological repeats were performed for all samples. Oil content was expressed as % per seed weight and fatty acid composition was expressed in mol% of total fatty acids detected.

[0151] NPC4-OE plants exhibited a higher oil content than WT under P- deficient P200 and P50 (FIG. 2B, bottom). With the increased oil content and seed yield, NPC4-OE increased oil yield per plant, with the greatest increase at P200 (62%), followed by P50 (37%) and P1000 (20%) in both NPC4-OE lines (FIG.2C).

[0152] In addition, NPC4-OE altered fatty acid composition (FIG.2D). Lipids were extracted and quantified as described previously (Welti et al., 2002; Li et al., 2024) with some modifications. The plants were grown under 1000 μM and 50 μM Pi treatments until flowering. Briefly, around 1 g of fresh samples of the senescing leaves and young 42 104866324.4Polsinelli Docket No.: 077875-849424 leaves were collected and immediately immersed into the pre-heated (75°C) extraction buffer (2-propanol:water:hexane=55:20:25) with 0.01% (v / v) BHT in screw-cap tubes at 75°C for 15 min. Then 1.5 ml of chloroform and 0.6 ml of water were added and mixed. The sample solution was transferred to another fresh glass tube after agitating (120 rpm) at room temperature for 1 hour. Lipids were extracted using chloroform: methanol (2:1 v / v) with 0.01% BHT repeatedly until the samples turned completely bleached (3~4 times), followed by a one-time wash with 1 ml of 1MKCL and 2 ml of water, respectively. The extracted lipids were dried under streams of nitrogen gas. The remaining tissues were dried in an oven at 105°C overnight and dry weight was measured. The dried lipid samples were dissolved in chloroform proportionally based on the dry weight of the samples. Lipid extracts with a mixture of solvents, chloroform / methanol / ammonium acetate (300 mM) in water (300:665:35, v / v / v), and internal standards were detected by ESI-MS / MS (API4000; Applied Biosystems, Foster City, CA). Data were processed and analyzed using Analyst software (v1.5.1) as described previously (Welti et al., 2002). DAG was quantified by a series of neutral loss scans that detected DAG species as [M + NH4]+ ions (Peters et al., 2010; Yuan et al., 2019). The corrected signals based on each DAG species were combined, and the levels of DAG species were calculated by comparison with the internal standard (di-14:0-DAG). The levels of DAG species were expressed as mass spectral signals relative to the internal standard because of variations of ionization and fragmentation efficiencies among different acyl groups.

[0153] At P50, NPC4-OE increased the linolenic acid (18:3) level by 20% with 18% decrease in linoleic acid (18:2) content. At P200, besides an increase in 18:3 and a decrease in 18:2, NPC4-OE seeds also displayed a 13% increase in oleic acid (18:1) with decreases in stearic acid (18:0), palmitic acid (16:0), arachidic acid (20:0), and eicosadienoic acid (20:2). At P1000, NPC4-OE seeds were increased in 18:3 (6%) with a decrease in 18:2 (16%), and also exhibited increases in 18:1 (19%) and gondoic acid (20:1) with a decrease in 20:2 (17%), but no decrease in 18:0 and 16:0 (FIG. 2D). At the three Pi levels tested, the common change is the increased 18:3 (12%) with a decrease in 18:2 (15%) in NPC4-OE seeds, and the magnitude of changes was affected by the level of Pi availability. Example 4. NPC4-OE increases P levels in young leaves and seeds.

[0154] For Pi remobilization, the germinated seedlings were cultured in ½ Hoagland solution with 100 μM Pi until leaf 6 appeared, then transferred to Hoagland 104866324.4Polsinelli Docket No.: 077875-849424 solution without Pi until the leaf 8 was fully expanded. Total P and free Pi contents were measured from root and leaf after 0 d, 5 d (leaf 7 of WT fully appeared), and 11 d (leaf 8 of WT fully appeared) transferring, respectively. For Pi uptake, three-day old seedlings were kept in hydroponic culture containing ½ Hoagland solution (Hoagland and Arnon, 1950) containing 50 μM Pi for 10 d, then transferred to ½ Hoagland solution without Pi for two weeks. Plant roots were placed in tubes with 45 ml Hoagland solution containing 100 μM Pi. Tubes were covered with Parafilm and covered by opaque plastic tarps. Five tubes without seedlings were also filled and covered as controls, and Pi levels in media were measured every 3 h.

