Enhanced nitrogen fixation with FUN

Abstract

The present disclosure relates to enhanced nitrogen fixation in legume plants growing under high nitrate or nitrate stress conditions. In particular, the present disclosure relates to genetically modified plants having altered FUN or FUN downstream target levels or expression, as well as methods of producing and growing the same. The disclosure further relates to nodulation senescence controlled by FUN and its downstream targets, and modulation of FUN activity or expression by cellular zinc.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 483,248, filed February 3, 2023, and U.S. Provisional Application No. 63 / 580,171, filed September 1, 2023, each of which is hereby incorporated by reference in its entirety. References to electronic sequence listings

[0002] The contents of the electronic sequence listing (794542002240SEQLIST.xml; size: 175,320 bytes; and creation date: February 1, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to enhancing nitrogen fixation in legumes grown under conditions of high nitrate or nitrate stress. Specifically, the present disclosure relates to genetically modified plants with altered levels or expression of FUN or FUN downstream targets, as well as methods for their production and growth. The present disclosure further relates to nodule senescence controlled by FUN and its downstream targets, and to the regulation of FUN activity by cellular zinc. Background Art

[0004] Plant growth and development depend on carbon dioxide and sunlight above ground level, as well as water and mineral nutrients in the soil. Nutrient availability in the soil depends on many factors, and nutrient availability varies across space and time. Local nutrient sensing, as well as awareness of the global nutrient status, shapes the plant's response to its nutritional environment and acts to coordinate plant development with microbial engagement to optimize nutrient capture and regulate plant growth. One of the major nutrients limiting plant productivity is nitrogen (N).

[0005] Nitrogen fixation is essential for the sustainable and profitable production of legumes. The symbiotic relationship between legumes and nitrogen-fixing microorganisms is controlled by the plants in various ways, including by the number of nodules allowed to form (Nishimura, R. et al., HAR1 mediates systemic regulation of symbiotic organ development. Nature 420, 426–429 (2002); Krusell, L., Madsen, LH, Sato, S. & Aubert, G. Shoot control of root development and nodulation is mediated by areceptor-like kinase. Nature 420, 422–426 (2002); Searle, IR et al., Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase. Science 299, 109–112 (2003); Tsikou, D. et al., Systemic control of legume susceptibility to rhizobial infection by a mobile microRNA. Science 362, 233–236 (2018), and the function of the resulting organs. Nitrogen fixation in legumes supports all of the plant's nitrogen needs, balanced by nitrogen acquisition from available soil resources. However, soil nitrogen supply fluctuates, and so does the plant's demand for nitrogen.

[0006] In high-intensity agriculture, nitrogen can be applied in high concentrations in the form of inorganic fertilizers to promote crop productivity. These concentrations are generally higher than what plants need or what the soil can store. This causes these nutrients to be released into the environment, affecting ecosystems and biodiversity, and leading to climate change (CJStevens,Nitrogen in the environment.Science363,578-580(2019);JA Foley et al., Solutions for a cultivated planet.Nature 478,337-342(2011);J.Rockstrom et al., A safe operating space for humanity.Nature 461,472-475(2009)). However, the presence of high nitrates in soil has been shown to suppress or inhibit the ability of legumes to fix nitrogen.

[0007] A ubiquitous need is to enhance the ability of legumes and other plants that are symbiotic with nitrogen-fixing bacteria to fix nitrogen under high nitrate or nitrate stress conditions, which otherwise inhibit, suppress or reduce nitrogen fixation. For example, it is necessary to improve the response of legumes or other plants to nitrate and the ability of corresponding nitrogen fixation regulation. Specifically, it is necessary to identify transcription factors that can regulate nitrogen fixation under high nitrate conditions, because these transcription factors provide a means for engineered legumes to grow and fix nitrogen even under high nitrate conditions. Further example, it is necessary to modify nodulation aging in legumes or other plants in response to nitrate levels and / or nitrogen stress. Specifically, it is necessary to identify transcription factors that can regulate nodulation aging under high nitrate and / or nitrogen stress conditions, because these transcription factors provide a further means for engineered legumes to grow and fix nitrogen even under high nitrate conditions or nitrogen stress. Summary of the Invention

[0008] To address these needs, the present disclosure provides the FUN transcription factor, which is a regulator of nitrogen fixation in legumes under high nitrate conditions. The present disclosure further provides downstream targets of FUN, which are also used to regulate nitrogen fixation under high nitrate conditions. Mutating or downregulating FUN or its downstream targets can be used to generate plants with resistance to nitrate nitrogen fixation. This provides an opportunity to increase biological nitrogen fixation in fields or cropping conditions with high levels of soil nitrate.

[0009] One aspect of the present disclosure includes a genetically modified plant or part thereof comprising one or more genetic alterations that result in reduced activity or expression of a FUN protein in the genetically modified plant or part thereof as compared to the activity or expression of the FUN protein in a control plant grown under the same conditions. In further embodiments of this aspect, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. NO:82 or its conserved domain, or a protein of the group of combinations thereof, is a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity. In additional embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82 or a conserved domain thereof, or a combination thereof.In yet other embodiments, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or a conserved domain thereof, or a combination thereof.

[0010] A further aspect of the present disclosure includes a genetically modified plant or part thereof comprising one or more genetic alterations that result in a decrease in the activity or expression of one or more of an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, a HO1 protein, an NRT2.1 protein, or an AS1 protein in the genetically modified plant or part thereof as compared to the activity or expression of an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein, a HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same conditions. In a further embodiment of this aspect, the protein is an NRT3.1 protein, and wherein the NRT3.1 protein is expressed in the NRT3.1 protein.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:74 or a conserved domain thereof; wherein the protein is a bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:75 or a conserved domain thereof; wherein the protein is a NAC domain-containing protein, and wherein the NAC domain-containing protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72、SEQ ID NO:73 or its conserved domain, or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a protein of a group comprising a combination thereof; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77 or its conserved domain; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 78 or a conserved domain thereof; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 79 or a conserved domain thereof. In another embodiment of this aspect, the NRT3.1 protein comprises SEQ ID NO:74 or a conserved domain thereof; wherein the bZIP28 protein comprises SEQ ID NO:75 or a conserved domain thereof; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73 or a conserved domain thereof; wherein the HO1 protein comprises SEQ ID NO:77 or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO:78 or a conserved domain thereof, or wherein the AS1 protein comprises SEQ ID NO:79. In still further embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:42 or a conserved domain thereof, or a combination thereof.

[0011]

[0015] Additional aspects of the present disclosure include genetically modified plants comprising one or more genetic alterations that result in reduced activity or expression of one or more of a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein in the genetically modified plant, as compared to the activity or expression of a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein in a control plant grown under the same conditions, wherein the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, a NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 or SEQ ID NO:84 or a conserved domain thereof, or a group of polypeptides having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity in combination thereof, and wherein the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein has enhanced expression in nodules in the absence of one or more genetic alterations.

[0012] In additional embodiments of this aspect, which may be combined with any of the preceding embodiments, the reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. In yet further embodiments of this aspect (which may be combined with any of the preceding embodiments), the reduction is due to knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out a domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out a domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out a domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, preferably, the binding site is a transcription activator protein binding site or a TATA box. In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the growth conditions comprise moderate nitrate levels, high nitrate levels, or nitrate levels around the plant that reduce or suppress nitrogen fixation. In yet other embodiments of this aspect, the nitrate level is between about 10 mM and about 250 mM nitrate, or includes at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate. In still other embodiments of this aspect, the genetically modified plants have increased nitrogen fixation compared to control plants when grown under the same growth conditions. In further embodiments of this aspect, nitrogen fixation is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In yet further embodiments of this aspect (which may be combined with any preceding embodiment), the plant forms nodules. In further embodiments of this aspect, the plant has an increased number of nodules, an increased hemoglobin content, or an increased acetylene reduction assay (ARA) activity compared to control plants grown under the same conditions.

[0013] Additional aspects of the present disclosure include methods of growing a genetically altered plant that has increased nitrogen fixation under conditions comprising nitrate levels surrounding the roots of the plant that suppress nitrogen fixation, the method comprising: (a) providing a genetically altered plant, wherein the plant or part thereof comprises one or more genetic alterations that result in decreased activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein, or any combination thereof, in the genetically altered plant or part thereof, as compared to the activity or expression of the FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions, and wherein the one or more genetic alterations reduces the suppression of nitrogen fixation by nitrate levels; and (b) growing the genetically altered plant under nitrate levels surrounding the roots of the plant, wherein the genetically modified plant has increased nitrogen fixation as compared to the control plant grown under the same conditions. In further embodiments of this aspect, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. NO:82 or its conserved domains, or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity to a protein of a group of combinations thereof. In another embodiment of this aspect,The FUN protein includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, or conserved domains thereof, or a combination thereof. In still further embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or a conserved domain thereof, or a combination thereof. In yet further embodiments of this aspect, the protein is NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 74, or a conserved domain thereof; wherein the protein is bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 75, or a conserved domain thereof; wherein the protein is a NAC domain-containing protein,And wherein the NAC domain-containing protein includes a protein selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77, or a conserved domain thereof; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77, or a conserved domain thereof; ID NO: 78 or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity thereof to a conserved domain thereof; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 79 or a conserved domain thereof. In further embodiments of this aspect,wherein the NRT3.1 protein comprises SEQ ID NO:74 or its conserved domain; wherein the bZIP28 protein comprises SEQ ID NO:75 or its conserved domain; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72 or SEQ ID NO: 73 or a conserved domain thereof, or a combination thereof; wherein the HO1 protein comprises SEQ ID NO: 77 or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO: 78 or a conserved domain thereof, or wherein the AS1 protein comprises SEQ ID NO: 79 or a conserved domain thereof. In still further embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO: 31, SEQ ID NO: 41 or SEQ ID NO: 42 or a conserved domain thereof, or a combination thereof. Additional embodiments of this aspect, which may be combined with any of the preceding embodiments, include the nitrate level in step (c) being between about 10 mM and about 250 mM nitrate,In some embodiments, the plant comprises at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate. Further embodiments of this aspect (which can be combined with any of the preceding embodiments) include an increase in nitrogen fixation of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In further embodiments of this aspect, the plant comprises an increase in the number of nodules or an increase in the hemoglobin content when compared to control plants when grown under the same growth conditions. In further embodiments of this aspect, increased nitrogen fixation is measured using a method selected from the group consisting of: measuring the number of pink nodules per plant compared to control plants, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour compared to control plants (acetylene reduction assay (ARA)), or measuring micrograms of hemoglobin per plant compared to control plants.

[0014] A further aspect of the present disclosure includes a method of growing a genetically modified plant capable of fixing nitrogen when grown under nitrogen fertilizer conditions, the method comprising: (a) providing a genetically modified plant, wherein the plant or part thereof comprises one or more genetic alterations that result in an increase in the activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in the genetically modified plant or part thereof as compared to the activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions; The invention relates to a method for producing a plant comprising: (a) reducing the activity or expression of a protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, an NRT2.1 protein, or an AS1 protein, or any combination thereof, and wherein the one or more genetic alterations reduce nitrate levels that inhibit nitrogen fixation; (b) growing the plant under conditions comprising standard nitrate levels surrounding the roots of the plant; and (c) applying nitrogen fertilizer, thereby generating conditions comprising nitrate levels surrounding the roots of the plant that inhibit nitrogen fixation, wherein the genetically modified plant has increased nitrogen fixation compared to a control plant grown under the same conditions. In further embodiments of this aspect, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. NO:82 or its conserved domains, or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity to a protein of a group of combinations thereof. In another embodiment of this aspect,The FUN protein includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, or conserved domains thereof, or a combination thereof. In still further embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or a conserved domain thereof, or a combination thereof. In yet further embodiments of this aspect, the protein is NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 74, or a conserved domain thereof; the protein is bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 75, or a conserved domain thereof; the protein is a NAC domain-containing protein,And wherein the NAC domain-containing protein includes a protein selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77, or a conserved domain thereof; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:78, or a conserved domain thereof; NO:78 or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity thereof to a conserved domain thereof; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:79 or a conserved domain thereof. In further embodiments of this aspect,wherein the NRT3.1 protein comprises SEQ ID NO:74 or its conserved domain; wherein the bZIP28 protein comprises SEQ ID NO:75 or its conserved domain; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73 or a conserved domain thereof, or a combination thereof; wherein the HO1 protein comprises SEQ ID NO:77 or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO:78 or a conserved domain thereof, or wherein the AS1 protein comprises SEQ ID NO:79 or a conserved domain thereof. In still further embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:42 or a conserved domain thereof, or a combination thereof. Additional embodiments of this aspect, which may be combined with any of the preceding embodiments, include the nitrate level in step (c) being between about 10 mM and about 250 mM nitrate,In some embodiments, the plant may have an increase in nitrogen fixation of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, the plant may have an increase in nitrogen fixation of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In some embodiments, the plant may have an increase in nodule number or an increase in hemoglobin content compared to control plants when grown under the same growth conditions. In additional embodiments of this aspect, increased nitrogen fixation is measured using a method selected from the group consisting of: measuring the number of pink nodules per plant compared to control plants, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour compared to control plants (acetylene reduction assay (ARA)), or measuring micrograms of hemoglobin per plant compared to control plants. In further embodiments of this aspect, which may be combined with any of the preceding embodiments, the genetically altered plant is grown in an intercropping system with a non-nitrogen fixing plant, or in a sequential system following a non-nitrogen fixing plant.

[0015] Additional aspects of the present disclosure include methods for delaying nodulation senescence, the methods comprising: (a) providing a genetically altered plant, wherein the plant or part thereof comprises one or more genetic alterations that result in reduced activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein, or any combination thereof, in the genetically altered plant or part thereof, as compared to the activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions, and wherein the one or more genetic alterations delay nodulation senescence; and (b) cultivating the genetically altered plant under stress conditions, wherein the genetically altered plant has delayed nodulation senescence compared to a control plant grown under the same conditions. Further embodiments of this aspect include stress conditions selected from the group consisting of moderate nitrate levels, high nitrate levels, nitrate levels around the plant that promote nodulation and senescence, moderate heat levels, high heat levels, heat levels around the plant that promote nodulation and senescence, moderate water deficit levels, high water deficit levels, water deficit levels around the plant that promote nodulation and senescence, moderate waterlogging levels, high waterlogging levels, or waterlogging levels around the plant that promote nodulation and senescence. In further embodiments of this aspect, which may be combined with any of the preceding embodiments, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. NO:82 or its conserved domains, or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity to a protein of a group of combinations thereof. In another embodiment of this aspect,The FUN protein includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, or conserved domains thereof, or a combination thereof. In still further embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or a conserved domain thereof, or a combination thereof. In yet further embodiments of this aspect (which may be combined with any of the aforementioned embodiments of a protein comprising a NAC domain), wherein the protein is NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 74, or a conserved domain thereof; wherein the protein is bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 75, or a conserved domain thereof; wherein the protein is a protein comprising a NAC domain,SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or a conserved domain thereof, or a combination thereof; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 77, or a conserved domain thereof; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 78, or a conserved domain thereof; NO:78 or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity thereof to a conserved domain thereof; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:79 or a conserved domain thereof. In further embodiments of this aspect,wherein the NRT3.1 protein comprises SEQ ID NO:74 or its conserved domain; wherein the bZIP28 protein comprises SEQ ID NO:75 or its conserved domain; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72 or SEQ ID NO: 73 or a conserved domain thereof, or a combination thereof; wherein the HO1 protein comprises SEQ ID NO: 77 or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO: 78 or a conserved domain thereof, or wherein the AS1 protein comprises SEQ ID NO: 79 or a conserved domain thereof. In still further embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO: 31, SEQ ID NO: 41 or SEQ ID NO: 42 or a conserved domain thereof, or a combination thereof. In further embodiments of this aspect, nodular senescence is delayed by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

[0016] Yet further aspects of the present disclosure include methods for inducing FUN protein filamentation, comprising: (a) providing a FUN protein; and (b) increasing the amount of zinc or manganese in the environment of the FUN protein, wherein the increased amount of zinc or manganese induces filamentation compared to a control FUN protein in an environment in which the amount of zinc or manganese is not increased. In another embodiment of this aspect, filamentation is induced under high nitrate conditions. In another embodiment of this aspect (which may be combined with any of the preceding embodiments), the method is performed in vitro.

[0017] Further aspects of the present disclosure include methods for inducing filamentation, comprising: (a) providing a plant comprising a FUN protein; and (b) cultivating the plant under conditions of increased zinc or manganese, wherein filamentation of the FUN protein in the plant is induced compared to the FUN protein in a control plant grown under conditions without increased zinc or manganese. In another embodiment of this aspect, the plant comprises a genetic alteration. In another embodiment of this aspect, filamentation is induced under high nitrate conditions. In yet another embodiment of this aspect (which may be combined with any of the aforementioned embodiments in which the plant comprises a genetic alteration), the genetic alteration reduces the activity of the FUN protein without eliminating the activity of the FUN protein. In a further embodiment of this aspect, the induction of filamentation results in an increase in nitrogen fixation in the genetically altered plant, or a reduction in or inactivation of the activity of the FUN protein, compared to a control plant grown under the same conditions. In still further embodiments of this aspect, which may be combined with any of the preceding embodiments or aspects of the method of inducing filamentation, the number of nodules is increased, the hemoglobin content is increased, or the acetylene reduction assay (ARA) activity is increased compared to control plants grown under the same conditions.

[0018] Yet further aspects of the present disclosure include methods for regulating nodulation function based on the amount of available nitrogen in the soil, the methods comprising: a) providing a genetically altered plant comprising a FUN protein with altered activation by nitrate; and b) cultivating the genetically altered plant under conditions of nitrate concentration, wherein the genetically altered plant has reduced FUN activity or expression and / or reduced FUN active form compared to a WT plant grown under the same nitrate conditions. In further embodiments of this aspect, altering FUN protein activation by nitrate comprises downregulating FUN, reducing FUN activity, knocking out FUN by mutation, knocking down FUN expression, knocking out a promoter element of FUN, or a combination thereof. In further embodiments of this aspect, altering FUN protein activation by nitrate comprises manipulating environmental or cellular zinc levels, wherein the manipulation results in the FUN protein being maintained in an inactive filamentous form. In still further embodiments of this aspect, altering FUN protein activation by nitrate comprises genetically modifying the FUN protein sequence to alter sensitivity to zinc.

[0019] In further embodiments of this aspect, which may be combined with any of the preceding embodiments or aspects, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 NO:82 or its conserved domain, or a protein of the group of combinations thereof, is a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity. In additional embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82 or a conserved domain thereof, or a combination thereof. In still further embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or a conserved domain thereof, or a combination thereof.

[0020] Further aspects of the present disclosure include methods of making a genetically altered plant having increased nitrogen fixation under conditions including nitrate levels around the plant's roots that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic alterations that reduce the activity or expression of a FUN protein as compared to the activity or expression of the FUN protein in a control plant grown under the same conditions.In another embodiment of this aspect, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. NO:82 or a group of proteins thereof or a conserved domain thereof or a combination thereof; wherein the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 ID NO:81 or SEQ ID NO:82 or a conserved domain thereof, or a combination thereof; or wherein the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8 or SEQ ID NO:9 or a conserved domain thereof, or a combination thereof.

[0021] Additional aspects of the present disclosure include methods for making a genetically altered plant having increased nitrogen fixation under conditions including nitrate levels around the plant's roots that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic alterations that reduce the activity or expression of one or more of the following: NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein, as compared to the activity or expression of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions. In yet another embodiment of this aspect, the protein is NRT3.1 protein, and wherein the NRT3.1 protein is NRT3.1 protein.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:74 or a conserved domain thereof; wherein the protein is a bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:75 or a conserved domain thereof; wherein the protein is a NAC domain-containing protein, and wherein the NAC domain-containing protein comprises a polypeptide selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQID NO:72、SEQ ID NO:73 or its conserved domain, or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a protein of a group comprising a combination thereof; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77 or its conserved domain; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 78 or a conserved domain thereof; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 79 or a conserved domain thereof; wherein the NRT3.1 protein comprises SEQ ID NO: 74 or a conserved domain thereof; wherein the bZIP28 protein comprises SEQ ID NO: 75 or a conserved domain thereof; wherein the NAC domain-containing protein comprises SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73 or a conserved domain thereof, or a combination thereof; wherein the HO1 protein comprises SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, NO:77 or its conserved domain; wherein the NRT2.1 protein comprises SEQ ID NO:78 or its conserved domain, or wherein the AS1 protein comprises SEQ ID NO:79; or wherein the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:42 or its conserved domain, or a combination thereof.

[0022] Yet additional aspects of the present disclosure include methods of making a genetically altered plant having increased nitrogen fixation under conditions comprising nitrate levels around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic alterations that reduce the activity or expression of one or more of a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein, as compared to the activity or expression of the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein in a control plant grown under the same conditions. In some embodiments of this aspect, the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein or AS1 protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30 NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 or SEQ ID NO:84 or a conserved domain thereof, or a group of polypeptides having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity in combination thereof, and wherein the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein has enhanced expression in nodules in the absence of one or more genetic alterations.

[0023] Additional aspects of the present disclosure include methods for making a genetically modified plant or part thereof according to any of the above embodiments, the methods comprising: introducing into a plant cell a genetic alteration that reduces or knocks out the activity or expression of a FUN protein, a FUN-like protein, a NAC domain-containing protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein. In further embodiments of this aspect, the genetic alteration comprises a first nucleic acid sequence that is capable of reducing or knocking out a second nucleic acid sequence encoding a FUN protein, a FUN-like protein, a NAC domain-containing protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein operably linked to a promoter. In another embodiment of this aspect (which may be combined with any of the preceding embodiments), the genetically modified plant is selected from one or more of the following groups: alfalfa, Bambara peanut, beans (e.g., kidney beans, black beans, etc.), blackcurrant, chickpea, clover, cowpea, forage legumes, legume trees, lentils, lotus, lupine, Medicago spp., peas, peanuts, pigeon peas, soybeans, Parasponia, alder, and elm. In another embodiment of this aspect, the nucleic acid comprises an RNA silencing-related short RNA, antisense RNA, siRNA, miRNA, dsRNA, tasiRNA, or a secondary siRNA. In yet another embodiment of this aspect, the promoter is a nodulation-specific promoter, a root-specific promoter, an inducible promoter, a constitutive promoter, or a combination thereof. In a further embodiment of this aspect, the promoter is a constitutive promoter, and wherein the promoter is selected from the group consisting of the CaMV35S promoter, a derivative of the CaMV35S promoter, a maize ubiquitin promoter, a polyubiquitin promoter, a vein mosaic cassava virus promoter, or an Arabidopsis thaliana UBQ10 promoter. In still further embodiments of this aspect, the nucleic acid sequence is inserted into the plant genome such that the nucleic acid sequence is operably linked to an endogenous promoter. In a further embodiment of this aspect, the endogenous promoter is a nodulation-specific promoter or a root-specific promoter.

[0024] Further aspects of the present disclosure include methods for preparing a genetically modified plant or part thereof of any of the foregoing embodiments, the method comprising genetically modifying a plant cell by transforming the plant cell with one or more gene editing components, wherein the gene editing components target an endogenous nuclear genomic sequence encoding a FUN protein, a FUN-like protein, a protein containing a NAC domain, a NRT3.1 protein, a bZIP28 protein, a protein containing a NAC domain, a HO1 protein, a NRT2.1 protein, or an AS1 protein, wherein the endogenous nuclear genomic sequence or a portion thereof is knocked out. In another embodiment of this aspect, the one or more gene editing components include a ribonucleoprotein complex targeting a nuclear genomic sequence; a vector comprising a TALEN protein coding sequence, wherein the TALEN protein targets the nuclear genomic sequence; a vector comprising a ZFN protein coding sequence, wherein the ZFN protein targets the nuclear genomic sequence; an oligonucleotide donor (OND), wherein the OND targets the nuclear genomic sequence; or a vector CRISPR / Cas enzyme coding sequence and a targeting sequence, wherein the targeting sequence targets the nuclear genomic sequence.

[0025] Yet another aspect of the present disclosure includes an expression vector or isolated DNA molecule comprising (i) one or more nucleotide sequences encoding a FUN protein, a FUN-like protein, a HO1 protein, a protein containing a NAC domain, a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence, (ii) a nucleotide sequence encoding a FUN protein, a FUN-like protein, a HO1 protein, a protein containing a NAC domain, a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence, and (iii) a nucleotide sequence encoding a FUN protein, a FUN-like protein, a HO1 protein, a protein containing a NAC domain, a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence, S1 protein or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence, or (iii) one or more nucleotide sequences comprising a mutation in a gene for a FUN protein, a FUN-like protein, a HO1 protein, a NAC domain-containing protein, a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein or a combination thereof, wherein the mutation reduces or knocks out the activity or expression of the protein, and the one or more nucleotide sequences are operably linked to at least one homologous nucleic acid sequence that hybridizes adjacent to the mutation site in the gene. In a further embodiment of this aspect, the protein is a FUN protein, and wherein the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82. The proteins of group NO:82 have polypeptides that are at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical;wherein the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or a conserved domain thereof, or a combination thereof; or wherein the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 NO:9 or its conserved domain, or a combination thereof; wherein the protein is a FUN-like protein, and wherein the FUN-like protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:83, and SEQ ID NO:84, or its conserved domain, or a combination thereof; wherein the FUN-like protein comprises SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:83, or SEQ ID NO:84, or its conserved domain, or a combination thereof; and / or wherein the protein is an NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:74 or its conserved domain; wherein the protein is a bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:74 or its conserved domain; A polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to NO:75 or a conserved domain thereof;wherein the protein is a NAC domain-containing protein, and wherein the NAC domain-containing protein comprises a protein selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or a conserved domain thereof, or a combination thereof; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77, or a conserved domain thereof; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77, or a conserved domain thereof; ID NO: 78 or a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity thereof to a conserved domain thereof; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 79 or a conserved domain thereof; wherein the NRT3.1 protein comprises SEQ ID NO: 74 or a conserved domain thereof;wherein the bZIP28 protein includes SEQ ID NO:75 or its conserved domain; wherein the protein containing the NAC domain includes SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73 or a conserved domain thereof, or a combination thereof; wherein the HO1 protein comprises SEQ ID NO:77 or a conserved domain thereof; wherein the NRT2.1 protein comprises SEQ ID NO:78; or wherein the AS1 protein comprises SEQ ID NO:79 or a conserved domain thereof; or wherein the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:42 or a conserved domain thereof, or a combination thereof. ;

[0026] Some aspects of the present disclosure relate to bacterial cells or Agrobacterium cells comprising the expression vector or isolated DNA molecule of any of the preceding embodiments.

[0027] Additional aspects of the present disclosure relate to a genetically modified plant, plant part, plant cell, or seed comprising the expression vector or isolated DNA molecule of any of the preceding embodiments.

[0028] A further aspect of the present disclosure relates to a kit comprising the expression vector or isolated DNA molecule of any preceding embodiment or the bacterial cell or Agrobacterium cell of the preceding embodiment.

[0029] Still further aspects of the present disclosure relate to methods of increasing nitrogen fixation, delaying nodulation senescence, or inducing FUN filamentation in plants, the methods comprising: (a) introducing a genetic alteration via the expression vector or isolated DNA molecule of any of the preceding embodiments; and optionally (b) treating the plants with zinc or manganese or growing the plants under high zinc, high manganese, or high nitrate conditions.

[0030] Additional aspects of the present disclosure relate to a genetically altered plant genome comprising (i) one or more genetic alterations in the genetically modified plant or part thereof of any of the preceding embodiments, or (ii) one or more genetic alterations in a genetically modified plant or part thereof produced by the method of any of the preceding embodiments.

[0031] Yet further aspects of the present disclosure relate to non-regenerable parts or cells of the genetically modified plant or part thereof of any one of the preceding embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] This 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] Figures 1A-1V We show the nodulation phenotype and nitrogen fixation activity of fun mutant Nelumbo plants under nitrate-limiting conditions, as well as the nodule-specific expression of Fun. Figure 1A Representative images of the nodulation phenotypes of wild-type (WT; Gifu) Nelumbo nucifera plants under KCl conditions (control; upper left), WT Nelumbo nucifera plants under 10 mM KNO3 conditions (upper middle), and fun mutant Nelumbo nucifera plants under 10 mM KNO3 conditions (upper right and lower rows), fun mutants including fun (upper right), fun-2 (lower left), fun-3 (lower middle), and fun-4 (lower right). Scale bar = 1 cm. Figure 1B Shows Figure 1A Diagram of the FUN gene and LORE1 insertion in various phenotypic genetic backgrounds. In fun and fun-4, LORE1 is inserted in the promoter region (light gray line). In fun-2, LORE1 is inserted at the end of the fourth intron (intron indicated by the black line). In fun-3 (30099638), LORE1 is inserted at the end of the seventh intron. Arrows indicate the insertion points of LORE1 along the gene. Figure 1CShown are the total number of nodules (white boxes, marked “total”) and the number of pink functional nodules (colored boxes, marked “pink”) formed on wild-type (WT; Gifu) Nelumbo nucifera plants and fun mutant Nelumbo nucifera plants after 2 weeks of 10 mM KNO3 exposure. Figure 1D Shown are the nitrogen fixation activities of wild-type (WT; Gifu) and fun mutant Nelumbo plants quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) after 2 weeks of 10 mM KNO3 exposure. Figure 1E Shown is a 14-day (top) and 20 mM KNO3 (bottom) follow-up process. Figure 1D ARA measured value (vertical axis).Light grey line and medium grey circle represent the ARA result of WT plant.At top, medium grey line and medium grey circle represent the ARA result of fun mutant plant, and black line and medium grey circle represent the ARA result of fun-3 mutant plant.In bottom, black line and medium grey circle represent the ARA result of fun mutant plant. Figure 1F Shown are the total number of nodules (white boxes, marked “total”) and the number of pink functional nodules (gray boxes, marked “pink”) formed on wild-type (WT; Gifu) Nelumbo nucifera plants, fun mutant Nelumbo nucifera plants, fun-2 mutant Nelumbo nucifera plants, fun-3 mutant Nelumbo nucifera plants, and fun-4 mutant Nelumbo nucifera plants after 2 weeks of 10 mM KNO3 exposure. Figure 1G Shown are the nitrogen fixation activities of wild-type (WT; Gifu) Nelumbo nucifera plants, fun mutant Nelumbo nucifera plants, fun-2 mutant Nelumbo nucifera plants, fun-3 mutant Nelumbo nucifera plants, and fun-4 mutant Nelumbo nucifera plants quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) after 2 weeks of 10 mM KNO3 exposure. Figure 1H Shown are the number of pink nodules per plant for wild type (WT), fun, fun-3, and fun-4 mutants with 5 mM nitrate applied before inoculation (grey boxes, labeled "5 mM") and without nitrate applied before inoculation (white boxes, labeled "0 mM"). Plants were grown on plates with 0 or 5 mM KNO3 and then inoculated with nodulation bacteria; pink nodules were measured 3 weeks after inoculation. Figure 1I Shown are the total number of nodules per plant for wild type (WT), fun, fun-3, and fun-4 mutants with 5 mM nitrate applied before inoculation (grey boxes, labeled "5 mM") and without nitrate applied before inoculation (white boxes, labeled "0 mM"). Plants were grown on plates with 0 or 5 mM KNO and then inoculated with rhizobia; nodules were measured 3 weeks after inoculation. Figure 1JNitrogen fixation activity quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) is shown for wild type (WT), fun, fun-3, and fun-4 mutants that were applied with 5 mM nitrate before inoculation (grey boxes, labeled "5 mM") and without nitrate application before inoculation (white boxes, labeled "0 mM"). Plants were grown on plates with 0 or 5 mM KNO3 and then inoculated with rhizobia; ARA was performed 3 weeks after inoculation. Figures 1F-1J , letters indicate significant differences among the comparison groups of plants (p<0.05). Figure 1K Shown are the leghemoglobin contents (μg leghemoglobin per plant) of wild-type (WT; Gifu) Nelumbo plants and fun mutant Nelumbo plants exposed to 10 mM KNO 3 for 2 weeks. Figure 1L Complementation of the fun mutant grown under 10 mM KNO3 exposure for 2 weeks is shown, where the gray boxes indicate empty vector (labeled "EV") transformed into the WT background (left) or transformed into the fun mutant background (center), and the white boxes (right) indicate proUbi:FUN-GFP transformed into the fun mutant background. Letters represent significant differences between the compared groups of plants. Figure 1M Representative images of nodulated plant roots from plants expressing the proFun:GUS reporter construct, used to visualize Fun gene expression in situ, are shown. Roots were stained with 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). Blue staining indicates expression of the GUS reporter gene; Scale bar = 2 cm. Figure 1N This is a median longitudinal section of a nodule imaged by light microscopy. Blue staining indicates expression of the GUS reporter gene; scale bar = 200 μm. Figure 1O yes Figure 1N Magnified image of the 200 μm tumor, where “ic” indicates infected cells, “uc” indicates uninfected cells, and “nc” indicates the nodule cortex; scale bar = 200 μm. Figures 1M-1O In the present invention, proFun:GUS is a construct in which the native Fun promoter sequence (proFUN) is fused to the coding sequence of β-glucuronidase (GUS), followed by a fusion with the native Fun terminator sequence (tFUN). Figure 1P Schematic diagram of the FUN protein showing the bZIP DNA-binding domain (gray oval) and the sensor domain (dark gray asterisk). Figure 1QShown is a bar graph comparing normalized RNA measurements of FUN transcripts across different plant tissues of Lotus japonicus Gifu, calculated by Kamal et al. (2020), “Insights into the evolution of symbiosis gene copy number and distribution from a chromosome-scale Lotus japonicus Gifu genome sequence.” DNA Res. 27(3). L. japonicus Gifu tissues are listed along the vertical axis. “Nodulation 21 days” and “Nodulation 10 days” refer to measurements taken from nodules 21 days and 10 days after inoculation with Mesorhizobium loti R7A, respectively. Figure 1R A histogram shows significantly differentially expressed genes that are upregulated (top) or downregulated (bottom) in WT plants (black boxes) or fun mutant plants (grey boxes). The number of DE genes is plotted along the horizontal axis. Figure 1S A bar graph showing the differential expression levels (expressed as log2 fold change relative to expression in WT plants, horizontal axis) of selected genes (vertical axis) upregulated in fun mutant plants (black boxes) and fun-3 mutant plants (grey boxes). Figure 1T A bar graph showing the differential expression levels (expressed as log2 fold change relative to expression in WT plants, horizontal axis) of downregulated selected genes (vertical axis) in fun mutant plants (black boxes) and fun-3 mutant plants (grey boxes). Figure 1U Ontology groups enriched in up-regulated genes (first three major branches from the top) and down-regulated genes (fourth to seventh major branches from the top) identified by GO-MWU are shown. Text size and weight indicate p-values. Figure 1V The relative expression of downstream targets of FUN is shown, which have an identified TGA motif within the promoter that is differentially expressed in fun relative to wild type. Relative expression is shown in the RNAseq time series from Wang et al. (Wang, L. et al. A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi: 10.1111 / nph.18896). Figures 1C-1D and 1F-1L, circles represent individual plants. Figures 1C-1EIn 1K-1L and 1K-1L, asterisks indicate significant differences between the compared groups; “**” refers to p-value < 0.01, and “*” refers to p-value < 0.05.

[0034] Figures 2A-2S We show that FUN controls the expression of downstream genes Nrt2.1, Ho1, NAC094, Nrt3.1, and AS1 to regulate nitrate signaling and nitrogen fixation in nodules. Figure 2A Shown are the expression levels of Nrt2.1 (left), Ho1 (center), and NAC094 (right) genes in nodules of wild-type Nelumbo nucifera (Gifu, white), fun mutant Nelumbo nucifera (gray), and fun-3 mutant Nelumbo nucifera (dark gray) after 0, 3, and 24 h of 10 mM KNO3 nitrate treatment. Figure 2B Shown are the expression levels of Nrt3.1 (left) and AS1 (right) genes in nodules of wild-type Nelumbo nucifera (Gifu; white), fun mutant Nelumbo nucifera (gray), and fun-3 mutant Nelumbo nucifera (dark gray) after 10 mM KNO3 nitrate treatment for 0, 3, and 24 hours. Figure 2C Schematic diagrams of the promoters of Nrt2.1 (proNRT2.1; top), Ho1 (proHO1; second from the top), NAC094 (proNAC094; middle), Nrt3.1 (proNRT3.1, second from the bottom), and AS1 (proAS1, bottom) are shown. The Nrt2.1 promoter has four putative FUN binding sites (FBSs), designated p1, p2, p3, and p4; the Ho1 promoter has two putative FBSs, designated p1 and p2; the NAC094 promoter has one putative FBS, designated p1; the Nrt3.1 promoter has three putative FBSs, designated p1, p2, and p3; and the AS1 promoter has one putative FBS, designated p1. Figure 2D Shown are gel images from EMSA assays showing the binding of FUN protein to DNA probes containing FBS p1, p2, p3, and p4 from the promoter of Nrt2.1 (left), DNA probes containing FBS p1 and p2 from the promoter of Hole (center), and DNA probes containing FBS p1 from the promoter of NAC094 (right). Figure 2E Shown are images of gels from EMSA assays showing binding of FUN protein to DNA probes containing FBS p1 from the promoter of Nrt2.1, FBS p1, p2, and p3 from the promoter of Nrt3.1, and FBS p1 from the promoter of AS1. Figure 2FShown in competition assay, for p1 (top) and p4 (bottom) FBS EMSA gel images of the promoter from Nrt2.1. Competing DNA is 50 times, 150 times and 500 times the concentration of WT DNA, and no label is placed on the probe. Label "m" corresponds to a DNA probe with a mutation in the TGACG core binding site. Figure 2G The results of a transcriptional activation assay of the Nrt2.1 (left), Ho1 (center), and NAC094 (right) promoters in N. benthamiana leaves by FUN are shown, where white bars represent GFP and gray bars represent pro35S:FUN-GFP. Pro35S:FUN-GFP was expressed as an effector, and GUS was driven by the Nrt2.1 promoter, the Ho1 promoter, or the NAC094 promoter as a reporter gene. Figure 2H The results of a transcriptional activation assay of the Nrt3.1 (left) and AS1 (right) promoters by FUN in Nicotiana benthamiana leaves are shown, where white bars represent GFP and gray bars represent pro35S:FUN-GFP. Pro35S:FUN-GFP was expressed as an effector, and the GUS reporter gene was driven by the Nrt3.1 promoter or the AS1 promoter. Figure 2I Representative images of the nodulation phenotype of WT (Gifu) Nelumbo nucifera, nrt2.1-3 mutant Nelumbo nucifera, ho1-4 mutant Nelumbo nucifera, or nac094-3 mutant Nelumbo nucifera after 2 weeks of 10 mM KNO 3 exposure are shown. The scale bar for all four figures is 1 cm. Figure 2J Shown are the total number of nodules (gray boxes, labeled “total”) and the number of pink functional nodules (white boxes, labeled “pink”) formed on wild-type (WT; Gifu) Nelumbo nucifera plants, nrt2.1-3 mutant Nelumbo nucifera plants, and nrt2.1-4 mutant Nelumbo nucifera plants exposed to 10 mM KNO3 for 2 weeks. Figure 2K Shown are the nitrogen fixation activities quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) on wild-type (WT; Gifu) Nelumbo plants, nrt2.1-3 mutant Nelumbo plants, and nrt2.1-4 mutant Nelumbo plants under 2 weeks of 10 mM KNO3 exposure. Figure 2L Shown are the leghemoglobin contents (μg leghemoglobin per plant) of wild-type (WT; Gifu) Nelumbo nucifera plants, nrt2.1-3 mutant Nelumbo nucifera plants, and nrt2.1-4 mutant Nelumbo nucifera plants exposed to 10 mM KNO 3 for 2 weeks. Figure 2MShown are the total number of nodules (white boxes, marked “total”) and the number of pink functional nodules (grey boxes, marked “pink”) formed on wild-type (WT; Gifu) Nelumbo nucifera plants, ho1-4 mutant Nelumbo nucifera plants, and ho1-5 mutant Nelumbo nucifera plants exposed to 10 mM KNO3 for 2 weeks. Figure 2N Shown are the leghemoglobin contents (μg leghemoglobin per plant) of wild-type (WT; Gifu) Nelumbo nucifera plants, nac094-3 mutant Nelumbo nucifera plants, and nac094-4 mutant Nelumbo nucifera plants under 10 mM KNO 3 exposure for 2 weeks. Figure 2O Shown are the total number of nodules (white boxes, marked “total”) and the number of pink functional nodules (grey boxes, marked “pink”) formed on wild-type (WT; Gifu) Nelumbo nucifera plants, nac094-3 mutant Nelumbo nucifera plants, and nac094-4 mutant Nelumbo nucifera plants exposed to 10 mM KNO3 for 2 weeks. Figure 2P Shown are the nitrogen fixation activities quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) on wild-type (WT; Gifu) Nelumbo plants, ho1-4 mutant Nelumbo plants, and ho1-5 mutant Nelumbo plants under 2 weeks of 10 mM KNO3 exposure. Figure 2Q Shown are the leghemoglobin contents (μg leghemoglobin per plant) of wild-type (WT; Gifu) Nelumbo nucifera plants, ho1-4 mutant Nelumbo nucifera plants, and ho1-5 mutant Nelumbo nucifera plants exposed to 10 mM KNO 3 for 2 weeks. Figure 2R Shown are the nitrogen fixation activities quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) on wild-type (WT; Gifu) Nelumbo plants, nac094-3 mutant Nelumbo plants, and nac094-4 mutant Nelumbo plants under 2 weeks of 10 mM KNO3 exposure. Figure 2S Shown are the wild-type plants that received mock treatment (labeled "WT Mock") and the wild-type, fun and fun-3 mutant plants 24 hours after nitrate exposure ( respectively Marked as "WT 24h", "fun 24h" and "fun-324h" ), Normalized read counts of downstream targets of FUN with identified TGA motifs within their promoters. The graph shows the normalized read counts of NRT2.1, HO1, NAC094, NRT3.1, and AS1 from left to right. Figures 2A-2B In , 2G-2H, and 2J-2R, circles represent individual plants. Figures 2A-2B , 2G-2H, and 2J-2R, asterisks indicate significant differences between the compared groups; “**” refers to p-value < 0.01, and “*” refers to p-value < 0.05.