[0155] Total P and soluble Pi concentrations were measured based on a modified ammonium molybdate method (Ames, 1966). For total P measurement (Lapis- Gaza et al, 2014), ten to twenty mg dry tissues were digested in 3 mL HNO3 at 100°C for 2 h. After cooling for 10 min, 1 mL of HClO4 was added, and the acid digests were heated to 120°C for 1 h until the tissues were broken down completely. After cooling, samples were diluted with 2 mL of double-distilled water. Aliquots (10-50 μl) of samples were diluted to g / L malachite green) was added and allowed to stand for 2 h. The absorbance of the reaction was measured at 650 nm and the total Pi concentrations were calculated from the standard curve constructed using standard solutions of KH2PO4. For soluble inorganic phosphorus concentration (Staudinger et al., 2022), dried leaves were ground to powders centrifuged at 5000 g for 2 min and aliquots of the supernatant were diluted to 650 μl. The ammonium para molybdate solution (w / v in 1 N H2SO4). The mixture was incubated at from the standard curve of KH2PO4. Experiments included five biological repeats.

[0156] To investigate the effect of NPC4 on P utilization, P levels in leaves from 3-week-old seedlings grown at P1000, P200, and P50 were assayed. In both WT and OE plants, the total P and free Pi level in leaves at P1000 was about 2- to 3-fold higher than those at P200 and P50 (FIG. 3A). NPC4-OE increased the total P and free Pi content at all three Pi levels tested, compared to WT leaves. The magnitude of increase for total P was 20% at P1000, 28% at P200, and 22% at P50 in NPC4-OE leaves. For free Pi levels, NPC4-OE leaves showed a greater increase at the moderate Pi-deficient P200 (67%) 104866324.4Polsinelli Docket No.: 077875-849424 whereas they had a 40% increase under Pi-sufficient P1000 and a 37% increase at severe Pi-deficient P50, compared with WT leaves. Like leaves, NPC4-OE increased both total P and free Pi in seeds at all three Pi levels tested (FIG.3B). Compared to WT seeds, NPC4- OE seeds were increased in total P by about 15% and free Pi by about 28% at Pi-sufficient and deficient conditions (FIG.3B).

[0157] To examine the effect of NPC4-OE on P distribution at different ages of leaves, we assayed P levels at different positions on maturing plants (FIG. 3C and FIG.9A- 9B). The free Pi and total P levels in top, young leaves (YL) were more than 2-fold higher than those in bottom, senescent leaves (SL; FIGs. 3C, 3D). NPC4-OE increased total P and free Pi in young leaves but decreased both in old senescent leaves at Pi-sufficient conditions. Compared to WT, the senescent leaves of NPC4-OE plants were about 36% and 26% lower in free Pi and total P levels, respectively (FIG.3D). Conversely, the young leaves of NPC4-OE plants contained about 26% and 17% higher in free Pi and total P, respectively, than those of WT, whereas no difference in total and free Pi in middle leaves (ML) between WT and NPC4-OE (FIG.3D). Example 5. NPC4-OE promotes Pi mobilization from old to young leaves.

[0158] The opposite effects of NPC4-OE on P levels in young and old leaves suggest that NPC4 may promote P remobilization from old to young leaves during plant growth. Thus, P remobilization was measured by measuring P levels at different stages of leaves after transferring plants grown at a Pi-sufficient condition to a Pi-free medium, followed by continued growth for 11 days (FIG.4A, 4B). In roots, the total P levels tended to increase at day 0, 5, and 11 (FIG.4A), whereas the free Pi level decreased from 34% at the day of the transfer (day 0) to 27% and 22% at days 5 and 11, respectively, after transfer to the Pi-free medium in WT and NPC4-OE roots (FIG.4B). These changes could mean that the free Pi might be used for organic P formation during P deficiency in roots. By comparison, the total P level in leaves decreased at day 0, 5, and 11 after transfer (FIG. 4A). At the day 0 of transfer, NPC4-OE and WT seedlings have similar total P levels in all leaves. However, NPC4-OE displayed at a higher level of total P in the young leaf at 5 and 11 days after transfer, and a lower total P level in older leaves that did WT (FIG.4B).

[0159] In the bottom leaves, the level of free Pi decreased from 0 (11%), 5 (7%), and 11 (6%) days after transfer to a Pi-free medium. At the day 0 of transfer, NPC4- OE and WT seedlings have similar Pi levels in all leaves, but NPC4-OE started to display lower Pi in older leaves (leaf 3) and a higher Pi in younger leaves (leaf 6) (FIG. 4B). Five 45 104866324.4Polsinelli Docket No.: 077875-849424 days after transfer, NPC4-OE showed a higher Pi level in the youngest leaf (leaf 7), no difference in other leaves (FIG. 4B). At 11 days after transfer, NPC4-OE displayed a higher Pi level in the youngest leaf (leaf 8), but a lower Pi level in old leaves (leaf 3) (FIG.4B). Under this condition, because no Pi was absorbed from the media, the higher Pi level in young leaves with a lower Pi level in old leaves NPC4-OE than WT supports that NPC4-OE promotes P remobilization from old to young leaves.

[0160] In addition, the effect of NPC4-OE on P uptake was measured (FIG. 4C). Three-day old seedlings grown hydroponically under P1000 were transferred to P100 for 10 days and then to a medium without Pi for 14 days. After that, seedlings were placed in a P100 medium to monitor the Pi uptake. NPC4-OE and WT displayed no difference in the first 3 hours, but NPC4-OE seedlings displayed 35-57% higher uptake than WT from 6 to 24 hours tested. Example 6. NPC4-OE increases the expression of Pi transport-related genes.

[0161] To probe further the effect of NPC4-OE on P utilization, transcript levels of selected genes involved in Pi remobilization, uptake, and responses in young, middle, and old leaves were monitored. Total RNA was isolated from plant tissues using TRIzol reagent (Invitrogen) according to the manufacturer’s procedure. DNA contamination in RNA samples was prevented with RNase-free DNase (Ambion, Inc. Austin, TX, USA). RNA was reverse-transcribed using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). qRT-PCR reactions were performed with MyiQ Sequence Detection System (Bio- Rad) using SYBR Green to monitor the expression of the target genes and reference genes. Ubiquitin 10 (UBQ10) or CsACT2 (Csa19g026200.1) was used as an internal reference to normalize the relative amount of mRNAs in all samples. The cDNAs were amplified, and PCR products were quantified by using gene-specific primers. The

[0162] Under P-sufficient conditions, all three camelina NPC4s displayed a higher level of expression in old leaves than in young leaves, and NPC4-OE further increased camelina NPC4 expression (FIG.5A). For Pi transporter genes, PHT1;9 controls Pi uptake in Arabidopsis and rice , two of the three CsPHT1;9 genes (Csa09g081170 and Csa16g040570) were more than 2- to 3-fold higher in the old leaves of NPC4-OE than WT, but there was no difference in the CsPHT1;9 transcript levels in middle and young leaves between NPC4-OE and WT (FIG.5B). Of CsPHT1;7 and CsPHT1;8, one copy in each of them (Csa09g064460 and Csa14g026060) exhibited a higher expression level in old leaves 104866324.4Polsinelli Docket No.: 077875-849424 of NPC4-OE (FIG. 5B). NPC4-OE increased the transcript level of all three PHT1;1s (Csa18g010480, Csa11g071540, Csa20g067300) by 1.5-fold in old leaves but had no effect on those genes in young leaves compared with WT (FIG.5B). In addition, xylem Pi exporter CsPHO1 in leaves were also analyzed, and Csa19g033930, Csa15g044550, and Csa01g028010 were also analyzed ranged 2 to 4-fold increases of the mRNA level in old leaves of NPC4-OE lines over WT (FIG.5B). Overall, compared to WT, NPC4-OE increased the transcript levels of Pi transporter genes in old leaves, whereas the expression level of those genes remained similar in young and middle leaves. The results are consistent with the measurements of P contents where old leaves of NPC4-OE were lower in total P and free Pi than those of WT (FIG.3D), supporting that NPC4 promotes Pi remobilization from old or source leaves to young leaves or sink tissues.