[0035] Figures 3A-3L It was shown that in the presence of physiological concentrations of zinc (Zn), the sensor domain of FUN forms filamentous structures. Figure 3A Shown are the results of dynamic light scattering (DLS) analysis on the FUN sensor domain exposed to 4 mM MgCl2, CaCl2, MnCl2, ZnCl2, NH4Cl, KNO3, KNO2, KCl or a blank sample ("FUN sensor alone", control). Figure 3B Shown are the results of DLS analysis of FUN(bZIP) with MnCl2 at concentrations of 8 mM, 4 mM, 2 mM, 1 mM, 500 μM, 250 μM, 62.5 μM, 15.6 μM, 3.9 μM, or 0 μM. Figure 3C Shown are the results of DLS analysis of FUN (bZIP) with ZnCl2 at concentrations of 125 μM, 62.5 μM, 31.3 μM, 15.6 μM, 7.8 μM, 3.9 μM, 2.0 μM, 0 μM, or 8 mM without FUN (8 mM without bZIP; control). Figure 3D Shown are the results of DLS analysis of FUN bZIP alone ("Fun sensor"), FUN with 100 μM ZnCl2 ("FUN sensor+Zn"), or FUN with 100 μM ZnCl2 and 5 mM ethylenediaminetetraacetic acid (EDTA) ("FUN sensor+Zn+EDTA"). Figure 3E The SAXS analysis graph is shown, where the vertical axis is the scattering intensity "I(q)" in cm -1 , as the horizontal axis The scattering of the FUN sensor (bZIP only) alone ("FUN sensor," gray), FUN bZIP bound to Zn ("FUN sensor + Zn," light gray), or FUN bZIP with Zn removed using EDTA ("FUN sensor + Zn + EDTA," black) is plotted. Figure 3F Shows the Figure 3E A histogram of the distances between pairs of points within the analyzed particle, with the pairwise distance distribution model p(r) plotted on the vertical axis and the distance in angstroms plotted on the horizontal axis. With arrow The maximum diameter (Dmax) of the zinc-binding FUNbZIP ("FUN sensor + Zn") is With arrow The Dmax of the measured values ​​of FUN bZIP only (“FUN sensor”) or FUN bZIP with Zn removed (“FUN sensor + Zn + EDTA”) is Figure 3G Shows the Figure 3E and 3FCalculated Guinier plot, where the radius of gyration is calculated from the scattering intensity as a function of the scattering vector q (vertical axis relative to the horizontal axis). Solid circles are the data used in the fit, and open circles indicate omitted data points. The p(r) function shows that the radius of gyration for the pure FUN sensor sample and the sample containing EDTA + zinc is The radius of gyration of the zinc-bound sample is In the Guinier analysis, the values ​​were slightly lower for all samples. Figure 3H Representative electron microscopy images of the FUN sensor domain alone (bZIP1 sensor; top), the FUN sensor domain with 300 μM ZnCl2 (bZIP1 sensor+300 μM Zn; middle); and FUN with 300 μM ZnCl2 and 5 mM EDTA (bZIP1 sensor+300 μM Zn+5 mM EDTA; bottom) are shown. Figure 3I Shown are the relative expression of Fun (vertical axis) over time (horizontal axis) in 3-week-old nodules exposed to 10 mM KNO 3 for 0, 0.5, and 3 hours (left) and 0, 1, 3, and 7 days (right). Figure 3J The purification and thermal stability of the FUN sensor domain are shown. The left side shows a size exclusion chromatogram of the FUN sensor domain (Superdex 200 increase 10 / 300), with absorbance (vertical axis) plotted against elution volume (horizontal axis). The right side shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of SEC fractions. Fractions 14-17 were pooled and stored, as indicated by the dotted line on the chromatogram and the horizontal line above the SDS-PAGE. Figure 3K The inflection temperatures (T) of the FUN sensors were measured alone (“FUN sensor only”) or in combination with different ions (i.e., MgCl2, CaCl2, MnCl2, ZnCl2, NH4Cl, KNO3, KNO2, or KCl) at a concentration of 4 mM (vertical axis). i Thermal stability of the purified FUN sensor domain (°C). Figure 3L Results of DLS analysis of FUN protein containing a zipper domain and a sensor domain in the absence (grey line) and presence (medium grey line) of 100 μM ZnCl2 are shown. The zinc-induced change in hydrodynamic radius was reversed with 5 mM EDTA (black line). Figures 3C-3GIn the figure, the gray "FUN sensor" represents the measurement value of the FUN sensor alone, the medium gray "FUN sensor + Zn" represents the measurement value of the FUN sensor treated with 100 μM ZnCl2, and the black "FUN sensor + Zn + EDTA" represents the FUN sensor treated with 100 μM ZnCl2 and then treated with 5 mM EDTA to remove Zn. Figures 3C-3G In the figure, the four-pointed star labeled "apo" represents the FUN sensor in the apo structure ("apo"). The chain of overlapping four-pointed stars labeled "Zn-bound" represents the combination of the FUN sensor bound to Zn in a larger oligomer.

[0036] Figures 4A-4H Shown are zinc-regulated subcellular localization and function of FUN. Figure 4A Representative images showing the subcellular localization of pro35S:FUN-GFP in Nicotiana benthamiana leaves. Figure 4B The fluorescence distribution of FUN-GFP subcellular localization in Nicotiana benthamiana leaves is shown. Figure 4C The ratio of punctate nuclei (dark grey) to total nuclei (homo; light grey) showing subcellular localization of FUN-GFP in Nicotiana benthamiana leaves is shown. Figures 4A-4C In Figure 5, the results were obtained at 500 μM MgCl2 (mock), MnCl2 (Mn), and ZnCl2 (Zn). Figure 4D Shown are the results of a transcriptional activation assay of the Nrt2.1 promoter by FUN in Nicotiana benthamiana leaves after 500 μM MgCl2 (mock) and ZnCl2 (Zn) treatment, where white bars represent GFP and gray bars represent pro35S:FUN-GFP. FUN-GFP was expressed as an effector, and GUS was driven by the Nrt2.1 promoter (proNrt2.1:GUS) as a reporter gene. Figure 4E Shown are the nitrogen fixation activities quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) of wild-type (WT; Gifu) Nelumbo plants exposed to 500 μM MgCl2 (mock; white) and ZnCl2 (Zn; dark gray) in combination with KCl (left) or 10 mM KNO3 (center and right). Figure 4F Shown are nitrogen fixation activities quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) on wild-type (WT; Gifu) Nelumbo plants and fun mutant Nelumbo plants exposed to 500 μM MgCl2 (mock; white) and ZnCl2 (Zn; dark gray) together with 10 mM KNO3. Figure 4GShown are the leghemoglobin contents (μg leghemoglobin per plant) of wild-type (WT; Gifu) Nelumbo nucifera plants and fun mutant Nelumbo nucifera plants exposed to 500 μM MgCl 2 (mock; white) and ZnCl 2 (Zn; dark gray) together with 10 mM KNO 3 . Figure 4H Shown are the leghemoglobin contents (μg leghemoglobin per plant) of wild-type (WT; Gifu) Nelumbo nucifera plants after 2 weeks of 10 mM KCl exposure (left), 10 mM KNO3 exposure (center), or 10 mM KNO3 plus ZnCl2 treatment (right). Figures 4D-4H In the figure, circles represent individual plants.

[0037] Figures 5A-5H We show that nitrate promotes zinc export in nodule cells via the zinc-sensitive dye Zinpyr-1. Figure 5A It was shown that the expression of two putative zinc transporter genes, Zip2 and Zip4, was induced in nodules after 24 h of treatment with 10 mM KNO3 (nitrate). Figure 5B Depicted are the mechanisms of FUN-regulated nodulation function. On the left, the mechanism operates under low soil nitrate levels, where zinc accumulates in nodules, retaining FUN in inactive filaments and allowing continued nitrogen fixation. On the right, the mechanism operates under high soil nitrate levels, where cellular zinc levels decrease, releasing active FUN from filaments and increasing target gene expression. Arrows indicate the directional effects of these conditions. "NAC094," "HO1," and "NRT2.1" represent the activity of these three target genes that induce nodule senescence. Dark gray indicates the presence of inactive FUN filaments and the corresponding pink nodulation phenotype. Light gray indicates the presence of active FUN. Figure 5C Shown are cellular zinc levels within nodules as indicated by Zinpyr-1 fluorescent dye 24 hours after mock treatment (left, KCl) and nitrate treatment (right, KNO3). Scale bar = 200 μm. Figure 5D Shows Figure 5C The average intensity of the fixation area indicated by the middle dashed circle. Figure 5E Images of nodule sections taken from an X-ray fluorescence (XRF) microscope 24 hours after mock treatment (left, KCl) or nitrate treatment (right, KNO3) are shown. Scale bar = 20 μm. Figure 5F Shows the Figure 5E Images of the 100-well plate, where the white box outlines the area used for quantitative zinc analysis. Scale bar = 20 μm. Figure 5G Fluorescence images generated by the FUN-GFP construct in Nelumbo nucifera roots treated with control (left, labeled "MgCl2") and zinc (right, labeled "ZnCl2") are shown. Scale bar = 20 μm. Chi-square test values ​​are shown below; *** = p value < 0.01. Figure 5H Confocal images of Nicotiana benthamiana leaves expressing the FUN-GFP construct and co-infiltrated with either MgCl2 (left) or ZnCl2 (right) two days prior to confocal observation are shown. Scale bar = 5 μm. Chi-square test values ​​are shown below; * = p value < 0.05.

[0038] Figures 6A-6E Shown are the phylogenetic tree of FUN proteins and the relative expression patterns of soybean FUN orthologs. Figure 6A The phylogeny of FUN orthologs identified using shoot.bio and FUN and LjFUN-like is shown. The support values ​​of the tree are plotted at the bifurcation points. Figure 6B The relative expression (vertical axis) patterns of the closest FUN soybean orthologs, Glyma.02G097900 and Glyma.01G084200, across different tissues (horizontal axis) are shown. For each tissue, the expression level of Glyma.02G097900 is shown on the left, and the expression level of Glyma.01G084200 is shown on the right. Figure 6C A schematic diagram of the LjFUN protein is shown. The DNA-binding bZIP domain is shown in the left box, and the zinc sensor domain is shown in the right box. Figure 6D The first half of a protein alignment of selected FUN and FUN-like protein orthologs is shown. Figure 6E The second half of the protein alignment of selected FUN and FUN-like protein orthologs is shown. Figure 6A 、 6DIn 6E and 6E, the plant species names correspond to the abbreviations used as follows: Prunus persica: Prupe; Lotus japonicus: Lj; Glycine max: Glyma; Manihot esculenta: Manes; Gossypium raimondii: Gorai; Eucalyptus grandis: Eucgr; Brassica oleracea: Bol; Arabidopsis thaliana: AT; Solanum lycopersicum: Solyc; Aquilegia coerulea: Aqcoe; Amborella trichopoda: AmTr; Spirodela polyrhiza: Spipo; Musa acuminata: GSMUA; Zea mays: Zea serrata; mays): GRMZM; ​​millet (Setaria italica): Seita; wheat (Triticum Aestivum): Traes; barley (Hordeum vulgare): HORVU; and rice (Oryza sativa): Os. Figures 6D-6E In the figure, from top to bottom, the aligned protein sequences are consensus sequence (SEQ ID NO: 150), Nipponia LjFUN (SEQ ID NO: 1), Glycine max Glyma.02G097900.1 (SEQ ID NO: 8), Glycine max Glyma.01G084200.1 (SEQ ID NO: 9), Glycine max Glyma.10G276100 (SEQ ID NO: 6), Glycine max Glyma.20G113600.1 (SEQ ID NO: 7), Lotus japonicus LjFUNL (SEQ ID NO: 83), and Arabidopsis thaliana AT1G68640.1 (SEQ ID NO: 4).

[0039] Figure 7 A phylogenetic tree showing the NAC domain-containing protein Nac094 (labeled "LjNAC094") from Lotus japonicus and orthologous NAC domain-containing proteins from other species is shown. Species names and protein identifiers are indicated at the ends of branches. Support values ​​for the tree are plotted at bifurcation points.

[0040] Figures 8A-8C showed evidence that FUN plays a role in drought and heat tolerance. Figure 8A Shown are RNAseq counts of NAC094 (left panel) and HO1 (right panel) in nodules of Medicago truncatula under the following conditions (from left to right): watered, 2 days of drought, and 4 days of drought. Figure 8B Shown are RNAseq counts of NAC094 (left panel) and HO1 (right panel) in nodules of Lotus japonicus under the following conditions (from left to right): watered, 2 days of drought, and 4 days of drought. Figure 8C Shown are nitrogen fixation activities quantified using the acetylene reduction assay (ARA; nmol C2H2 / h / plant) of wild-type plants (left) and fun-3 mutant plants (right) before (grey boxes) and after 7 days of heat stress (white boxes). 'ns' indicates no significant difference. Details

[0041] The following description sets forth exemplary methods, parameters, etc. However, it should be recognized that these descriptions are not intended to limit the scope of the present disclosure, but are provided as descriptions of exemplary embodiments. FUN protein

[0042] One aspect of the present disclosure includes the FUN (Fixation under Nitrate) proteins disclosed throughout the text and their related uses, wherein FUN is a transcriptional regulator expressed in nodulation. Example 1 describes the identification of the basic leucine zipper transcription factor FUN as a novel master regulator of nitrogen fixation in legumes. Examples of FUN proteins include, but are not limited to, the originally identified Lotus japonicus FUN protein (LotjaGi2g1v0279100; SEQ ID NO: 1), as well as orthologs of the Lotus japonicus FUN protein (which are also expressed in nodules of the corresponding plants), including, but not limited to, soybean FUNa protein and FUNb protein (Glyma.02G097900; SEQ ID NO: 8, and Glyma.01G084200; SEQ ID NO: 9), Vicia faba FUN protein (Vfaba.Hedin2.R1.1g203360.1; SEQ ID NO: 81), and V. unguiculata (Cowpea) FUN (Vigun02g036100.1.p; SEQ ID NO: 82). FUN proteins that exhibit activity, expression, or enhanced activity or enhanced expression in nodules are readily distinguished from FUN-like proteins (paralogs of FUN proteins) that can be distinguished by their lack of enhanced expression or activity in nodules. FUN-like proteins also form independent paralogous branches on the phylogenetic tree. Exemplary FUN-like proteins include lotus plant FUN-like protein (LotjaGi5g1v0341400; SEQ ID NO: 83); cowpea FUN-like protein (Vigun07g272100.1.p; SEQ ID NO: 84); soybean FUN-like A (G.max Wm82.a2.v1|Glyma.10G276100.1.p; SEQ ID NO: 6); soybean FUN-like B (G.max Wm82.a2.v1|Glyma.20G113600.1.p; SEQ ID NO: 7) and Arabidopsis PAN (A.thalianaAraport11|AT1G68640.1; SEQ ID NO: 4). The FUN protein can be a protein overexpressed in nodulation. FUN or FUN-like protein can be in an inactive filamentous form (e.g., large filamentous form) or an active oligomeric non-filamentous form. FUN or FUN-like genes encode proteins of the TGA transcription factor family. TGA transcription factors belong to the bZIP transcription factor family, wherein the bZIP transcription factor family is characterized by the presence of a leucine zipper (bZIP) DNA binding domain at the N-terminus and a DOG1 domain at the C-terminus. Figure 1P The sensor domain ( Gruden, K. & Coll, A. TGA transcription factors-Structural characteristics as basis for functional variability. Front. Plant Sci. 13, 935-819 (2022). For the FUN and FUN-like protein sequences disclosed herein, the protein domains and detailed locations were analyzed and listed in Table 1. Table 1. Protein domains and locations of identified FUN, FUN-like, NAC, NRT3.1, NRT2.1, AS1, and HO1 protein sequences.

[0043] Protein domains and motifs are both conserved sequence patterns. A domain is an independently folded unit of a protein. A motif is a small region or group of small regions of three-dimensional structure or nucleotide or amino acid sequence that is shared between proteins. Conserved domains or motifs are recurring units in molecular evolution, and their extent can be determined through sequence and structure analysis. Conserved domains or motifs can contain conserved sequence patterns or sequence motifs, which allow for the detection of domains or motifs in polypeptide sequences. Downstream targets of FUN protein

[0044] FUN is a transcriptional regulator that controls the expression of downstream genes, including high-affinity nitrate transporter 2.1 (NRT2.1), heme oxygenase (HO1), NAC domain-containing protein 94 (NAC transcription factor 94 or NAC094), high-affinity nitrate transporter 3.1 (NRT3.1), basic leucine zipper transcription factor 28 (bZIP28), and asparagine synthase 1 (AS1), to regulate nitrate signaling and nitrogen fixation in nodules. Exemplary NRT2.1 proteins include SEQ ID NO: 78. Exemplary HO1 proteins include SEQ ID NO: 77. Exemplary NRT3.1 proteins include SEQ ID NO: 74. Exemplary bZIP28 proteins include SEQ ID NO: 75. Exemplary AS1 proteins include SEQ ID NO: 79. Lotus japonicus NAC094 (SEQ ID NO: 31; also known as FEZ protein), which is a downstream target of FUN ( Figure 7 ). Exemplary NAC094 homologs (NAC domain-containing proteins) include SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:76. Genetically modified plants and related methods

[0045] Aspects of the present disclosure include genetically modified plants, or parts thereof, comprising one or more genetic alterations that result in reduced activity or expression of a FUN protein in the genetically modified plant, or part thereof, as compared to the activity or expression of the FUN protein in a control plant grown under the same conditions. In further embodiments of this aspect, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. The proteins of group NO:82 have polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a protein, or polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof, e.g., a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof recited in Table 1 (e.g., a bZIP1, bZIP2, or DOG1 domain thereof) or a combination thereof.In another embodiment of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, or respective domains thereof recited in Table 1, or a combination thereof. In yet other embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or domains thereof as described in Table 1, or combinations thereof.

[0046] A further aspect of the present disclosure includes a genetically modified plant or part thereof comprising one or more genetic alterations that result in a decrease in the activity or expression of one or more of an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein (also known as a FEZ protein), a HO1 protein, an NRT2.1 protein, or an AS1 protein in the genetically modified plant or part thereof as compared to the activity or expression of an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein (also known as a FEZ protein), a HO1 protein, an NRT2.1 protein, or an AS1 protein in a control plant grown under the same conditions. In a further embodiment of this aspect, the protein is an NRT3.1 protein, and wherein the NRT3.1 protein is expressed in the NRT3.1 protein.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:74; wherein the protein is a bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:75; wherein the protein is a NAC domain-containing protein, and wherein the NAC domain-containing protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 and SEQID The protein of the group of NO:73 has a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 78; or wherein the protein is AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 78; NO:79 having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity, or a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a domain thereof, such as a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to each of the domains recited in Table 1 or a combination thereof. In further embodiments of this aspect, NRT3.1 protein includes SEQ ID NO:74; wherein the bZIP28 protein includes SEQ ID NO:75; wherein the NAC domain-containing protein includes SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO: In yet another embodiment of this aspect, the NAC domain-containing protein comprises SEQ ID NO: 31, SEQ ID NO: 41, or SEQ ID NO: 42, or a combination thereof. For the NRT3.1, NRT2.1, AS1, and HO1 protein sequences disclosed herein, the protein domains and their detailed locations are analyzed and listed in Table 1. .

[0047] Additional aspects of the present disclosure relate to a genetically altered plant genome comprising (i) one or more genetic alterations in the genetically modified plant or part thereof of any preceding embodiment, or (ii) one or more genetic alterations in a genetically modified plant or part thereof produced by the method of any preceding embodiment. In yet further embodiments of this aspect, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. The proteins of group NO:82 have polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a protein of group NO:82, or polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof, such as polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to each of the domains recited in Table 1 or a combination thereof. In further embodiments of this aspect,The FUN protein includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, or domains thereof as described in Table 1, or combinations thereof. In further embodiments of this aspect, wherein the protein is NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 74; wherein the protein is bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 75; wherein the protein is a NAC domain-containing protein,And wherein the NAC domain-containing protein includes a protein selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:78; NO:78 has a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO:79,or a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof, such as a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to each of the domains recited in Table 1 or a combination thereof. In another embodiment of this aspect, the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:79, or respective domains thereof recited in Table 1, or combinations thereof. In still further embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:42, or respective domains thereof listed in Table 1, or combinations thereof.

[0048] In further embodiments of this aspect, which may be combined with any of the preceding embodiments, the reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. In yet further embodiments of this aspect (which may be combined with any of the preceding embodiments), the reduction is due to knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out a domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out a domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out a domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, preferably the binding site is a transcription activator protein binding site or a TATA box. In further embodiments of this aspect (which may be combined with any of the preceding embodiments), the growth conditions comprise moderate nitrate levels, high nitrate levels, or nitrate levels around the plant that reduce or suppress nitrogen fixation. In yet other embodiments of this aspect, the nitrate level is between about 10 mM and about 250 mM nitrate, or includes at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate. In still other embodiments of this aspect, the genetically modified plants have increased nitrogen fixation compared to control plants when grown under the same growth conditions. In further embodiments of this aspect, nitrogen fixation is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In yet further embodiments of this aspect (which may be combined with any preceding embodiment), the plant forms nodules. In further embodiments of this aspect, the plant has an increased number of nodules, an increased hemoglobin content, or an increased acetylene reduction assay (ARA) activity compared to control plants when grown under identical conditions.

[0049] In certain embodiments of any of the foregoing aspects and various embodiments thereof, the plant part can be a seed, pod, fruit, leaf, flower, stem, root, any of the foregoing parts or cells thereof, or a non-renewable part or cell of a genetically modified plant part. As used herein, a "non-renewable" part or cell of a genetically modified plant or part thereof is a part or cell that cannot be induced to form a complete plant by itself, or cannot be induced to form a complete plant capable of sexual and / or asexual reproduction. In certain embodiments, the non-renewable part or cell of a plant part is a part of a transgenic seed, pod, fruit, leaf, flower, stem or root, or a cell thereof. In other embodiments, the non-renewable part or cell of a plant part is part of a processed plant product.

[0050] Also provided are processed plant products containing detectable amounts of nucleotide segments, expressed RNA and / or proteins comprising the genetic modifications disclosed herein. Such processed products include, but are not limited to, plant biomass, oil, meal, animal feed, flour, flakes, bran, cotton wool, shells, and processed seeds. Processed products may be non-renewable. Plant products may include commodities or other commercial products derived from transgenic plants or transgenic plant parts, wherein by detecting nucleotide segments, expressed RNA and / or proteins comprising the distinguishing parts of the genetic modifications disclosed herein, commodities or other products may be tracked throughout a commercial process.

[0051] Additional aspects of the present disclosure include methods of growing a genetically altered plant that has increased nitrogen fixation under conditions comprising nitrate levels surrounding the roots of the plant that suppress nitrogen fixation, the method comprising: (a) providing a genetically altered plant, wherein the plant or part thereof comprises one or more genetic alterations that result in decreased activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also known as a FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein, or any combination thereof, in the genetically altered plant or part thereof, as compared to the activity or expression of the FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also known as a FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions, and wherein the one or more genetic alterations reduces the suppression of nitrogen fixation by nitrate levels; and (b) growing the genetically altered plant under nitrate levels surrounding the roots of the plant, wherein the genetically modified plant has increased nitrogen fixation compared to a control plant grown under the same conditions. In yet another embodiment of this aspect, the reduced activity or expression is due to knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, preferably the binding site is a transcription activator protein binding site or a TATA box. In a further embodiment of this aspect,FUN proteins include those selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81 or SEQ ID The proteins of group NO:82 have polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a protein of group NO:82, or polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof, such as a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof recited in Table 1 (e.g., a bZIP1, bZIP2, or DOG1 domain thereof), or a combination thereof. In further embodiments of this aspect,The FUN protein includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, or domains thereof as described in Table 1, or combinations thereof. In still further embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or a domain thereof as recited in Table 1, or a combination thereof. In yet further embodiments of this aspect, wherein the protein is NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 74; wherein the protein is bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 75; wherein the protein is a NAC domain-containing protein,and wherein the NAC domain-containing protein comprises a protein selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:78; NO:78 has a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO:78; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO:79,or a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof, such as a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to each of the domains recited in Table 1 or a combination thereof. In a further embodiment of this aspect, wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:79, or the respective domains thereof recited in Table 1, or a combination thereof. In still further embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:42,or any of the respective domains recited in Table 1 or a combination thereof. Additional embodiments of this aspect, which may be combined with any of the preceding embodiments, include the nitrate level in step (c) being between about 10 mM and about 250 mM, or at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, or at least about 250 mM. Further embodiments of this aspect, which may be combined with any of the preceding embodiments, include an increase in nitrogen fixation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In further embodiments of this aspect, the number of nodules is increased or the hemoglobin content is increased compared to control plants when grown under the same growth conditions. In further embodiments of this aspect, the increased nitrogen fixation is measured using a method selected from the group consisting of: measuring the number of pink nodules per plant compared to control plants, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour compared to control plants (acetylene reduction assay (ARA)), or measuring micrograms of hemoglobin per plant compared to control plants (e.g., as described in Example 1).

[0052] Further aspects of the present disclosure include methods of growing genetically modified plants that are capable of fixing nitrogen when grown under nitrogen fertilizer conditions, the methods comprising: (a) providing a genetically modified plant, wherein the plant or part thereof comprises one or more genetic alterations that result in reduced activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also known as a FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein, or any combination thereof, as compared to the activity or expression of the FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein (also known as a FEZ protein), HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions, and wherein the one or more genetic alterations reduce nitrate level repression of nitrogen fixation; (b) growing the plant under conditions comprising a standard nitrate level surrounding the roots of the plant; and (c) applying nitrogen fertilizer, thereby generating conditions comprising a nitrate level surrounding the roots of the plant that represses nitrogen fixation, wherein the genetically modified plant has increased nitrogen fixation compared to a control plant grown under the same conditions. In another embodiment of this aspect, the activity or expression that is reduced is due to knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, preferably the binding site is a transcription activator protein binding site or a TATA box. In a further embodiment of this aspect,The FUN protein includes a protein selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81 or SEQ ID The proteins of group NO:82 have polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a protein of group NO:82, or polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof, such as a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof recited in Table X. In further embodiments of this aspect,The FUN protein includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, or domains thereof as described in Table 1, or combinations thereof. In still further embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or a domain thereof as recited in Table 1, or a combination thereof. In yet further embodiments of this aspect, wherein the protein is NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 74; wherein the protein is bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 75; wherein the protein is a NAC domain-containing protein,And wherein the NAC domain-containing protein includes a protein selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:78; NO:78 has a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO:79. In further embodiments of this aspect,wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:79, or respective domains thereof recited in Table 1 or combinations thereof. In still further embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:42,or their respective domains recited in Table 1 or a combination thereof. Additional embodiments of this aspect, which may be combined with any of the preceding embodiments, include the nitrate level in step (c) being between about 10 mM and about 250 mM nitrate, or at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate. Further embodiments of this aspect, which may be combined with any of the preceding embodiments, include an increase in nitrogen fixation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%. In further embodiments of this aspect, the number of nodules is increased or the hemoglobin content is increased compared to control plants when grown under the same growth conditions. In additional embodiments of this aspect, the increased nitrogen fixation is measured using a method selected from the group consisting of: measuring the number of pink nodules per plant compared to control plants, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour compared to control plants (acetylene reduction assay (ARA)), or measuring micrograms of hemoglobin per plant compared to control plants (e.g., as described in Example 1). In further embodiments of this aspect, which may be combined with any of the preceding embodiments, the genetically altered plants are grown in an intercropping system with non-nitrogen fixing plants, or in a sequential system following non-nitrogen fixing plants.

[0053] Additional aspects of the present disclosure include methods of delaying nodulation senescence, the methods comprising: (a) providing a genetically altered plant, wherein the plant or part thereof comprises one or more genetic alterations that result in reduced activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein, or any combination thereof, in the genetically altered plant or part thereof, as compared to the activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions, and wherein the one or more genetic alterations delay nodulation senescence; and (b) cultivating the genetically altered plant under stress conditions, wherein the genetically altered plant has delayed nodulation senescence compared to a control plant grown under the same conditions. In yet another embodiment of this aspect, the reduced activity or expression is due to knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, preferably the binding site is a transcription activator protein binding site or a TATA box. Further embodiments of this aspect include stress conditions selected from the group consisting of: moderate nitrate levels, high nitrate levels, nitrate levels around the plant that promote nodulation and senescence, moderate heat levels, high heat levels, heat levels around the plant that promote nodulation and senescence, moderate water shortage (i.e., drought) levels, high water shortage levels, water shortage levels around the plant that promote nodulation and senescence, moderate waterlogging levels, high waterlogging levels, waterlogging levels around the plant that promote nodulation and senescence. For each of these conditions, the stress level is considered to be sufficient to inhibit nitrogen fixation in a particular plant species. In further embodiments of this aspect (which may be combined with any of the preceding embodiments),The FUN protein includes a protein selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ IDNO:81 or SEQ ID The proteins of group NO:82 have polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a protein of group NO:82, or polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof, such as polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to each of the domains recited in Table 1 or a combination thereof. In further embodiments of this aspect,The FUN protein includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 8, or domains thereof as described in Table 1 or combinations thereof. In still further embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or a domain thereof as recited in Table 1, or a combination thereof. In yet further embodiments of this aspect (which may be combined with any of the foregoing embodiments of proteins having NAC domains), wherein the protein is NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 74; wherein the protein is bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 75; wherein the protein is a protein having NAC domains,And wherein the NAC domain-containing protein includes a protein selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:78; NO:78 has a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO:78; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO:79,or a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a domain thereof, such as a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to each of the domains recited in Table 1, or a combination thereof. In a further embodiment of this aspect, wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:79, or respective domains thereof recited in Table 1, or a combination thereof. In still further embodiments of this aspect, the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or respective domains thereof recited in Table 1, or a combination thereof. In further embodiments of this aspect,Nodule senescence is delayed by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

[0054] Yet further aspects of the present disclosure include methods for inducing filamentous formation of a FUN protein, comprising: (a) providing a FUN protein; and (b) increasing the amount of zinc or manganese in the environment of the FUN protein, wherein the increased amount of zinc or manganese induces filamentous formation compared to a control FUN protein in an environment in which the amount of zinc or manganese is not increased. In another embodiment of this aspect, filamentous formation is induced under high nitrate conditions. In another embodiment of this aspect (which may be combined with any of the foregoing embodiments), the method is performed in vitro.

[0055] Still further aspects of the present disclosure include methods of inducing filamentation, comprising: (a) providing a plant comprising a FUN protein; and (b) cultivating the plant under conditions of increased zinc or manganese, wherein filamentation of the FUN protein in the plant is induced compared to the FUN protein in a control plant grown under conditions without increased zinc or manganese. In another embodiment of this aspect, the plant comprises a genetic alteration. In another embodiment of this aspect, filamentation is induced under high nitrate conditions. In yet another embodiment of this aspect (which may be combined with any of the aforementioned embodiments in which the plant comprises a genetic alteration), the genetic alteration reduces the activity of the FUN protein without eliminating the activity of the FUN protein. In a further embodiment of this aspect, the induction of filamentation results in an increase in nitrogen fixation in the genetically altered plant, or a reduction in the activity of the FUN protein or its inactivation, compared to a control plant grown under the same conditions. In still further embodiments of this aspect, which may be combined with any of the preceding embodiments or aspects of the method of inducing filamentation, the number of nodules is increased, the hemoglobin content is increased, or the acetylene reduction assay (ARA) activity is increased compared to control plants when grown under the same conditions.

[0056] Yet further aspects of the present disclosure include methods for regulating nodulation function based on the amount of available nitrogen in the soil, the methods comprising: a) providing a genetically modified plant comprising a FUN protein whose activation is altered by nitrate; and b) cultivating the genetically modified plant under conditions of nitrate concentration, wherein the genetically modified plant has reduced FUN activity or expression and / or a reduced active form of FUN compared to a WT plant grown under the same nitrate conditions. In further embodiments of this aspect, altering FUN protein activation by nitrate comprises downregulating FUN, reducing FUN activity, knocking out FUN by mutation, knocking down FUN expression, knocking out a promoter element of FUN, or a combination thereof. In further embodiments of this aspect, altering FUN protein activation by nitrate comprises manipulating environmental or cellular zinc levels, wherein the manipulation results in the FUN protein being maintained in an inactive filamentous form. In still further embodiments of this aspect, altering FUN protein activation by nitrate comprises genetically modifying the FUN protein sequence to alter sensitivity to zinc.

[0057] In further embodiments of this aspect, which may be combined with any of the preceding embodiments and aspects, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. The proteins of group NO:82 have polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to, or polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to, their domains, e.g., polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to, their respective domains recited in Table 1 or a combination thereof.In another embodiment of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, or a domain thereof recited in Table 1, or a combination thereof. In still further embodiments of this aspect, the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8, or SEQ ID NO: 9, or domains thereof as described in Table 1, or combinations thereof.

[0058] Further aspects of the present disclosure include methods of making a genetically altered plant having increased nitrogen fixation under conditions including nitrate levels around the plant's roots that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic alterations that reduce the activity or expression of a FUN protein as compared to the activity or expression of the FUN protein in a control plant grown under the same conditions. In yet another embodiment of this aspect, the reduced activity or expression is due to knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, preferably the binding site is a transcription activator protein binding site or a TATA box. In another embodiment of this aspect, the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO: 82. The proteins of group NO:82 have polypeptides that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical;Among them, FUN protein includes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81 or SEQ ID NO:82; or wherein the FUN protein includes SEQ ID NO:1, SEQ ID NO:8 or SEQ ID NO:9, or a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a domain thereof, such as a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to each of the domains recited in Table 1 or a combination thereof.;

[0059] Additional aspects of the present disclosure include methods of making a genetically altered plant having increased nitrogen fixation under conditions including nitrate levels around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic alterations that reduce the activity or expression of one or more of an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein (also known as a FEZ protein), a HO1 protein, an NRT2.1 protein, or an AS1 protein, as compared to the activity or expression of the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein in a control plant grown under the same conditions. In yet another embodiment of this aspect, the reduced activity or expression is due to knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out a binding site in the promoter region of the gene, preferably the binding site is a transcription activator protein binding site or a TATA box. In another embodiment of this aspect, the protein is NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:74; wherein the protein is a bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:75; wherein the protein is a NAC domain-containing protein, and wherein the NAC domain-containing protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 and SEQ ID The protein of the group of NO:73 has a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:78; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:79; wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; wherein NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:79; or wherein the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41, or SEQ ID NO:42, or a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a domain thereof, such as a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% or 100% identity to each of the domains recited in Table 1, or a combination thereof.

[0060] Yet additional aspects of the present disclosure include methods of making a genetically altered plant having increased nitrogen fixation under conditions comprising nitrate levels around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or a part thereof one or more genetic alterations that reduce the activity or expression of one or more of a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein (also known as a FEZ protein), a HO1 protein, a NRT2.1 protein, or an AS1 protein, as compared to the activity or expression of the FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein in a control plant grown under the same conditions. In yet another embodiment of this aspect, the reduced activity or expression is due to knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, and / or the genetic alteration comprises knocking out the gene for the protein, introducing a premature stop codon in the coding sequence of the gene for the protein, RNAi silencing, knocking out the domain of the protein, introducing a transcriptional repressor protein binding site, or knocking out the binding site in the promoter region of the gene, preferably the binding site is a transcription activator protein binding site or a TATA box. In some embodiments of this aspect, the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein or AS1 protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 or SEQID NO:84 A polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity, or a group of domains thereof recited in Table 1 or a combination thereof, and wherein the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein or AS1 protein has enhanced expression in nodules in the absence of one or more genetic alterations.

[0061] Additional aspects of the present disclosure include methods of making a genetically modified plant or part thereof according to any of the above embodiments, the methods comprising: introducing a genetic alteration into a plant cell to reduce or knock out the activity or expression of a FUN protein, a FUN-like protein, a NAC domain-containing protein (also known as a FEZ protein), a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein. In further embodiments of this aspect, the genetic alteration comprises a first nucleic acid sequence that is capable of reducing or knocking out a second nucleic acid sequence encoding a FUN protein, a FUN-like protein, a NAC domain-containing protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein operably linked to a promoter. In another embodiment of this aspect (which may be combined with any of the preceding embodiments), the genetically modified plant is selected from one or more of the group consisting of alfalfa, Bambara peanut, beans (e.g., kidney beans, black beans, etc.), blackcurrant, chickpea, clover, cowpea, forage legumes, legume trees, lentils, lotus plants, lupines, Medicago species, peas, peanuts, pigeon peas, soybeans, Sophora flavescens, alder, and elm. In another embodiment of this aspect, the nucleic acid comprises an RNA silencing-associated short RNA, antisense RNA, siRNA, miRNA, dsRNA, tasiRNA, or a secondary siRNA. In yet another embodiment of this aspect, the promoter is a nodulation-specific promoter, a root-specific promoter, an inducible promoter, a constitutive promoter, or a combination thereof. In another embodiment of this aspect, the promoter is a root-specific promoter, and wherein the promoter is selected from the group consisting of: NFR1 promoter, NFR5 promoter, LYK3 promoter, CERK6 promoter, NFP promoter, Lotus japonicus NFR5 promoter (SEQ ID NO:85), Lotus japonicus NFR1 promoter (SEQ ID NO:89), Lotus japonicus CERK6 promoter (SEQ ID NO:87), Medicago truncatula NFP promoter (SEQ ID NO:86), Medicago truncatula LYK3 promoter (SEQ ID NO:88), maize metallothionein promoter, chitinase promoter, maize ZRP2 promoter, tomato LeExt1 promoter, glutamine synthase soybean root promoter, RCC3 promoter, rice antiquitin promoter, LRR receptor kinase promoter and Arabidopsis thaliana pCO2 promoter. In a further embodiment of this aspect, the promoter is a constitutive promoter, and wherein the promoter is selected from the group comprising: CaMV35S promoter, a derivative of CaMV35S promoter, maize ubiquitin promoter, polyubiquitin promoter, vein mosaic cassava virus promoter or Arabidopsis UBQ10 promoter.In still other embodiments of this aspect, the nucleic acid sequence is inserted into the genome of the plant such that the nucleic acid sequence is operably linked to an endogenous promoter. In further embodiments of this aspect, the endogenous promoter is a nodulation-specific promoter or a root-specific promoter.