[0163] In addition, the effect of NPC4-OE on the transporter expression in roots and leaves under Pi-sufficient and deficient conditions were monitored (FIG.5C). Under sufficient Pi, NPC4-OE showed almost no effect on increasing PHT expression in roots, with only a slight increase in PHT1;8, but the leaves of NPC4-OE displayed increased all these transporters examined. The lack of increase in PHT1 expression in roots under sufficient Pi could mean that does not promote Pi uptake under sufficient Pi (FIG. 5C). The increased PHT1 expression in NPC4-OE leaves, particularly in old leaves, is consistent with the NPC4 promotion of Pi remobilization. However, under Pi deficiency, NPC4-OE increased the expression of PHT1;1, PHT1;8, and PHT1;9 (not PHT1;7) in roots, suggesting that NPC4-OE increases Pi uptake under P-limited conditions. In leaves, NPC4- OE increased all the transporters tested, and the magnitude of increase is greater in NPC4- OE leaves over WT leaves under P-deficient than -sufficient conditions (FIG.5C). These results suggest that NPC4 promotes Pi remobilization more under P-limited than sufficient growth conditions. Example 7. NPC4-OE promotes the decrease in phospholipids under P deficiency.

[0164] Under sufficient Pi, young leaves had higher levels of phospholipids and galactolipids, but a lower level of DAG than old leaves (FIG.6A). This trend held under Pi deficiency except the PA level in young leaves was lower than that in old leaves. Compared with leaves from sufficient Pi, Pi deficiency greatly decreased the level of phospholipids, including PC, PE, PG, PS, and PA (FIGs. 6A, 6B). In contrast, there was no significant decrease in DGDG in old or young leaves under Pi deficiency. Instead, the 104866324.4Polsinelli Docket No.: 077875-849424 DGDG level increased in old leaves, compared with that under sufficient Pi (FIG.6A). The MGDG level decreased in young leaves, but not in old leaves under Pi deficiency (FIG.6A). The WT plants displayed about 3-fold decrease in the phospholipid to galactolipid ratio, comparing the Pi sufficient to Pi deficient condition (FIG.6A, bottom). When mol% changes of lipids were considered, P deficiency decreased greatly in all phospholipids with an increase in mol% of DGDG (FIG. 9A-9B). The decrease in phospholipids with an increase in galactolipids in camelina is consistent with the membrane lipid remodeling under Pi deficiency observed in other plant species.

[0165] Compared to WT leaves, NPC4-OE increased DAG levels in old and young leaves, with a more increase in old leaves under sufficient Pi (FIG. 6A). NPC4-OE decreased PC and PE levels in old and young leaves (FIG. 6B). NPC4-OE decreased PS and PG levels in young leaves, with no effect on PG in old leaves. NPC4-OE had no major effect on PA levels, even though PA tended to be lower in OE leaves. At Pi deficiency, NPC4-OE increased the DAG level in old and young leaves, but the DAG level in old leaves was only about 50% of that in the old leaves at sufficient Pi (P1000). NPC4-OE decreased PC and PE levels in old and young leaves. NPC4-OE decreased PS and PG levels in young leaves, with no effect on PG in old leaves. NPC4-OE had no major effect on PA level in old or young leaves. The magnitude of decreases in the phospholipid to galactolipid ratio under Pi deficiency was greater in NPC4-OE than WT leaves (FIG.6A, bottom panel; FIG. 9A-9B). Compared with WT, the increased DAG and decreased PC and PE, which are potential product and substrates, respectively, of NPC4 hydrolysis, indicate that the overexpressed NPC4 hydrolyzes glycerophospholipids.

[0166] In camelina leaves, the major molecular species of DAG were 18:3 / 16:0-, 16:0 / 16:0-, and 18:2 / 16:0-, followed by 18:3 / 18:3-, 18:3 / 18:1-, and 18:3 / 18:0- DAG species (FIG.10A-10B). At sufficient Pi, old leaves had a higher level of the major molecular species of DAG than young leaves. NPC4- OE plants had higher levels of major DAG species in old and young leaves than WT under Pi- sufficient and deficient conditions. The major molecular species of DGDG, MGDG, PA, PC, PE, PS, and PG were all decreased under Pi deficiency, but the magnitude of decreases in phospholipid species was greater relative to that of galactolipid species. NPC4-OE further decreased the major molecular species of PC, PE, PS, and PG in old and young leaves compared to those of WT in Pi sufficiency and deficiency (FIG.10A-10B), suggesting that NPC4 may not display selectivity to specific acyl species of phospholipids. 104866324.4Polsinelli Docket No.: 077875-849424 Example 8. Discussion of results of Examples 1-7

[0167] The expression of NPC4 is induced greatly by P deficiency, but the effect of NPC4 on plant growth and seed production remained largely unknown till a recent study showing that NPC4-OE enhanced, whereas its knockout decreased, rapeseed growth and seed production under P limitation . The present study examined the effect of NPC4 on camelina response to P deficiency. The results indicate that NPC4-OE improves camelina seed and seed oil production, and the improvement is greater under P-deficient than sufficient conditions. This study provides further support for the use of increased NPC4 expression to promote plant growth and seed oil production under P-limited growth conditions.