[0062] Further aspects of the present disclosure include methods for preparing a genetically modified plant or part thereof of any of the foregoing embodiments, the method comprising genetically modifying a plant cell by transforming the plant cell with one or more gene editing components, wherein the gene editing components target an endogenous nuclear genomic sequence encoding a FUN protein, a FUN-like protein, a protein containing a NAC domain (also known as a FEZ protein), a NRT3.1 protein, a bZIP28 protein, a protein containing a NAC domain, a HO1 protein, a NRT2.1 protein, or an AS1 protein, wherein the endogenous nuclear genomic sequence or a portion thereof is knocked out. In another embodiment of this aspect, the one or more gene editing components include a ribonucleoprotein complex targeting a nuclear genomic sequence; a vector comprising a TALEN protein coding sequence, wherein the TALEN protein targets the nuclear genomic sequence; a vector comprising a ZFN protein coding sequence, wherein the ZFN protein targets the nuclear genomic sequence; an oligonucleotide donor (OND), wherein the OND targets the nuclear genomic sequence; or a vector CRISPR / Cas enzyme coding sequence and a targeting sequence, wherein the targeting sequence targets the nuclear genomic sequence.

[0063] Contrast as herein described can be control sample or reference sample from wild type, homozygote or invalid separation plant, species or sample or its population.Control plant as herein described can also be the plant identical with gene change or genetic modification but do not have change or modification, or the wild-type plant under identical growth conditions, soil and / or growth medium.Reference value can be used for replacing control sample or reference sample, and it previously obtained from wild type, homozygote or invalid separation plant, species or sample or its population or one group of wild type, homozygote or invalid separation plant, species or sample.Control sample or reference sample can also be the sample or the spiked-in sample with the detectable composition of known quantity.

[0064] The recitation of each discrete value recited herein is understood to include the range between each value. The recitation of a range of values ​​recited herein is understood to include the discrete values ​​within that range. Expression vector or isolated DNA molecule, cell or kit containing the same and related methods

[0065] Yet another aspect of the present disclosure includes an expression vector or isolated DNA molecule comprising (i) one or more nucleotide sequences encoding a FUN protein, a FUN-like protein, a HO1 protein, a protein containing an NAC domain (also known as a FEZ protein), a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence; and (ii) a vector or isolated DNA molecule capable of reducing or knocking out the expression of a protein encoding a FUN protein, a FUN-like protein, a HO1 protein, a protein containing an NAC domain (also known as a FEZ protein), a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof. 1 protein, AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence; or (iii) one or more nucleotide sequences comprising a mutation in a gene for FUN protein, FUN-like protein, HO1 protein, NAC domain-containing protein, bZIP28 protein, NRT2.1 protein, NRT3.1 protein, AS1 protein, or a combination thereof, wherein the mutation reduces or knocks out the activity or expression of the protein, and the one or more nucleotide sequences are operably linked to at least one homologous nucleic acid sequence that hybridizes adjacent to the mutation site in the gene. In another embodiment of this aspect, the expression control sequence comprises a nodulation-specific promoter, a root-specific promoter, an inducible promoter, a constitutive promoter, or a combination thereof. In still further embodiments of this aspect, wherein the promoter is a root-specific promoter, and wherein the promoter is selected from the group consisting of: an NFR1 promoter, an NFR5 promoter, a LYK3 promoter, a CERK6 promoter, an NFP promoter, a Lotus japonicus NFR5 promoter (SEQ ID NO:85), a Lotus japonicus NFR1 promoter (SEQ ID NO:89), a Lotus japonicus CERK6 promoter (SEQ ID NO:87), a Medicago truncatula NFP promoter (SEQ ID NO:86), a Medicago truncatula LYK3 promoter (SEQ ID NO:88), a maize metallothionein promoter, a chitinase promoter, a maize ZRP2 promoter, a tomato LeExt1 promoter, a glutamine synthase soybean root promoter, a RCC3 promoter, a rice antiquitin promoter, a LRR receptor kinase promoter, and an Arabidopsis thaliana pCO2 promoter;Or wherein the promoter is a constitutive promoter, and wherein the promoter is selected from the group consisting of a CaMV35S promoter, a derivative of the CaMV35S promoter, a maize ubiquitin promoter, a polyubiquitin promoter, a vein mosaic cassava virus promoter, and an Arabidopsis UBQ10 promoter. In a further embodiment of this aspect, the protein is a FUN protein, and wherein the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82. NO:82, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical proteins; wherein the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 NO:30, SEQ ID NO:80, SEQ ID NO:81 and SEQ ID NO:82;or wherein the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 8 or SEQ ID NO: 9; wherein the protein is a FUN-like protein, and wherein the FUN-like protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a protein selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 83 and SEQ ID NO: 84; wherein the FUN-like protein comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 83 or SEQ ID NO: 84; and / or wherein the protein is an NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to a protein selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 83 or SEQ ID NO: 84; NO:74 has a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity; wherein the protein is a bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:75;wherein the protein is a NAC domain-containing protein, and wherein the NAC domain-containing protein comprises a protein selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:78; ID NO:78 having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:79;wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78; or wherein the AS1 protein comprises SEQ ID NO:79; or wherein the NAC domain-containing protein comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:42, or a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a domain thereof, such as a polypeptide having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to each of the domains recited in Table 1 or a combination thereof.;

[0066] Some aspects of the present disclosure relate to bacterial cells or Agrobacterium cells comprising the expression vector or isolated DNA molecule of any of the preceding embodiments.

[0067] Other aspects of the present disclosure relate to a genetically modified plant, plant part, plant cell, or seed comprising the expression vector or isolated DNA molecule of any of the preceding embodiments.

[0068] A further aspect of the present disclosure relates to a kit comprising the expression vector or isolated DNA molecule of any of the preceding embodiments or the bacterial cell or Agrobacterium cell of the preceding embodiments.

[0069] Still further aspects of the present disclosure relate to methods for increasing nitrogen fixation, delaying nodulation senescence, or inducing FUN filamentation in plants, the methods comprising: (a) introducing a genetic alteration via an expression vector or isolated DNA molecule of any of the preceding embodiments; and optionally (b) treating the plant with zinc or manganese, or growing the plant under high zinc, high manganese, or high nitrate conditions. High manganese, high zinc, or high nitrate conditions can be above the soil environmental conditions for plant growth, above the optimal conditions for plant growth, in any non-natural or non-naturally occurring amount, or at a level that induces or maintains filamentation or aggregation of the FUN protein. In some embodiments, the high level can be 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 250%, or 500% higher than such environmental conditions, such optimal conditions, or such naturally occurring amounts. Plant breeding methods

[0070] Plant breeding begins with an analysis of existing germplasm, identifying problems and weaknesses in the existing germplasm, developing project goals, and defining specific breeding objectives. The next step is to select germplasm with traits that meet the project goals. The selected germplasm is hybridized to recombinantly develop the desired traits and to grow varieties or parental lines through selection. The goal is to integrate improved combinations of desired traits from the parental germplasm into a single variety or hybrid. These important traits can include higher yield, field performance, improved fruit and agronomic quality, resistance to biotic stresses (such as diseases and pests), and tolerance to environmental stresses (such as drought and heat).

[0071] Every breeding program should include regular, objective evaluations of the efficiency of the breeding program. Evaluation criteria will vary depending on goals and objectives but should include annual selection returns based on comparison with appropriate standards, the overall value of advanced breeding lines, and the number of successful cultivars produced per unit of input (e.g., per year, per dollar spent, etc.). Promising advanced breeding lines are thoroughly tested for at least three years in environments representative of the commercial target area and compared to appropriate standards. The best lines are candidates for new commercial cultivars; those lines still lacking certain traits are used as parents to generate new populations for further selection. These processes, which lead to eventual marketing and distribution, typically take five to ten years from the time of the first cross or selection.

[0072] The choice of breeding or selection method depends on the way the plant is propagated, the heritability of the trait to be improved, and the type of cultivar used commercially (e.g., F1 hybrid cultivars, inbred cultivars, etc.). For highly heritable traits, selecting superior individual plants evaluated at a single location will be effective, while for traits with low heritability, selection should be based on average values ​​obtained from repeated evaluations of related plant families. Genetic complexity also affects the choice of breeding method. Backcross breeding is used to transfer one or several genes for highly heritable traits into the desired cultivar (e.g., for breeding disease-resistant cultivars); while recurrent selection techniques are used for quantitative heritable traits controlled by multiple genes, using various recurrent selection techniques. Commonly used selection methods include pedigree selection, improved pedigree selection, mass selection, and recurrent selection.

[0073] Pedigree selection is commonly used to improve inbred lines of self-pollinating crops or cross-pollinating crops. Two parents with good complementary traits are crossed to produce F1. The F2 population is produced by selfing one or several F1s or by crossing two F1s (inbreeding). Selection of the best individuals usually starts with the F2 population; then, starting with F3, the best individuals in the best families are selected. Families or hybrid combinations involving individuals in these families are often tested repeatedly in the F4 generation to improve the selection effect on low heritable traits. In the later stages of inbreeding (i.e., F6 and F7), the best lines or mixtures of phenotypically similar lines are tested for potential promotion as new cultivated varieties.

[0074] Mass selection and recurrent selection can be used to improve populations of self- or cross-pollinated crops. By crossing several different parents, populations of heterozygous individuals with genetic variation are identified or constructed. The best plants are selected based on individual vigor, superior progeny, or excellent combining ability. Selected plants are then crossed to create new populations, where further cycles of selection continue.

[0075] Backcross breeding (i.e., recurrent selection) can be used to transfer genes for simple, highly heritable traits into a desired homozygous cultivar or line that is the recurrent parent. The source of the trait to be transferred is called the donor parent. The resulting plants are expected to have the properties of the recurrent parent (e.g., cultivar) and the desired traits transferred from the donor parent. After the initial hybridization, individuals with the donor parent phenotype are selected and repeatedly hybridized (backcrossed) with the recurrent parent. The resulting plants are expected to have the properties of the recurrent parent (e.g., cultivar) and the desired traits transferred from the donor parent.

[0076] Strictly speaking, the single-seed descent procedure refers to planting a separate population, harvesting a sample of one seed from each plant, and using that seed sample to plant the next generation. As the population develops from the F2 to the desired level of inbreeding, the plants that derive the line will each be traced back to a different F2 individual. Because some seeds fail to germinate or some plants fail to produce at least one seed, the number of plants in the population decreases with each generation. Therefore, when the generations are completed, not all of the F2 plants originally sampled in the population are represented by the progeny.

[0077] In addition to phenotypic observation, the genotype of a plant can also be examined. There are many laboratory-based techniques that can be used to analyze, compare, and characterize plant genotypes; among them are isozyme electrophoresis, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), arbitrarily primed polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), sequence-characterized amplified regions (SCARs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs, also known as microsatellites), fluorescently labeled inter-simple sequence repeats (ISSRs), single nucleotide polymorphisms (SNPs), genotyping by sequencing (GbS), and next-generation sequencing (NGS).

[0078] Molecular markers or "markers" can also be used for qualitative trait selection in the breeding process. For example, markers that are tightly linked to an allele or that contain sequences within the actual allele of interest can be used to select plants containing the allele of interest. The use of markers in the selection process is generally referred to as genetic marker-enhanced selection or marker-assisted selection. Methods for carrying out marker analysis are generally known to those skilled in the art.

[0079] Mutation breeding can also be used to introduce new traits into plant varieties. Spontaneous or artificially induced mutations can provide useful sources of variation for plant breeders. The goal of artificial mutagenesis is to increase the mutation rate of the desired characteristic. The mutation rate can be increased in a variety of different ways, including: temperature, long-term seed storage, tissue culture conditions, radiation (such as X-rays, gamma rays, neutrons, beta radiation or ultraviolet radiation), chemical mutagens (such as base analogs, such as 5-bromo-uracil), antibiotics, alkylating agents (such as sulfur mustard, nitrogen mustard, epoxides, ethyleneamines (ethyleneamines), sulfates, sulfonates, sulfones or lactones), azides, hydroxylamines, nitrous acid or acridines. Once the desired trait is observed through mutagenesis, the trait can then be integrated into the existing germplasm through traditional breeding techniques. Detailed information on mutation breeding can be found in Principles of Cultivar Development: Theory and Technique, Walter Fehr (1991), Agronomy Books, 1( https: / / lib.dr.iastate.edu / agron_books / 1 ) is found in

[0080] The production of doubled haploids can also be used to develop homozygous lines in breeding programs. Doubled haploids are produced by doubling one set of chromosomes of a heterozygous plant, thereby producing completely homozygous individuals. For example, see Wan et al., Theor. Appl. Genet., 77:889-892, 1989.

[0081] Additional non-limiting examples of breeding methods that can be used include, but are not limited to, those described in Principles of Plant Breeding, John Wiley and Son, pp. 115-161 (1960); Principles of Cultivar Development: Theory and Technique, Walter Fehr (1991), Agronomy Books, 1 ( https: / / lib.dr.iastate.edu / agron_books / 1 ) (which is incorporated herein by reference). Gene modification Methods in molecular biology of plant cells, plant parts and plants

[0082] One aspect of the present disclosure provides genetically altered or modified plants or parts thereof comprising one or more genetic alterations that result in reduced activity or expression of a FUN protein. Additional aspects of the present disclosure include genetically modified plants or parts thereof comprising one or more genetic alterations that result in reduced activity or expression of one or more of an NRT3.1 protein, a bZIP28 protein, an NAC domain-containing protein (also known as a FEZ protein), a HO1 protein, an NRT2.1 protein, or an AS1 protein.

[0083] FUN is a member of the TGA transcription factor family. TGA transcription factors can be characterized by a DNA binding bZIP domain at the N-terminus, a DOG1 domain at the C-terminus ( Gruden, K. & Coll, A. TGA transcription factors-Structural characteristics as basis for functional variability. Front. Plant Sci. 13, 935819 (2022)). The present disclosure redefines the DOG1 domain as a sensor domain ( Figure 1P ) (e.g., in Lotus japonicus FUN (SEQ ID NO: 1)), this domain senses zinc. FUN is highly conserved in legumes, as demonstrated by the fact that all legumes analyzed carry FUN proteins and FUN-like paralogs in the PAN orthogroup ( Figure 6A ). Exemplary FUN homologs include SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30. Exemplary FUN-like homologs include SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84. Additional TGA transcription factors related to FUN proteins are FUN-like proteins. FUN-like proteins can be distinguished from FUN proteins by the lack of enhanced expression in nodules (there may be some expression in roots (potentially including nodules)) and the fact that they form independent paralogous branches on the phylogenetic tree.

[0084] Lotus japonicus NAC094 (SEQ ID NO: 31; also known as FEZ protein) is a downstream target of FUN ( Figure 7 ). Exemplary NAC094 homologues include SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73.

[0085] The transformation and generation of genetically altered monocot and dicot cells are well known in the art. See, for example, Weising et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Patent 5,679,558; Agrobacterium Protocols, ed: Gartland, Humana Press Inc. (1995); Wang et al., Acta Hort. 461:401-408 (1998); and Broothaerts et al., Nature 433:629-633 (2005). The choice of method varies depending on the type of plant to be transformed, the specific application, and / or the desired outcome. One skilled in the art can readily select an appropriate transformation technique.

[0086] Any method for deleting, inserting or otherwise modifying cellular DNA (for example, genomic DNA and organelle DNA) known in the art can be used for practicing compositions, methods and techniques disclosed herein.For example, by, for example, removing repressor binding sites or introducing enhancer binding sites, CRISPR / Cas-9 systems and related systems (for example, TALEN, ZFN, ODN etc.) can be used for inserting heterologous genes into the target site in genomic DNA, or substantial editing endogenous genes to express heterologous genes, or modifying promoters to increase or otherwise change the expression of endogenous genes.For example, the disarmament Ti plasmid (which contains a gene construct for deleting or inserting a target gene) in Agrobacterium tumefaciens can be used for transforming plant cells, and then, the program described in the art (for example EP 0116718, EP 0270822, PCT open WO 84 / 02913 and published European patent application (" EP") 0242246) can be used to regenerate the transformed plant cells from the transformed plant cells. Each Ti plasmid vector contains a gene between the border sequences of the T-DNA of the Ti plasmid or at least to the left of the right border sequence. Of course, other types of vectors can also be used to transform plant cells using procedures such as direct gene transfer (e.g., as described in EP 0 233 247), pollen-mediated transformation (e.g., as described in EP 0 270 356, PCT Publication WO 85 / 01856, and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (e.g., as described in EP 0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (e.g., as described in U.S. Pat. No. 4,536,475), and other methods, such as those used to transform certain maize lines (e.g., U.S. Pat. No. 6,140,553; Fromm et al., Bio / Technology (1990) 8, 833-839; Gordon-Kamm et al., The Plant For cotton transformation, the method described in PCT Patent Publication WO 00 / 71733 can be used. For soybean transformation, reference can be made to methods known in the art, for example, the methods of Hinchee et al. (Bio / Technology, (1988) 6, 915) and Christou et al. (Trends Biotech, (1990) 8, 145) or WO 00 / 42207.

[0087] The genetically modified plants of the present disclosure can be used in conventional plant breeding programs to produce more genetically modified plants with the same characteristics, or genetically modified plants are introduced into other varieties in the same or related plant species. The seeds obtained from the modified plants preferably include genetic modifications, as stable insertions in chromosomal DNA or modifications to endogenous genes or promoters. Plants comprising genetic modifications according to the present disclosure include plants comprising or derived from root stocks of plants (such as fruit trees or ornamental plants) comprising genetic modifications of the present disclosure. Therefore, any non-transgenic grafted plant parts inserted into transformed plants or plant parts are included in the present disclosure.

[0088] The genetic alterations of the present disclosure (included in expression vectors or expression cassettes) that result in expression of the introduced gene or altered endogenous gene expression will typically utilize a plant-expressible promoter. As used herein, a "plant-expressible promoter" refers to a promoter that ensures expression of the genetic alterations of the present disclosure in plant cells. Examples of constitutive promoters frequently used in plant cells are the cauliflower mosaic virus (CaMV) 35S promoter (Kay et al., Science, 236, 4805, 1987), the minimal CaMV 35S promoter (Benfey & Chua, Science, (1990) 250, 959-966), various other derivatives of the CaMV 35S promoter, the figwort mosaic virus (FMV) promoter (Richins et al., Nucleic Acids Res. (1987) 15:8451-8466), the maize ubiquitin promoter (Christensen & Quail, Transgenic Res, 5, 213-8, 1996), the polyubiquitin promoter (Ljubql, Maekawa et al., Mol Plant Microbe Interact. 21, 375-82, 2008), the vein mosaic virus promoter (International Application WO 20080001), the cassava mosaic virus promoter (International Application WO 200800104 ... 97 / 48819) and the Arabidopsis UBQ10 promoter (Norris et al., Plant Mol. Biol. 21, 895-906, 1993).

[0089] Additional examples of promoters that direct constitutive expression in plants are known in the art and include: the strong constitutive 35S promoter ("35S promoter") of, for example, cauliflower mosaic virus (CaMV) isolates CM 1841 (Gardner et al., Nucleic Acids Res, (1981) 9, 2871-2887), CabbB S (Franck et al., Cell (1980) 21, 285-294), and CabbB JI (Hull and Howell, Virology, (1987) 86, 482-493); promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., Plant Mol Biol, (1992) 18, 675-689); the gos2 promoter (de Pater et al., The Plant J (1992) 2, 834-844); the emu promoter (Last et al., Theor Appl Genet, (1990) 81, 581-588); actin promoters such as those described by An et al. (The Plant J, (1996) 10, 107), the rice actin promoter described by Zhang et al. (The Plant Cell, (1991) 3, 1155-1165); the promoter of figwort mosaic virus (FMV) (Richins et al., Nucleic Acids Res. (1987) 15:8451-8466); the promoter of cassava vein mosaic virus (WO 97 / 48819; Verdaguer et al., Plant Mol Biol, (1998) 37, 1055-1067); the pPLEX series of promoters from subterranean clover stunt virus (WO 96 / 06932, in particular the S4 or S7 promoter); alcohol dehydrogenase promoters, such as pAdh1S (GenBank Accession Nos. X04049, X00581); and the TR1' promoter and TR2' promoter that drive gene expression from the 1' and 2' ends of the TDNA, respectively ("TR1' promoter" and "TR2' promoter," respectively) (Velten et al., EMBO J, (1984) 3, 2723-2730).