[0168] Moreover, this study reveals a mechanism by which NPC4 enhances P utilization and plant growth response to P deficiency. That is the NPC4 promotion of P remobilization from old, senescent tissues to young, growing tissues. The first line of evidence supporting the remobilization effect of NPC4 came from the observation that the P distribution in leaves of different ages. NPC4-OE plants have a significantly higher P level in young leaves, but a lower P level in old leaves than do WT ones. Secondly, assaying P redistributions in leaves after transferring plants from a P- sufficient to a P-free condition supports the positive role of NPC4 in P remobilization. Compared with WT, NPC4-OE plants displayed a more P decrease in old leaves, but a greater P increase in young leaves. Under this assay condition, because no P was taken up from the media, the P level changes in leaves at different ages indicates the amount of P remobilized within a plant. Thirdly, the gene expression data from different ages of leaves and under P sufficient and deficient conditions provide further support for the role of NPC4 in P remobilization. Camelina NPC4s are expressed at a higher level in old leaves than young leaves, and NPC4-OE further increases the expression of camelina endogenous NPC4s in old leaves. NPC4-OE increased the expression of multiple PHT1 Pi transporters in old leaves, but not in young leaves. PHT1 transporters play key roles in P acquisition, transport, and remobilization from old and mature to young tissues. For example, PHT1;7 and PHT1;8 regulate Pi distribution in Arabidopsis and rice , and the expression level of some PHT1;7 and PHT1;8 homologs was higher in old leaves of NPC4- OE than WT plants. NPC4-OE displayed increased expression of xylem P exporter involved in P remobilization more in old leaves over WT. The increased expression of those transporters in old leaves supports that NPC4 promotes P remobilization from old to young tissues. In addition, the magnitude of increase of the P transporters is greater under P-deficient than -sufficient conditions in 104866324.4Polsinelli Docket No.: 077875-849424 NPC4-OE leaves over WT leaves. These results suggest that NPC4 plays a greater role in promoting P remobilization under P limited than sufficient conditions.

[0169] In addition to P remobilization, NPC4-OE also increases P uptake under limited P conditions as indicated by the higher depletion of P from the medium in NPC4-OE than WT seedlings. Under sufficient P, NPC4-OE showed almost no effect on increasing the expression of PHT1 in roots, with only a slight increase in PHT1;8, but NPC4-OE displayed increased expression of all these transporters in leaves. For example, PHT1;9 mediates Pi uptake in Arabidopsis and rice, but its expression was not increased in NPC4-OE roots under sufficient P. In contrast, under P deficiency, NPC4-OE increased the expression of PHT1;1, PHT1;8, and PHT1;9 in roots. The transporter expression patterns in roots suggest that NPC4-OE increases P uptake under P-limited conditions but less under sufficient P.

[0170] The present results raise the intriguing question of how NPC4 promotes P remobilization from senescing to young tissues. NPC4 displayed higher levels of expression in senescent leaves than young leaves and its expression is induced further by P deficiency. NPC4 hydrolyzes phospholipids to release P-containing head groups. NPC4-OE increases phospholipid declines under P deficiency, indicating more phospholipid hydrolysis and remodeling. The effect of NPC4 on decreasing glycerophospholipids is apparent in camelina, which was also observed in another oil seed crop rapeseed (Yang et al., 2023). So, one way by which NPC4 improves P remobilization and plant growth is via promoting phospholipid degradation to release P. However, the effect of NPC4 is not limited to lipid remodeling because the present results showed increased expression of P transporters in NPC-4-OE leaves and roots particularly under P-deficient conditions. PHR1 (Phosphate Response 1) is a Myb transcription factor positively regulating the expression of many genes in plant response to P limitation, including those for P transporters and lipid remodeling enzymes (Rubio et al., 2001; Pant et al., 2015). While the expression of PHR itself is response to P availability, PHR1 regulates the expression of P starvation-induced genes via modulation by the SPX domain-containing proteins, which control the cytoplasm to the nucleus translocation of PHR1 (Lv et al., 2014) or compete with the PHR1 binding to its target promoter P1BS element (Wang et al., 2014; Puga et al., 2014). Recently, the inventors showed that PA binds to several Myb transcriptional factors and affects the intracellular translocation of proteins from the cytoplasm to the nucleus (Kim et al., 2020, 2022). In addition, recently PA has been shown to bind to an ammonium transporter and regulate its transporter activity (Cao et al., 2023). Thus, besides its role in lipid remodeling, 50 104866324.4Polsinelli Docket No.: 077875-849424 NPC4 and its mediated lipid changes may modulate the transcriptional regulation of P deficiency induced genes and P transporter activities.

[0171] In summary, this study shows that increased expression of NPC4 promotes camelina growth and seed oil production and the enhancement is greater under Pi-limited growth conditions. Moreover, it reveals a mechanism by which NPC4 enhances P remobilization from old, senescing tissues to young tissues to promote growth and seed production. The strategy of NPC4-OE is potentially applicable to enhance camelina resilience to Pi limitation and seed production with a decreased need of P fertilizers. Example 9. Knockout and overexpression of NPC4 in rice to test its effect on rice response to phosphate deficiency

[0172] The present results have shown that NPC4 is a positive regulator for improving crop production under low P, and its OE increased plant growth and seed oil production particularly under P deficiency in Camelina sativa. To explore whether NPC4’s effect is conserved in monocot crops, CRISPR-Cas9 editing was used to knockout rice NPC4 (Oryza sativa NPC4). NPC4 expression was also increased in rice by overexpressing it under a constitutive promoter (ZmUbi, a maize ubiquitin promoter). As shown in FIG. 11A, the knockout of NPC4 compromised rice response to low P whereas overexpression of NPC4 increased rice root growth (FIG.11A). Under P starvation conditions, the NPC4- CRISPR knockout (CR) rice plants had shorter roots and fewer numbers of roots, whereas NPC4-overexpression (OE) displayed greater root growth and root numbers than WT control (FIG.11B and FIG.11C).

[0173] PA binds to the Myb transcription factor Phosphate Response 1 (PHR1), a core positive regulator for plant response to P deficiency. The results presented herein demonstrate that NPC4 promotes P remobilization from senescing to young tissues and increases the expression of P transporters under low P and in senescing leaves. PHR1 activates the expression of many P starvation response (PSR) genes, including those for P transporters via its binding to the promoter P1BS element. To probe the mechanism of NPC’s action, the interaction between rice PHR and phosphatidic acid (PA) that can be produced from DAG, the lipid product of NPC’s reaction, was tested. The rice genome has 3 PHR1 orthologs, PHR1, 2, and 3, and they together activate the expression of most P- starvation response genes. but they differ in DNA binding affinities and expression patterns in different tissues and growth stages, suggesting some functional diversification. PHR1, 2, and 3 proteins were produced, and initial results indicate that PHRs from rice bind PA but 104866324.4Polsinelli Docket No.: 077875-849424 not phosphatidylcholine (PC), or acidic phosphatidylinositol (PI) (FIGs. 12A-12D, only PHR1 data shown). This result reveals a new mechanism regulating PHR function in plants responding to low P. 104866324.4

Claims

Polsinelli Docket No.: 077875-849424 CLAIMS What is claimed is:

1. A method of improving phosphorus redistribution in a plant, the method comprising: introducing to the plant a recombinant DNA construct comprising a polynucleotide sequence encoding a nonspecific phospholipase C4 (NPC4) protein operably linked to a heterologous promoter, wherein one or more of the remobilization of phosphorus from old, senescing tissues to young, growing tissues and seeds is enhanced, plant growth and yield is enhanced, or an alteration in the fatty acid composition in the plant compared to a wild type plant.

2. The method of claim 1, wherein the expression of one or more phosphorus transporter genes is enhanced in the old tissues.

3. The method of claim 1 or claim 2, wherein the plant is cultivated under phosphorus- limited conditions.

4. The method of claim 3, wherein the growth and yield are enhanced in the plant under phosphorus-limited conditions when compared to phosphorus-sufficient conditions.

5. The method of claim 4, wherein the seed yield and oil production are increased in the plant under a phosphorus-limited condition when compared to a wild-type plant cultivated under similar conditions.

6. The method of claim 4, wherein the fatty acid composition is altered in the plant when compared to a wild-type plant cultivated under similar conditions.

7. The method of claim 6, wherein altered fatty acid composition is selected from the group consisting of increased the linolenic acid (18:3) content, decreased linoleic acid (18:2) content, increased oleic acid (18:1) content, decreased stearic acid (18:0), palmitic acid (16:0), arachidic acid (20:0), and eicosadienoic acid (20:2), increased 18:1 and gondoic acid (20:1), a decreased 20:2 fatty acid, and any combination thereof.