[0090] In another embodiment, the plant-expressible promoter can be a tissue-specific promoter, i.e., a promoter that directs higher levels of expression in some cells or tissues (e.g., root epidermal cells or root cortical cells) of the plant. In a preferred embodiment, a LysM receptor promoter will be used. Non-limiting examples include NFR1 promoter, NFR5 promoter, LYK3 promoter, NFP promoter, Lotus japonicus NFR5 promoter (SEQ ID NO:27), Lotus japonicus NFR1 promoter (SEQ ID NO:27), Medicago truncatula NFP promoter (SEQ ID NO:29), Lotus japonicus CERK6 promoter (SEQ ID NO:46), and Medicago truncatula LYK3 promoter (SEQ ID NO:28). In another preferred embodiment, a root-specific promoter will be used. Non-limiting examples include the maize metallothionein promoter (De Framond et al., FEBS 290, 103.-106, 1991; application EP 452269), the chitinase promoter (Samac et al., Plant Physiol 93, 907-914, 1990), the glutamine synthase soybean root promoter (Hirel et al., Plant Mol. Biol. 20, 207-218, 1992), the RCC3 promoter (PCT application WO 2009 / 016104), the rice antiquitin promoter (PCT application WO 2007 / 076115), the LRR receptor kinase promoter (PCT application WO 02 / 46439), the maize ZRP2 promoter (U.S. Pat. No. 5,633,363), the tomato LeExt1 promoter (Bucher et al., Plant Physiol 93, 907-914, 1990), the soybean root promoter (Hirel et al., Plant Mol. Biol. 20, 207-218, 1992), the RCC3 promoter (PCT application WO 2009 / 016104), the rice antiquitin promoter (PCT application WO 2007 / 076115), the LRR receptor kinase promoter (PCT application WO 02 / 46439), the maize ZRP2 promoter (U.S. Pat. No. 5,633,363), the tomato LeExt1 promoter (Bucher et al., Plant Physiol. 128, 911-923, 2002) and the Arabidopsis pCO2 promoter (Heidstra et al., Genes Dev. 18, 1964-1969, 2004). These plant promoters can be combined with enhancer elements, they can be combined with minimal promoter elements, or they can contain repeat elements to ensure the desired expression profile.

[0091] Examples of constitutive promoters frequently used in plant cells are the cauliflower mosaic virus (CaMV) 35S promoter (Kay et al., Science, 236, 4805, 1987) and various derivatives of this promoter, the cassava vein mosaic virus promoter (International Application WO 97 / 48819), the maize ubiquitin promoter (Christensen and Quail, Transgenic Res, 5, 213-8, 1996), polyubiquitin (Ljubql, Maekawa et al., Mol Plant Microbe Interact. 21, 375-82, 2008) and Arabidopsis UBQ10 (Norris et al., Plant Mol. Biol. 21, 895-906, 1993).

[0092] In some embodiments, further genetic alterations can be utilized to increase expression in plant cells. For example, introns at the 5' end or 3' end of the introduced gene, or introns in the coding sequence of the introduced gene, such as the hsp70 intron. Other such genetic elements may include, but are not limited to, promoter enhancer elements, two or three promoter regions, 5' leader sequences that are different from other transgenic or from endogenous (plant host) gene leader sequences, 3' trailing sequences that are different from other transgenic or from endogenous (plant host) trailing sequences used in the same plant.

[0093] The disclosed introduced gene can be inserted into the host cell DNA so that the inserted gene portion is located upstream (i.e. 5') of a suitable 3' end transcriptional regulatory signal (i.e., transcript formation and polyadenylation signal). This is preferably achieved by inserting the gene into the plant cell genome (nucleus or chloroplast). Preferred polyadenylation and transcript formation signals include nopaline synthase gene (Depicker et al., J. Molec Appl Gen, (1982) 1, 561-573), octopine synthase gene (Gielen et al., EMBO J, (1984) 3: 835-845), SCSV or malic enzyme terminator (Schunmann et al., Plant Funct Biol, (2003) 30: 453-460) and T DNA gene 7 (Velten and Schell, Nucleic Acids Res, (1985) 13, 6981-6998), which serve as 3' non-translated DNA sequences in transformed plant cells. In some embodiments, one or more introduced genes are stably integrated into the nuclear genome. When the nucleic acid sequence remains integrated into the nuclear genome and continues to express (i.e., produce detectable mRNA transcripts or proteins) throughout subsequent plant generations, there is stable integration. Stable integration into the nuclear genome can be achieved by any known method in the art (e.g., microprojectile bombardment, Agrobacterium-mediated transformation, CRISPR / Cas9, protoplast electroporation, microinjection, etc.).

[0094] The term recombinant or modified nucleic acid refers to a polynucleotide prepared by combining two otherwise separate sequence segments, which is achieved by artificial manipulation of separate polynucleotide segments through genetic engineering techniques or by chemical synthesis. By doing so, one can link together polynucleotide segments with desired functions, thereby producing a desired functional combination.

[0095] As used herein, the term "overexpression" refers to an increase in expression (e.g., mRNA, polypeptide, etc.) relative to that of a wild-type organism (e.g., a plant) as a result of genetic modification, and may refer to an expression of a heterologous gene at a sufficient level to achieve a desired result such as an increase in yield. In some embodiments, the increase in expression is a slight increase of about 10% higher than that in the wild-type. In some embodiments, the increase in expression is an increase of 50% or more (e.g., 60%, 70%, 80%, 100%, etc.) relative to that in the wild-type. In some embodiments, an endogenous gene is upregulated. In some embodiments, an exogenous gene is upregulated due to being expressed. Upregulation of genes in plants can be achieved by any method known in the art, including but not limited to the use of constitutive promoters to which inducible response elements have been added, inducible promoters, high expression promoters to which inducible response elements have been added (e.g., the PsaD promoter), enhancers, transcriptional and / or translational regulatory sequences, codon optimization, modified transcription factors, and / or mutated or modified genes that control expression of genes that are upregulated in response to stimuli such as cytokinin signaling.

[0096] When recombinant nucleic acid is intended to express, clone or replicate a specific sequence, the DNA construct prepared for introduction into the host cell will generally comprise a replication system (e.g., a vector) of an expected DNA fragment comprising the desired polypeptide encoded by the host identification, and may also comprise a transcription and translation initiation regulatory sequence operably connected to the polypeptide coding segment. In addition, such constructs may comprise cell localization signals (e.g., plasma membrane localization signals). In a preferred embodiment, such DNA constructs are introduced into the genomic DNA, chloroplast DNA, or mitochondrial DNA of the host cell.

[0097] In some embodiments, a non-integrating expression system can be used to induce expression of one or more introduced genes. The expression system (expression vector) may include, for example, an origin of replication or an autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, transcription terminator sequences, and mRNA stabilizing sequences. Where appropriate, a signal peptide of a secretory polypeptide from the same or related species may also be included, which allows the protein to pass through the cell membrane, cell wall, and / or remain in the cell membrane, cell wall, or be secreted from the cell.

[0098] The selectable marker for practicing the method disclosed herein can be a positive selectable marker. Usually, positive selection refers to the situation that only the recombinant polynucleotide of interest is present in the cell, and the cell of genetic modification can survive in the presence of toxic substances. Negative selection markers and screenable markers are also well known in the art and are considered by the present disclosure. Those skilled in the art will recognize that any available related markers can be used in practicing the compositions, methods and techniques disclosed herein.

[0099] Screening and molecular analysis of the recombinant strains disclosed herein can be performed using nucleic acid hybridization techniques. Hybridization procedures can be used to identify polynucleotides, such as polynucleotides modified using the techniques described herein, which have sufficient homology to the target regulatory sequences taught herein. Specific hybridization techniques are not crucial to the present disclosure. As hybridization techniques improve, those skilled in the art can readily apply hybridization techniques. Hybridization probes can be labeled with any suitable marker known to those skilled in the art. Hybridization conditions and washing conditions, such as temperature and salt concentration, can be varied to change the stringency of the detection threshold. For further guidance on hybridization conditions, see, for example, Sambrook et al. (1989) (see below) or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY).

[0100] In addition, the polymerase chain reaction (PCR) can be used to screen and molecularly analyze genetically altered strains, as well as to create desired isolated nucleic acids. PCR is a repetitive, enzymatic, primer-based method for synthesizing nucleic acid sequences. This procedure is well known and commonly used by those skilled in the art (see Mullis, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science 230:1350-1354). PCR is based on the enzymatic amplification of a DNA fragment of interest, which is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with their 3' ends facing each other. Repeated cycles of thermal denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase result in the amplification of the segment defined by the 5' end of the PCR primer. Since the extension product of each primer can serve as a template for another primer, each cycle actually doubles the amount of DNA template generated in the previous cycle. This results in an exponential accumulation of specific target fragments, reaching millions of times within a few hours. The amplification process can be fully automated by using a thermostable DNA polymerase such as Taq polymerase isolated from the thermophilic bacterium Thermus aquaticus. Other useful enzymes are known to those skilled in the art.

[0101] Nucleic acids and proteins disclosed herein can also encompass homologs of specifically disclosed sequences. Homology (e.g., sequence identity) can be 50%-100%. In some cases, such homology is greater than 80%, greater than 85%, greater than 90% or greater than 95%. Those skilled in the art can easily determine the homology or identity degree required for any intended use of a sequence. As used herein, the sequence identity percentages of two nucleic acids are determined using algorithms known in the art, such as those disclosed in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268 (which is modified as Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877). Such algorithms have been incorporated into the BLASTN, BLASTP, and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches are performed using the BLASTN program (score = 100, word length = 12) to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (BLASTN and BLASTX) are used. See www.ncbi.nih.gov. One skilled in the art can readily determine positions in a sequence of interest that correspond to amino acids or nucleic acids in a reference sequence by aligning the sequence of interest with the reference sequence using an appropriate BLAST program with default settings (e.g., for BLASTP: Gap Open Penalty: 11, Gap Extension Penalty: 1, Expectation: 10, Word Length: 3, Maximum Score: 25, Maximum Alignments: 15, and Matrix: blosum62; for BLASTN: Gap Open Penalty: 5, Gap Extension Penalty: 2, Nucleic Acid Matches: 1, Nucleic Acid Mismatches -3, Expectation: 10, Word Length: 11, Maximum Score: 25, and Maximum Alignments: 15).

[0102] Preferred host cells are plant cells. In this article, recombinant host cells refer to cells that have been genetically modified to contain isolated nucleic acid molecules, one or more deleted or otherwise non-functional genes that normally exist and function in the host cell, or one or more genes for producing at least one recombinant protein. Nucleic acids encoding proteins of the present disclosure can be introduced by any method known in the art suitable for use in specific cell types (including but not limited to transformation, lipofection, electroporation, or any other method known to those skilled in the art).

[0103] "Isolated," "isolated DNA molecule," or equivalent terms or phrases are intended to refer to a DNA molecule or other portion that exists alone or in combination with other components, but is different from or not in its natural environment. For example, nucleic acid elements naturally found in the DNA of an organism's genome (such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc.) are not considered "isolated" as long as the elements are within the genome of the organism and are located at the position within the genome in which they are naturally found. However, within the scope of the present disclosure, each of these elements and subparts of these elements will be "isolated" from their natural environment as long as the elements are not within the genome of the organism in which they are naturally found, the elements are different from their natural form, or the elements are not located at the position within the genome in which they are naturally found. Similarly, a nucleotide sequence encoding a protein or any naturally occurring variant of the protein will be an isolated nucleotide sequence as long as the nucleotide sequence encoding the protein is not in the DNA of the organism in which the sequence is naturally found in its natural location, or if the nucleotide sequence is different from its natural form. For the purposes of the present disclosure, a synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring protein will be considered isolated. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence that is inserted into the genome of a cell of a plant, algae, fungus, or bacterium, or that is present in DNA on an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether present in a plasmid or similar construct used to transform a cell, in the genome of a plant or bacterium, or in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from a plant or bacterium. plant

[0104] Compositions as described herein and methods can be used for plant, plant cell, plant part or its progeny, such as can form nodulation and have the plant (for example, leguminous plant) of endogenous FUN.Leguminous plant is the plant belonging to Fabaceae (Leguminosae) family, and feature can be that they fix the ability of nitrogen in soil by the symbiotic relationship of the nitrogen-fixing bacteria in its root nodule.Therefore, plant as described herein, plant cell, plant part or its progeny can be selected from following group: clover, Bambara peanut, beans (for example, kidney bean, black bean etc.), blackcurrant, chickpea, clover, cowpea, fodder legume, leguminous trees, lentil, lotus plant, lupin, Medicago species, pea, peanut, pigeon pea, soybean, Sophora flavescens, alder or elm.

[0105] Plants for which FUN orthologs have been identified may include Prunus persica (peach), Lotus japonica (e.g., Japanese lotus or bird's-foot trefoil), Glycine max (soybean), Manihot esculenta (cassava), Gossypium raimondii (wild cotton), Eucalyptus grandis (e.g., Eucalyptus grandis or Eucalyptus rosea), Brassica oleracea (e.g., wild cabbage, species may include various cultivated forms such as broccoli, cauliflower, cabbage, etc.), Arabidopsis thaliana (Shepherd's purse), Solanum lycopersicum (tomato), Aquilegia coerulea (Colorado blue columbine), Amborella trichopoda, Spirodela oleracea (Spirodela oleracea). polyrhiza (purple-backed duckweed), Musa acuminata (banana), Zeamays (maize, corn), Setaria italica (millet), Triticum aestivum (common wheat), Hordeum vulgare (barley), or Oryza sativa (rice).

[0106] Cover crops and combinations of cover crops can be used to add nitrogen to the soil and can benefit from reducing the expression of FUN or its downstream targets by increasing nitrogen content. For example, the cover crop can be a legume. In some embodiments, the legume can be soybean, cowpea, clover (e.g., red clover, white clover, crimson clover, balansa clover, Egyptian clover, Bersian clover, arrowleaf clover, ball clover, underground clover), vetch (e.g., common vetch, hairy vetch) or pea (e.g., Austrian winter pea, field pea). For another example, the cover crop can be a grass. In some embodiments, the grass can be rye (e.g., winter rye, cereal rye, Italian ryegrass), triticale, fescue (e.g., tall fescue, oxtail grass), sudangrass (e.g., sudangrass, sorghum-sudangrass hybrid) or alfalfa. For another example, the cover crop can be Brassica. In some embodiments, the Brassica can be mustard (e.g., white mustard), radish (e.g., white radish, oilseed radish), turnip (e.g., purple-top turnip, fodder turnip), or rapeseed (e.g., canola), campestris, sunflower, sunflower, kale, or a cereal (e.g., oats, buckwheat, or millet (e.g., pearl millet)).

[0107] Having generally described the compositions, methods, and processes of the present disclosure, the same will be better understood by reference to certain specific examples, which are included herein to further illustrate the disclosure and are not intended to limit the scope of the invention as defined by the claims. Example

[0108] The following examples further describe the present disclosure in detail, but these examples are not intended to limit the scope of the present disclosure in any way. The accompanying drawings should be considered an integral part of the specification and description of the present disclosure. The following examples are for illustration only and are not intended to limit the claimed disclosure. Example 1: FUN Controlled Nitrogen Fixation by Nitrate Repression

[0109] The following examples describe the identification of the Fun gene as a regulator of nitrogen fixation in Lotus japonicus. Further, experiments are described to characterize the expression pattern of the Fun gene. Materials and methods Mutagenesis screening

[0110] Use lotus root Gifu ecotype background, for forward gene screening, to identify despite restrictive nitrate conditions, but maintain nitrogen fixation mutants. Before applying restrictive nitrate conditions, allow functional nodulation to form, which means that the screening specifically identifies mutants whose nodulation function is impaired. Utilize the unique color of nitrogen-fixing nodules (functional nodules are pink) to screen out mutants that maintain nodulation function when watered for two weeks with water containing 10mM KNO . Under these growth conditions, most nodules on wild-type plants turn green and age, but fixation under nitrate (fun) mutant plants also continue to form pink nodules even under these high concentrations of nitrate. Plant strains and growing conditions

[0111] The Gifu ecotype of Lotus japonicus was used as the wild type (WT). LORE1 insertion mutants were sorted by LotusBase (lotus[dot]au[dot]dk), and homozygotes were isolated for phenotypic analysis, and higher-order mutants were generated as described in (Emms, DM & Kelly, S. SHOOT: phylogenetic gene search and orthologinference. Genome Biol. 23, 85 (2022). Mutant strains fun, fun-2, fun-3, and fun-4 were tested, and the specific insertions that generated the mutant genotypes were Figure 1B The line numbers and genotyping primers used are provided in Table 2 below. Table 2. Strain numbers and genotyping primers

[0112] All plants were grown at 21°C under 16-hour light / 8-hour dark conditions. For germination, Nelumbo nucifera seeds were scratched with sandpaper and surface-sterilized with 1% sodium hypochlorite for 10 minutes. Seedlings were washed five times with sterile water and germinated in an upright position on moist filter paper (AGF 651; Frisenette ApS) in sterile square Petri dishes at 21°C for two days. The seedlings were then transplanted into a substrate mixture (ceramsite:vermiculite = 3:1). Mutant screening and sequence analysis

[0113] A pool of LORE1 mutants, each with a random LORE1 insertion in its genome, was germinated in a mixed matrix (ceramsite:vermiculite 3:1) and inoculated with Mesorhizobium loti (M. loti) NZP2235. Four weeks after inoculation, the plants were watered with 10 mM KNO3 for three weeks. Most nodules turned green or black, and plants with pink nodules were isolated for rescreening in subsequent generations. DNA was isolated from mutant plants, and the LORE1 flanking sequences were sequenced to identify the LORE1 insertion sites, as previously described (Urbański, DF, et al., 2012). A., Stougaard, J. & Andersen, SUGenome-wide LORE1 retrotransposon mutagenesis and high-throughputinsertion detection in Lotus japonicus. Plant J. 69, 731–741 (2012)). Bacterial strains and culture conditions

[0114] Chemically competent Escherichia coli TOP10 (ThermoFisher Scientific) was used for molecular cloning and grown in LB medium at 37°C.

[0115] Agrobacterium rhizogenes strain AR1193 (Stougaard, J. Methods Mol Biol 1995 49:49-61) was used for all hairy root transformation experiments and cultured in LB medium at 28°C. Generation of plant expression vectors

[0116] To verify the function of the Fun gene, expression constructs were generated to express FUN fused to green fluorescent protein (GFP) under the control of the ubiquitin promoter (pUbi), and the constructs were named pUbi:FUN, proUbi:FUN-GFP, or FUN-GFP. For tobacco assays, the 35S promoter (pro35S) was used, and the constructs were named pro35S:FUN, pro35S:FUN-GFP, or FUN-GF.

[0117] To investigate the expression pattern of the Fun gene in situ, the coding sequence of β-glucuronidase (GUS) was assembled with the native Fun promoter sequence (proFUN) and the native Fun terminator sequence (tFUN), and the construct was named proFun:GUS. Hairy root transformation

[0118] To transform hairy roots of Lotus japonicus, the pIV10 expression vector (Hansen, J. et al., Plant Cell Rep 1989 8:12-15) was used. This expression vector contains sequences encoding a triple YFP fused to a nuclear localization signal (pIV10_tYFP-NLS), which served as a transformation control. In addition, the Nelumbo nucifera ubiquitin promoter and 35S terminator were cloned into the pIV10 expression vector.