8. The method of claim 4, wherein the phosphorus use efficiency (PUE) is enhanced in the plant under phosphorus-limited conditions.

9. The method of any of the preceding claims, wherein the plant is selected from a camelina plant or a rice plant. 104866324.4Polsinelli Docket No.: 077875-849424 10. The method of claim 9, wherein seed yield and oil content is enhanced when compared to a wild-type plant cultivated under the same conditions.

11. The method of claim 9, wherein the fatty acid composition is altered in the plant when compared to a wild-type camelina plant cultivated under the same conditions.

12. The method of claim 11, wherein altered fatty acid composition is selected from the group consisting of increased the linolenic acid (18:3) content, decreased linoleic acid (18:2) content, increased oleic acid (18:1) content, decreased stearic acid (18:0), palmitic acid (16:0), arachidic acid (20:0), and eicosadienoic acid (20:2), increased 18:1 and gondoic acid (20:1), a decreased 20:2 fatty acid, and any combination thereof.

13. The method of any one of the preceding claims, wherein the promoter is a constitutive promoter.

14. The method of claim 1, wherein the NPC4 protein is selected from AtNPC4, OsNPC3, and OsNPC4.

15. The method of claim 14, wherein the polynucleotide encoding NPC4 encodes an amino acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1, 7, 9, 10, 11, 12, or 13.

16. The method of claim 14, wherein the polynucleotide encoding NPC4 comprises a nucleic acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2, 6, or 8.

17. The method of claim 1, wherein the recombinant DNA construct comprises a CaMV promoter operably linked to a polynucleotide sequence encoding an AtNPC4 protein.

18. The method of claim 18, wherein the recombinant DNA construct comprises a nucleic acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO:

3.

19. A method of improving yield and oil content or altering fatty acid composition in a plant, the method comprising: introducing to the plant a recombinant DNA construct comprising a polynucleotide sequence encoding an NPC4 protein, wherein the plant is a camelina plant or a rice plant.

20. A genetically modified plant expressing a recombinant DNA construct comprising a polynucleotide sequence encoding a nonspecific phospholipase C4 (NPC4) protein 104866324.4Polsinelli Docket No.: 077875-849424 operably linked to a heterologous promoter, wherein one or more of remobilization of phosphorus from old, senescing tissues to young, growing tissues, yield or oil content are enhanced or in the plant or the fatty acid composition is altered, and wherein the plant is a camelina plant or a rice plant.

21. The genetically modified plant of claim 20, wherein the promoter is a constitutive promoter.

22. The genetically modified plant of claim 21, wherein the NPC4 protein is selected from AtNPC4, OsNPC3, and OSNPC4.

23. The genetically modified plant of claim 21, wherein the heterologous promoter is a CaMV promoter.

24. The genetically modified plant of claim 19, wherein growth, seed yield or oil production is enhanced, or fatty acid composition is altered in the plant when compared to a wild-type plant cultivated under similar conditions.

25. The genetically modified plant of claim 22, wherein the polynucleotide encoding NPC4 encodes an amino acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 1, 7, 9, 10, 11, 12, or 13.

26. The genetically modified plant of claim 22, wherein the polynucleotide encoding NPC4 comprises a nucleic acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2, 6, or 8.

27. A plant cell, seed, plant product thereof, of the genetically modified plant of claim 22.

28. A recombinant DNA construct comprising a heterologous promoter operably linked to a polynucleotide encoding a NPC4 comprising a nucleic acid sequence with at least 90% sequence identity to the sequence of SEQ ID NO: 2, 6, or 8.

29. A transgenic plant cell having in its genome the recombinant DNA construct of claim 28.

30. A transgenic plant comprising the transgenic plant cell of claim 29.

31. A crop product produced from the transgenic plant of claim 30.

32. A transgenic progeny seed or propagatable plant part of the transgenic plant of claim104866324.4Polsinelli Docket No.: 077875-849424 33. A kit for improving yield and oil content and altering fatty acid composition in a plant, the kit comprising one or more nucleic acid constructs for increasing the expression of a NPC protein in a plant, a plant comprising the one or more nucleic acid constructs for increasing expression of the NPC in the plant, or any combination thereof. 104866324.4

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