[0119] Lotus japonicus seeds were scratched with sulfuric acid for 15 minutes, washed 5 times in ddH2O, and then spread on wet filter paper for germination. The 3-day-old seedlings were transferred to a square plate containing solid 1 / 2B5 medium. The hairy rhizobium AR1193 strain (Stougaard, 1987#432) carrying the construct of interest was grown on LB agar containing ampicillin, rifampicin and spectinomycin for two days. For each construct, the cells grown on a plate were resuspended in 4ml YMB medium. Then, using a needle ( The bacterial suspension was used to transform the hypocotyls of 6-day-old seedlings using a 1ml injector (0.40x20mm). A hole was punched in the hypocotyl and a drop was placed on the wound. The square plate containing the transformed seedlings was sealed and placed in the dark for two days, and then moved to 21°C, 16 / 8 hours light / dark conditions. After three weeks, the untransformed roots were removed and the seedlings were transferred to the above-mentioned matrix mixture or to 1 / 4×B&D plates. After transformation, as described above, the plants were inoculated with rhizobia and watered with nitrate. All plants were grown at 21°C, 16 / 8 hours light / dark conditions. Nodulation assay

[0120] After 2 weeks of exposure to 10 mM KNO₃, the number of pink functional nodules and total nodules per plant was counted. Images were taken using a Leica M165FC fluorescence stereomicroscope equipped with a Leica DFC310 FX digital color camera. Mean values ​​between treatment groups were compared using analysis of variance and Tukey's post hoc test. Nitrogen fixation determination

[0121] Nitrogen fixation activity was quantified using the acetylene reduction assay (ARA), which measures the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour per lotus plant after 2 weeks of 10 mM KNO3 exposure, as previously described (Reid, DE, Heckmann, AB, Novák, O., Kelly, S. & Stougaard, J. CYTOKININ OXIDASE / DEHYDROGENASE3 maintains cytokinin homeostasis during root and nodule development in Lotus japonicus. Plant Physiol. 170, 1060–1074 (2016)). Nodulated roots of individual plants were placed in 5 ml glass GC vials. 500 μl of air in the vial was replaced with 2% acetylene using an injector. Samples were incubated at room temperature for 30 min, and ethylene was quantified using a SensorSense (Nijmegen, The Netherlands) ETD-300 ethylene detector operated in sampling mode with a flow rate of 2.5 L / h and a detection time of 6 min. Curves were integrated using the SensorSense valve controller software to calculate the total ethylene production for each sample. Mean values ​​between treatment groups were compared using analysis of variance and Tukey's post hoc test. Determination of leghemoglobin content

[0122] Leghemoglobin content was determined after 2 weeks of 10 mM KNO3 exposure as previously described (Du, M., Gao, Z., Li, X., and Liao, H. (2020). Excess nitrate induces nodule greening and reduces transcript and protein expression levels of soybean leghemoglobin. Ann. Bot. 126:61–72). Fresh nodules from each individual plant were first ground and homogenized in 16 volumes of 0.1 M pre-chilled PBS (Na2HPO4-NaH2PO4 buffer, pH 6.8 at 5°C). The resulting slurry was then centrifuged at 12,000 g for 15 min, and the supernatant was measured spectrophotometrically at wavelengths of 540, 520, and 560 nm. Bovine Hb was used as a protein standard, and leghemoglobin content was calculated from the standard curve. Mean values ​​between treatment groups were compared using analysis of variance and Tukey's post hoc test. GUS staining

[0123] Three weeks after inoculation, hairy roots were placed in GUS staining buffer containing 0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc), 100 mM potassium phosphate buffer (pH 7.0), 10 mM EDTA (pH 8.0), 1 mM potassium ferrocyanide, 1 mM potassium ferrocyanide, and 0.1% Triton X-10. Roots were incubated overnight at 37°C. Roots were washed twice with 70% ethanol before image acquisition. Statistical analysis

[0124] Means between treatment groups were compared using analysis of variance and Tukey's post hoc test. result FUN controls repression of nitrogen fixation by nitrate

[0125] To identify environmental regulators of nodulation, it was hypothesized that mutants with specific impairments in regulating nodulation function could be identified by imposing restrictive conditions after functional nodules were formed. LORE1 in the model legume Lotus japonicus (Lotus) was targeted using the unique pink, rather than green, senescent nodules of nitrogen-fixing nodules (Fukai, E. et al., Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1. Plant J. 69, 720-730 (2012); Urbanski, DF, et al., ... A., Stougaard, J. & Andersen, SUGenome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus. Plant J. 69, 731-741 (2012); A. et al., The LORE1 insertion mutant resource. Plant J. 88, 306-317 (2016)) were screened to identify genotypes that retained nodulation function despite nitrate repression. The mutants were identified as retaining a higher number of pink nodules relative to the wild type (WT) and were named fixation under nitrate (fun) ( Figure 1A and 1C Other LORE1 insertion mutants were also identified ( Figure 1BWhen the number of functional pink nodules per plant and the total number of nodules in the fun mutant Nelumbo nucifera plants were determined and compared with WT (Gifu) Nelumbo nucifera plants, it was found that the number of pink nodules retained by fun, fun-2, fun-3, and fun-4 plants was significantly higher than that of WT ( Figure 1C and 1F ).

[0126] The functionality of these pink nodules was confirmed by increased nitrogen fixation rates when measured by the acetylene reduction assay (ARA). Figure 1D 、 1E and 1G). In addition, fun mutant Nelumbo nucifera plants had increased leghemoglobin content compared to WT Nelumbo nucifera plants ( Figure 1K Analysis of the abundance of FUN transcripts in the available expression profile of Gifu (Kamal et al., DNA Res. 7 (2020)) revealed that FUN activity is highly upregulated in nodules where nitrogen fixation occurs ( Figure 1Q ). FUN protein structure

[0127] A LORE1 retrotransposon insertion was identified in the promoter region of a bZIP-type transcription factor (which was named FUN as described above). Figure 1B As shown, additional LORE1 insertions were subsequently identified in the promoter and gene regions of FUN. The FUN gene encodes a protein of the TGA family transcription factor, which is most similar to the Arabidopsis PERIANTHIA (PAN) transcription factor (Running, MP & Meyerowitz, E.M. Mutations in the PERIANTHIA gene of Arabidopsis specifically alter floral organ number and initiation pattern. Development 122, 1261–1269 (1996); Maier, A.T., Stehling-Sun, S., Offenburger, S.-L. & Lohmann, J.U. The bZIP Transcription Factor PERIANTHIA: A Multifunctional Hub for Meristem Control. Front. Plant Sci. 2, 79 (2011)). The TGA family belongs to the D group bZIP transcription factor ( -Laser, W., Snoek, BL, Snel, B. & Weiste, C. The Arabidopsis bZIP transcriptionfactor family-an update. Curr. Opin. Plant Biol. 45, 36-49 (2018)), and is characterized by the presence of a basic leucine zipper (bZIP) DNA binding domain in the N-terminus and a DOG1 domain of unknown function in the C-terminus ( Gruden, K. & Coll, A. TGA transcription factors-Structural characteristics as basis for functional variability. Front. Plant Sci. 13, 935819 (2022)). As described in more detail below, the DOG1 domain is called the sensor domain of FUN. The protein structure of FUN is as follows Figure 1P Shown, it depicts the bZIP domain and the sensor domain. Fun is specifically expressed in nodules

[0128] In Nelumbo nucifera, Fun transcripts were detected at high levels in nodules ( Figure 1Q ), and promoter activity was evident in nodules ( Figure 1M 、 1N To evaluate Fun expression in situ, a GUS transcriptional reporter gene construct driven by the native Fun promoter was transformed into wild-type (Gifu) Nelumbo plants ( Figures 1M-1O After GUS staining of nodule-bearing Nelumbo roots, it was found that Fun was only expressed in nodules ( Figure 1M The stained nodules were sectioned and imaged using an optical microscope to determine the cellular localization of Fun expression. This showed that Fun was expressed primarily in uninfected cells within the nodules ( Figures 1N-1O ). Complementation of the fun mutant in Nelumbo nucifera plants

[0129] By complementing the fun mutation with constitutively expressed FUN ( Figure 1L ) and by confirming that the nodulation phenotype is consistent in three independent LORE1 mutant alleles that reduce gene expression via promoter insertions ( fun and fun-4 ) or disrupt function via exon insertions ( fun-3 ). Figure 1A and 1E -1G), thus verifying that FUN is the causative gene. The intron insertion allele (fun-2) is not damaged compared to the wild type ( Figures 1F-1GFUN regulation is restricted to mature, functional nodules, as pre-inoculation nitrate application inhibits nodulation in fun mutant plants to the same extent as in wild-type plants ( Figures 1H-1J When cloned together with the Fun genomic sequence, the Fun promoter was also shown to be sufficient to complement the fun mutation (data not shown).

[0130] These results demonstrate that the Fun gene is specifically expressed in nodules and controls the repression of nitrogen fixation by nitrate. Example 2: FUN induces nodule senescence via multiple pathways

[0131] The following examples describe the identification of FUN target genes and FUN binding sites in FUN target gene promoters. In addition, the phenotypes of loss-of-function mutants of FUN targets in Nelumbo nucifera are described. Materials and methods Gene expression

[0132] For RNA-seq, three weeks after inoculation, plants were acclimated to the environment by soaking overnight in ¼ Long Ashton liquid medium before treatment and then treated with 0 or 10 mM KNO for 24 h. Mature nodules were harvested. mRNA was isolated using the NucleoSpinRNA Plant Kit (Macherey-Nagel) and RNA sequencing was performed by Novogene (PE-150 bp Illumina sequencing). RNAseq analysis was performed by mapping reads to a reference transcriptome using Salmon 39 and quantifying with DEseq2 (Love, MI, Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). Publicly available time series of nitrate-treated nodule formation (Wang, L. et al., A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi: 10.1111 / nph.18896) were obtained from GEO using accession number GSE197362. GO enrichment was performed using GO_MWU for GO terms obtained from lotus[dot]au[dot]dk.

[0133] In order to express the target gene, RevertAid reverse transcriptase (Thermo) was used to synthesize the first chain cDNA. qRT-PCR was performed using a LightCycler480 instrument and LightCycler480 SYBR Green I Master (Roche Diagnostics). Ubiquitin conjugating enzyme was used as a reference. The cDNA concentration of the target gene was calculated using amplicon PCR efficiency calculation using LinRegPCR (Ramakers, C., Ruijter, JM, Deprez, RHL & Moorman, AFM Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62-66 (2003)). For each of 5 biological replicates (each consisting of 8 to 10 nodules), the target gene was compared with the reference. At least two technical replicates were performed in each analysis. The primers used are listed in Table 3 below. Table 3. Primers used in qPCR. Plant strains and growing conditions

[0134] Plant lines and growth conditions were as described in Example 1. Bacterial strains and culture conditions

[0135] Bacterial strains and culture conditions were as described in Example 1. Hairy root transformation

[0136] Hairy root transformation was as described in Example 1. Nodulation assay

[0137] Nodulation assays were performed as described in Example 1. Nitrogen fixation determination

[0138] Nitrogen fixation activity was quantified as described in Example 1. Determination of leghemoglobin content

[0139] Leghemoglobin content was determined as described in Example 1. Electrophoretic mobility shift assay (EMSA)

[0140] DNA probes labeled with 6-FAM at the 5' end were synthesized by Eurofins and are provided in Table 4 below. Purified FUN DNA binding domain (residues 178-237) was incubated with the probe in EMSA buffer (25 mM Tris-HCl pH 8.0, 80 mM NaCl, 35 mM KCl, 5 mM MgCl2) at 37°C for 60 min. After incubation, the reaction mixture was electrophoresed in a 6% non-denaturing polyacrylamide gel, and the labeled DNA was then detected using a Typhoon scanner (Fujifilm). The probe without the 6-FAM label served as a competitor, while the probe with a mutation in the core binding site (TGACG) served as a mutant. Table 4. Probes used for EMSA. transient activation assay

[0141] The promoters of the FUN candidate target genes (NRT2.1, HO1, NAC094, NRT3.1, and AS1), the glucuronidase (GUS) CDS, and the 35S terminator were cloned into a compatible Golden Gate vector as reporter genes; while the 35S promoter, FUN CDS, eGFP, and 35S terminator were cloned as effectors. The reporter genes and effectors were cloned into the p50507 GoldenGate binary vector. These constructs were then transformed into Agrobacterium tumefaciens strain AGL1. These Agrobacterium tumefaciens were diluted to an OD of 0. 600 =0.2 and infiltrated into N. benthamiana leaves. Three days after infiltration, approximately 20 mg of samples were collected for protein extraction. GUS activity was measured using a Thermo Scientific Varioskan flash with 4-methylumbelliferyl-β-D-glucuronide as a substrate (Sigma-Aldrich). For zinc treatment, N. benthamiana leaves were infiltrated with 500 μM MgCl2 (mock), 500 μM ZnCl2, or 2.5 mM EDTA 2 days after A. tumefaciens infiltration. GUS activity was measured 1 day after treatment. result FUN is a master regulator of nodule senescence

[0142] Since FUN is a transcriptional regulator, RNAseq was performed to search for directly controllable Gene targets related to nitrate signaling or nodulation function. RNAseq analysis identified 587 genes whose expression changed by more than 2-fold in WT nodules exposed to nitrate. Comparison with the fun mutant revealed that 106 of these genes were differentially regulated in fun nodules ( Figure 1R-1TRNAseq analysis identified multiple downstream targets of FUN, some of which were upregulated and others of which were downregulated, and multiple gene ontology groups were detected in both the upregulated and downregulated gene sets by GO-MWU (Nielsen, R. et al., A scan for positively selected genes in the genomes of humans and chimpanzees. PLoS Biol. 3, e170 (2005)) ( Figure 1U Of these, 29 up-regulated genes and 22 down-regulated genes predicted to be targets of FUN are provided in Tables 5A and 5B below. Table 5A. Downstream targets of upregulated FUN Table 5B. Downstream targets of downregulated FUN Gene ID Gene annotation LotjaGi1g1v0049800 MLP-like protein LotjaGi4g1v0362400 protein-tyrosine sulfotransferase-like protein LotjaGi3g1v0087300 Cytochrome P450, family 82, subfamily G, polypeptide 1 LotjaGi6g1v0067000_LC B3 domain-containing transcription factor VRN1-like protein LotjaGi2g1v0240500_LC Plasma membrane fusion protein PRM1 LotjaGi1g1v0501300 Leucine-rich repeat receptor-like protein kinase family proteins LotjaGi1g1v0480900 Cytochrome P450 LotjaGi1g1v0403800 5'-Methylthioadenosine / S-adenosine homocysteine ​​nucleotidase LotjaGi5g1v0043800_LC 50S ribosomal protein L1 LotjaGi1g1v0643500 5'-Adenosylsulfate reductase-like 5 LotjaGi2g1v0048400 Aging-related proteins LotjaGi2g1v0028500 Aging-related proteins LotjaGi1g1v0723500 Multi-colored, UV-B-insensitive 4 LotjaGi3g1v0443800_LC N-α-acetyltransferase 16, NatA auxiliary subunit LotjaGi3g1v0039400 Trehalose-6-phosphate synthase LotjaGi3g1v0303400 Glycosyltransferase LotjaGi6g1v0271500 Transmembrane protein, putative LotjaGi5g1v0215400 Benzyl alcohol O-benzoyltransferase LotjaGi3g1v0329600 FAM136A family proteins LotjaGi1g1v0171200_LC LotjaGi5g1v0316900 Fantastic Four-like proteins LotjaGi3g1v0183200 FAF-like protein, chloroplast

[0143] From this long list, six upregulated genes were selected for more detailed study. These six genes are provided in Table 6 below. Of particular note among these genes are heme oxygenase HO1, which degrades leghemoglobin during nodulation aging (Wang, L. et al., CRISPR / Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation. New Phytol. 224, 818-832 (2019); Zhou, Y. et al., Heme catabolism mediated by hemeoxygenase in uninfected interstitial cells enables efficient symbiotic nitrogen fixation in Lotus japonicus nodules. New Phytol. (2023) doi: 10.1111 / nph.19074), nitrate transporter, NRT3.1, and asparagine synthase 1 (AS1) genes (which are important for nitrogen assimilation) ( Figure 1V). In addition, many putative TGA-type binding motifs (TGACG; Bartlett, A. et al., Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat. Protoc. 12, 1659-1672 (2017)) were identified in the promoter regions of two genes that showed phenotypes similar to fun when mutated: nitrate transporter NRT2.1 (Misawa, F. et al., Nitrate transport via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression of root nodulation in Lotus japonicus. Plant Cell 34, 1844-1862 (2022)) and NAC transcription factor NAC094 that triggers nodulation senescence (Wang, L. et al., A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi:10.1111 / nph.18896). Table 6. Six genes studied in more detail

[0144] To test whether the expression of these genes is controlled by FUN, the relative expression of these genes in nodules of fun mutants was examined after nitrate treatment. Figures 2A-2B ) and by RNAseq ( Figure 2S ) analysis showed that nitrate induction of all of these genes was attenuated in the fun mutant. FUN is co-expressed with NAC094 and HO1 in uninfected cells, while nitrate regulation of NAC094, which also occurs in infected cells (Wang, L. et al., A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi: 10.1111 / nph.18896), may require additional regulatory factors.

[0145] The promoter region of Nrt2.1 has four putative FBSs (P1-P4), the promoter region of Ho1 has two FBSs (P1 and P2), the promoter region of NAC094 has one FBS (P1), the promoter region of Nrt3.1 has three FBSs (P1, P2, and P3), and the promoter region of AS1 has one FBS (P1), all of which are explained in Figure 2C EMSA was performed to test whether the FUN DNA binding domain binds to the probe representing FBS. Figure 2D As can be seen in Figure 3, the FUN DNA binding domain binds to P1 and P4 of the Nrt2.1 promoter, P1 and P2 of the Ho1 promoter, and P1 of the NAC094 promoter. Figure 2E As can be seen in Figure 3, the FUN DNA binding domain additionally binds to P1, P2, and P3 in the Nrt3.1 promoter region and P1 in the AS1 promoter region. Competition experiments with excess unlabeled probe demonstrated the specificity of this interaction ( Figure 2F ).

[0146] To verify the in vivo relevance of the EMSA binding results, transient activation experiments were performed in Nicotiana benthamiana for the NRT2.1, HO1, NAC094, NRT3.1, and AS1 promoters, and it was shown that all promoters coupled to the GUS reporter gene were significantly induced by FUN in this system. The FUN-GFP construct was expressed as an effector, and the GUS driven by the promoter was expressed as a reporter gene. Figure 2G As shown, each of the Nrt2.1, HO1, and NAC094 promoters coupled to the GUS reporter gene was significantly induced by FUN in this system. Figure 2H The same effect was shown for Nrt3.1 and AS1.

[0147] Further supporting the idea that FUN is a master regulator controlling these pathways, mutants obtained in nrt2.1, ho1, and nac094 displayed nodulation phenotypes similar to those of the original fun mutant, including enhanced nitrogen fixation and leghemoglobin content. Figure 2I Shown are the nodulation phenotypes of nrt2.1-3, ho1-4, and nac094-3 mutants compared with WT. Figures 2J-2L Shown are the results of nodule number, ARA activity, and leghemoglobin content assays for nrt2.1-3 and nrt2.1-4 mutants compared with WT. Figures 2M-2Q Shown are the results of nodule number, ARA activity, and leghemoglobin content assays for the ho1-4 and ho1-5 mutants and the nac094-3 and nac094-4 mutants compared with WT. Figure 2RShown are the results of an ARA activity assay for nac094-3 and nac094-4 mutants compared to the wild-type plant. Together, these data suggest that FUN targets nodule senescence and nitrate signaling pathways, thereby regulating nodule function relative to the environment. Modulating nitrate signaling pathways in this way by FUN may contribute to altering the sensitivity of nodules to nitrate relative to other root tissues. Example 3: The oligomeric state of FUN is regulated by zinc

[0148] The following examples describe the structural characterization of the FUN sensor domain and experiments evaluating the contribution of manganese and zinc as ligands for the sensor domain. Materials and methods Protein production and purification

[0149] The FUN sensor domain (residues 244-480) with a 3C-cleavable N-terminal tag (consisting of 10x histidine, 7x arginine, and a SUMO tag) and the FUN sensor construct with a zipper domain (residues 178-480) N-terminally tagged with 7x-histidine and a GB1 tag were ordered from GenScript. The plasmids were transformed into E. coli LOBSTR cells (Andersen, KR, Leksa, NC & Schwartz, TU Optimized E. coli expression strain LOBSTR eminates common contaminants from His-tag purification. Proteins 81, 1857-1861 (2013)). The expression culture was grown in LB medium containing 0.1 mg / mL ampicillin and 0.034 mg / mL chloramphenicol at 37°C and 110 rpm to an OD of 0. 600=0.6. Cells were cold-shocked on ice for 30 min and then induced with 0.4 mM IPTG overnight at 18°C. Cells were pelleted (4400 g, 4°C, 10 min), resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 5 mM β-mercaptoethanol, and 1 mM benzamidine), and lysed by sonication. The lysate was clarified by centrifugation (30600 g, 4°C, 30 min), and protein was purified from the clarified lysate using a Protino Ni-NTA 5 mL column (Machery-Nagel). Protein was eluted with high imidazole buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5% glycerol, 500 mM imidazole, 5 mM β-mercaptoethanol). The zippered FUN sensor was not further purified, but the FUN sensor was dialyzed overnight against 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol at a 1:50 molar ratio with 3C protease. The cleaved tag and protease were then removed by a second Ni-IMAC step. The FUN sensor was further purified by SEC on a Superdex 200 Increase 10 / 300 GL (GE Healthcare) in minimal buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol).

[0150] For SAXS analysis, the FUN sensor was further purified on ResourceQ 1 mL (GE Healthcare) and eluted with a linear gradient of 10-500 mM NaCl and 10 mM Tris-HCl pH 8.0 with 5 mM β-mercaptoethanol. The eluted fractions were collected and dialyzed against minimal buffer. Dynamic light scattering (DLS) and nanodifferential scanning fluorimetry (nanoDSF) analysis

[0151] The changes in thermal unfolding (nanoDSF) and size (DLS) of FUN protein after adding ligand were analyzed on a Prometheus Panta instrument (NanoTemper Technologies). 0.8 mg / mL of purified protein was incubated with 4 mM of different potential ligands or 0-4 mM ZnCl2 for 20 min, followed by the addition of 5 mM EDTA to the sample for analysis of reversible filamentation. Before addition, ZnCl2 was filtered using a VivaSpin MWCO5kDa and immediately added to the protein sample. Ten continuous DLS measurements were performed on each sample at 25 ° C with 100% laser power, and then nanoDSF experiments were performed, which were measured from 25-90 degrees with a temperature ramp of 1 ° C / min, with an excitation power of 100%. All measurements were repeated three times. Small-angle X-ray scattering (SAXS) analysis

[0152] SAXS measurements were performed on a NanoSTAR instrument (Pedersen, JSA flux- and background-optimized version of the NanoSTAR small-angle X-ray scattering camera for solution scattering. J. Appl. Crystallogr. 37, 369-380 (2004); J. & Pedersen, JSA high-flux automated laboratory small-angle X-ray scattering instrument optimized for solution scattering. J. Appl. Crystallogr. 54, 295-305 (2021)). The instrument uses a Cu rotating anode with a scattering-free pinhole in front of the sample and a two-dimensional position-sensitive gas detector (Vantec500, Bruker AXS). The sample and buffer are measured in a homemade flow capillary. The intensity I (q) is expressed as a function of the scattering vector modulus Q (and Q = 4π sin (2θ) / λ), where 2θ is the scattering angle, and λ is the X-ray wavelength. Buffer scattering was subtracted from the scattering from the sample, and the intensity was converted to an absolute scale and corrected for variations in detector efficiency by normalizing to the scattering of pure water (Pedersen, JSA flux- and background-optimized version of the NanoSTAR small-angle X-ray scattering camera for solution scattering. J. Appl. Crystallogr. 37, 369-380 (2004)). 2 Plot the data in Guinier to determine the radius of gyration R g , and perform indirect Fourier transform (IFT) (Glatter, OA new method for theevaluation of small-angle scattering data. J. Appl. Crystallogr. 10, 415-421 (1977); Pedersen, JS, Hansen, S. & Bauer, R. The aggregation behavior of zinc-freeinsulin studied by small-angle neutron scattering. Eur. Biophys. J. 22, 379-389 (1994)) to obtain the pairwise distance distribution function p(r), which is a histogram of the distances between pairs of points within the particle, weighted by the excess scattering length density at the point. Note that the resolution of the SAXS data is about And therefore the total length of the fibrils induced by zinc cannot be resolved.In this case, the p(r) function is related to the cross-sectional structure of the filaments. Negative stain electron microscopy

[0153] For electron microscopy, 0.1 mg / mL of the purified FUN sensor domain was incubated at room temperature for 20 min with or without the addition of 100 μM ZnCl and with or without the addition of 5 mM EDTA. Samples for negative staining were prepared on 400 copper grids, which were manually covered with a carbon-coated collodion support film using a Leica EM SCD 500 High Vacuum Sputter Coater. Before staining, the grids were glow-discharged with a negative polarity of 25 mA for 45 s using a PELCO easiGlow glow discharge system. 3 μL of FUN sensor was deposited on the grids, incubated for 30 s, and then excess sample was removed from the grids using Whatman paper. After blotting, the grids were floated three times in a 2% uranyl formate solution for 15 s and then dried. Negative staining micrographs were recorded at the cryo-EM facility using a Tecnai G2 Spirit microscope equipped with a TemCam-F416 (4kx4k) TVIPS CMOS camera and a Veleta (2kx2k) CCD camera, operated at 120 kV. Micrographs were recorded at magnifications of 42,000x and 52,000x. Microscopy and confocal imaging

[0154] For FUN expression patterns, GUS-stained roots were observed using a Leica M165FC fluorescence stereomicroscope. Nodules were embedded in 3% agarose and cut into 100 μm sections using a vibratome. Nodule sections were observed using a Zeiss Axioplan 2 light microscope. For FUN subcellular localization, hairy roots of Nelumbo nucifera and Nicotiana benthamiana leaves expressing FUN-GFP were treated with 500 μM ZnCl2 (Zn) or MgCl2 (mock) for 3 days and then observed for fluorescence using a 491-535 nm filter on a Zeiss LSM 710 confocal microscope. For zinc biosensor (eCALWY and eCALWYnls) assays, nodules were embedded in 3% agarose and cut into 75 μm sections using a vibratome. Sky blue was excited at a wavelength of 458 nm, and the citrine fluorophore was captured using a 514-550 nm filter on a Zeiss LSM 710 confocal microscope. A 587-665 nm filter was used to detect Mesorhizobium lotus DsRed. Zinpyr-1 imaging and quantification

[0155] Plants with pink nodules (3wpi) were acclimatized before treatment by soaking overnight in 1 / 4Long Ashton liquid medium, and then treated for 24 hours with 0 or 10mM KNO. Ripe nodules were embedded in 3% agarose and cut into 80 μm sections using a vibrating microtome. Slices (slider) were stained for 3 hours with 5 μM Zinpyr-1 and rinsed three times with water. Fluorescence was observed using a Zeiss LSM 710 confocal microscope using the excitation at 488nm and the emission at 505-550nm. Fluorescence density was determined using ImageJ software. Micro X-ray fluorescence microscopy

[0156] Micro X-ray (mXRF) images were obtained using a scanning X-ray microscope equipped with a liquid nitrogen passively cooled cryogenic stage (Cotte, M. et al., The ID21 X-ray and infrared microscopy beamline at the ESRF: status and recent applications to artistic materials. J. Anal. At. Spectrom. 32, 477-493 (2017)). The samples were prepared as described in Escudero et al. (Escudero, V. et al., Medicago truncatula Ferroportin2 mediates iron import into nodule symbiosomes. New Phytol. 228, 194-209 (2020).). Briefly, nodules were embedded in OCT culture medium and then cryofixed by plunging them into liquid nitrogen-cooled isopentane. 20 mm sections of frozen samples were obtained using a Leica LN22 cryomicroscope with a microtome and mounted in a liquid nitrogen-cooled sample holder between two sheets of Ultralene (Spex SamplePrep, Rickmansworth, UK) foil. The beam was focused to 0.9 x 0.6 mm using a Kirkpatrick-Baez mirror optical system. 2 The emitted fluorescence signal is detected by an energy dispersive large area (80mm) 2 )SDD detector (XFlashSGX, RaySpec, High Wycombe, UK). 2The sample was raster scanned with a step size of 100 ms and a dwell time of 220 ms to collect images. The element distribution was calculated using the PyMca software package (Solé, VA et al., A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra. Spectrochim. Acta Part BAt. Spectrosc. 62, 63-68 (2007)). result The sensor domain of FUN forms filamentous structures in the presence of physiological concentrations of zinc

[0157] The FUN sensor domain has distant homology with metal binding proteins (Trepreau, J. et al., Structural basis for metal sensing, Cnr X. J. Mol. Biol. 408, 766-779 (2011)), and since transcriptional regulation of FUN was not observed in nodulation ( Figure 3I ), it is speculated that the activity may be regulated at the protein level. To understand the mechanism, the FUN sensor domain ( Figure 3J ) and screened with common cellular metal ions and nitrogen compounds to see if these substances affected the FUN sensor. It was found that the thermal stability of FUN (nanoDSF; Figure 3K ) and molecular size by dynamic light scattering (DLS) ( Figures 3A-3C ) were increased, while responses to other test compounds remained unchanged. Dose-response experiments revealed that zinc increased the molecular size of the FUN sensor at low physiologically relevant concentrations (3.9-7.8 μM), while only unnatural high concentrations of manganese (2-4 mM) increased its size, indicating that zinc is a relevant ligand ( Figure 3A and 3B ).

[0158] When zinc is chelated with EDTA, the changes induced by zinc are reversible ( Figure 3D Proteins containing both DNA binding and sensor domains also demonstrated similar zinc sensitivity and reversibility ( Figure 3L Further investigations using small-angle X-ray scattering (SAXS) experiments, which provided scattering data and pairwise distance distribution functions (histograms of distances between pairs of points within the structure), confirmed that in the presence of zinc, the FUN sensor switches from a smaller molecular size to a larger oligomeric form, and that this effect is reversible when zinc is removed with EDTA ( Figures 3E-3G ).

[0159] The structure of oligomeric forms of the FUN sensor was investigated using electron microscopy. Negative staining of samples revealed that when the FUN sensor was zinc-bound, large filamentous structures formed, and when zinc was removed using EDTA, these filaments disintegrated ( Figure 3H ).

[0160] Together, these results show that FUN binds low physiological concentrations of zinc, converting its oligomeric form into large filaments, and that this process is dynamic and reversible, which may be a mechanism for regulating activity. Example 4: Zinc regulation of FUN is relevant in vivo.

[0161] The following examples describe in vivo testing of zinc regulation of FUN. Materials and methods Subcellular localization assay

[0162] The subcellular localization of the FUN-GFP construct described in Example 1 was examined in Nicotiana benthamiana (tobacco) leaves. Tobacco leaves expressing the pro35S:FUN-GFP protein were infiltrated with 500 μM mock, Mn, and Zn. Fluorescence images were captured by confocal microscopy (Zeiss SP5), and nuclei were counted based on punctate or uniform distribution. Plant strains and growing conditions

[0163] Plant lines and growth conditions were as described in Example 1. Bacterial strains and culture conditions

[0164] Bacterial strains and culture conditions were as described in Example 3. Hairy root transformation

[0165] Hairy root transformation was as described in Example 1. Nodulation assay

[0166] Nodulation assays were performed as described in Example 1. Nitrogen fixation determination

[0167] Nitrogen fixation activity was quantified as described in Example 1. Determination of leghemoglobin content

[0168] Leghemoglobin content was determined as described in Example 1. Instant activation

[0169] The transient activation assay was as described in Example 2. Zinc biosensors

[0170] The FRET-based Zn biosensors eCALWY (Lanquar, V., Grossmann, G., Vinkenborg, JL, Merkx, M., Thomine, S., and Frommer, WB (2014). Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology. New Phytol. 202: 198–208) and eCALWYnls were used to measure the response of nodule cells to Zn treatment. The biosensor eCALWYnls contains a nuclear localization signal (nls). Expression analysis

[0171] RNAseq was used to compare the expression of putative zinc transporter genes Zip1 and Zip2 under normal and nitrate stress growth conditions. The original data were from the RNAseq analysis described in Example 3. result Zinc is a second messenger regulating FUN activity

[0172] The identification of zinc-induced FUN filaments raises the possibility that they may play a role in regulating protein activity. Specifically, zinc infiltration triggered changes in the nuclear fluorescence of FUN-GFP in Nicotiana benthamiana leaves. Subcellular localization assays revealed altered fluorescence, such as Figures 4A-4C As shown in , where zinc administration promoted the aggregation of FUN.

[0173] Using the NRT2.1 promoter as a readout for FUN activity, co-infiltration with zinc significantly reduced FUN activity in Nicotiana benthamiana leaves compared to mock (MgCl2) ( Figure 4D This suggests that the zinc-bound filamentous state of FUN is the inactive form of the protein. Considering the phenotype of the fun mutant, this suggests that zinc acts as a messenger, linking nitrate to FUN activity and nodulation regulation.

[0174] To test whether nitrate affects cellular zinc levels, experiments were performed using the zinc-sensitive zincpyr-1 dye (Sinclair, SA et al. The use of the zinc-fluorophore, Zinpyr-1, in the study of zinc homeostasis in Arabidopsis roots. New Phytol. 174, 39-45 (2007)) to assess the homeostasis of zinc in sections of Nelumbo nucifera nodules from plants grown in the absence of nitrate and from nodules exposed to 10 mM KNO for 24 h. Figures 5C-5D This revealed a significant reduction in zinc levels, particularly within the nitrogen-fixing zones of nitrate-treated nodules. This reduction was independently confirmed by micro-X-ray fluorescence microscopy of nodule sections treated with 10 mM KNO3 for 24 hours, which revealed a circular distribution of zinc in infected cells, associated with a radial distribution and dense packing of symbionts ( Figures 5E-5F Density measurements of 10 cells per condition confirmed that zinc was reduced by half compared to untreated nodules (0.54 ± 0.06; Figure 5F To confirm the in vivo relevance of zinc-dependent FUN filament formation, a FUN-GFP construct was expressed in Nelumbo roots. Under control conditions (500 μM MgCl2), FUN-GFP showed a dispersed localization in the nucleus, whereas zinc addition (500 μM ZnCl2) triggered its relocalization to distinct subnuclear condensates ( Figure 5G Consistent with the effect of zinc on protein activity in N. benthamiana, a zinc-dependent increase in aggregate frequency was also detected in leaves infiltrated with zinc together with the FUN-GFP construct ( Figure 5H Further confirming the link between nitrate, zinc, and FUN activity, the addition of 500 μM zinc significantly increased nodulation function in nitrate-exposed WT plants, as evidenced by acetylene reduction ( Figure 4E ) and leghemoglobin content ( Figure 4H ) assay, reproducing the phenotype of the fun knockout mutant. This increase was dependent on the presence of FUN, as no further increase in nodulation function was observed in the fun mutant ( Figure 4F and 4G ).at last, Figure 5A Shown are the results of expression analysis of two putative zinc transporters, Zip2 and Zip4, both of which were induced in nodules after nitrate treatment.

[0175] Together, these results show that changes in Zn concentration in response to soil nitrate are sufficient to alter FUN activity and, consequently, the nitrogen-fixing phenotype of nodules. discuss

[0176] The genetic screen described in Example 1 identified the basic leucine zipper transcription factor FUN as a novel master regulator of nitrogen fixation in legumes. The sensor domain within FUN was identified as key to its activity, and it was demonstrated that intracellular zinc levels determine protein activity via ligand-dependent protein filament formation. In Examples 2-4, it was shown that FUN forms inactive filaments at high zinc concentrations, which act as molecular reservoirs from which active proteins can be released when zinc levels decrease (Figure 5J). Cellular zinc levels exhibit an inverse relationship with nitrate, and zinc is shown to act as a second messenger, signaling nitrate availability and controlling the transition between the filamentous (inactive) and active states of the FUN protein.

[0177] In plants, it has been demonstrated that changes in zinc concentration affect the activity and nodulation function of the FUN protein, thereby acting to link soil nitrate supply with transcriptional regulation of nodulation metabolism. It is believed that this post-translational regulation of FUN activity allows plants to respond to nitrate concentration gradients by gradually reducing zinc levels, thereby releasing more active FUN to adapt nodulation function to the environment. The exact mechanism by which nitrate affects intracellular zinc concentration (e.g., via transporter regulation, organelle sequestration, or cellular export) is unclear. FUN is a transcription factor in the TGA family, whose members regulate a range of important plant traits, including nitrate uptake (Alvarez, JM et al., Systems approach identifies TGA1 and TGA4 transcription factors as important regulatory components of the nitrate response of Arabidopsis thaliana roots. Plant J. 80, 1-13 (2014), Ruffel, S. et al., Genome-wide analysis in response to nitrogen and carbon identifies regulators for root AtNRT2 transporters. Plant Physiol. 186, 696-714 (2021)), pathogen response (Kumar, S. et al., Structural basis of NPR1 in activating plant immunity. Nature 1-6 (2022)) and floral development (Maier, AT, Stehling-Sun, S., Offenburger, S.-L. & Lohmann, JU The bZIP Transcription Factor PERIANTHIA: A Multifunctional Hub for Meristem Control. Front. Plant Sci. 2, 79 (2011). Given that the identified sensor domain is present in TGA family homologs, it seems plausible that zinc or other metal ions and their metabolites could provide similar graded responses to environmental stimuli, thereby enabling the connection between the environment and plant development through metal ion signaling. Manipulating metal ion accumulation or protein filamentation in response to these metal ions could provide new approaches for optimizing these important plant traits.

[0178] Nitrogen fixation is an energy-demanding process that requires the provision of fixed carbon to the symbiotic rhizobia. A regulated senescence program allows limiting the carbon supply to nodules and resupplying nutrients to support plant growth and reproduction (Puppo, A. et al., Legumenodule senescence: roles for redox and hormone signaling in the orchestration of the natural aging process. New Phytol. 165, 683-701 (2005)). Recently, several NAC transcription factors have been shown to regulate pathways required for nodulation and senescence (Yu, H. et al., GmNAC039 and GmNAC018 activate the expression of cysteine ​​protease genes to promote soybean nodule senescence. Plant Cell (2023) doi: 10.1093 / plcell / koad129; Wang, L. et al., A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence. New Phytol. (2023) doi: 10.1111 / nph.18896). The identification of FUN as a new regulator of senescence-related processes through multiple pathways (including through NAC094) opens new avenues for fine-tuning these pathways to enhance soil nitrate tolerance in legumes and provides opportunities for increasing the delivery of fixed nitrogen to important agricultural crops.Importantly, the specificity of the identified regulatory pathway for nodulation function ensures that the mutants do not exhibit interactions with other genetic pathways such as nodule number regulation (Krusell, L., Madsen, LH, Sato, S. & Aubert, G. Shoot control of root development and nodulation is mediated by a receptor-like kinase. Nature 420, 422-426 (2002); Nishimura, R. et al., HAR1 mediates systemic regulation of symbiotic organ development. Nature 420, 426-429 (2002); Huault, E. et al., Local and systemic regulation of plant root system architecture and symbiotic nodulation by areceptor-like kinase. PLoS Genet. 10, e1004891 (2014)) or nitrate acquisition and signaling (Lin, J.-S. et al., NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula.Nat Plants 4,942-952(2018);Misawa,F. et al., Nitratetransport via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression of root nodulation in Lotus japonicus.Plant Cell 34,1844-1862(2022);Jiang,S. et al., NIN-like protein transcription factors regulate leghemoglobin genes in legumenodules.Science 374,625-628(2021)). Example 5: Identification of FUN and NAC094 orthologs and construction of phylogenetic tree

[0179] The following examples describe the identification of FUN and NAC094 orthologs in other plant species and the construction of phylogenetic trees using these sequences. Materials and methods Identification of FUN and NAC094 orthologs and construction of phylogenetic tree

[0180] A BLAST query was performed against the target species using the FUN protein sequence. Similarly, a BLAST query was performed against the target species using the NAC094 protein sequence. Candidate BLAST hits were aligned with the phylogenetic tree using Shoot.bio (Emms, DM, Kelly, S. SHOOT: phylogenetic gene search and ortholog inference. Genome Biol 23, 85 (2022)).

[0181] The FUN orthologous protein sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.

[0182] The NAC094 orthologous protein sequences are SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73.

[0183] Protein sequences were aligned with MAFFT 7.490 and a tree was constructed using FastTree 2.1.11. The tree was visualized using iTOL 6.7.3 (Letunic, I. & Bork, P. Interactive Tree of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293-W296 (2021)). result

[0184] Phylogenetic analysis showed that FUN is highly conserved in legumes, with legumes carrying both FUN and FUN-like paralogs in the PAN orthologous group ( Figure 6AThe duplication of FUN in legumes resulted in two copies of the gene in Lotus japonicus: FUN, which is expressed in nodules and described in Example 1; and FUN-like, which is not expressed in nodules and has non-legume orthologs. A second duplication occurs in soybean (Glycine max), with two orthologs for FUN and two orthologs for FUN-like.

[0185] FUN orthologs are those within the same phylogenetic clade as Lotus FUN (LotjaGi2g1v0279100; SEQ ID NO: 1) and Glycine max (soybean) FUNa (Glyma.02G097900; SEQ ID NO: 8) and FUNb (Glyma.01G084200; SEQ ID NO: 9). The closest non-legume ortholog member is Arabidopsis PAN (AT1G68640.1; SEQ ID NO: 4). Orthologs were further confirmed by gene expression in root nodules as determined by RNAseq (data not shown).

[0186] To evaluate whether soybean (Glycine max) FUN orthologs also exhibit nodulation-specific expression, the expression levels of two orthologs, FUNa and FUNb, in different soybean tissues were measured ( Figure 6B In fact, both orthologs are predominantly expressed in symbiotic nodules. These observations suggest that the function of FUN may be conserved in soybean and other legumes.

[0187] FUN paralogs that do not function in nodulation regulation are those in the phylogenetic clade with Lotus FUN-like (LotjaGi5g1v0341400; SEQ ID NO:83) and soybean FUN-like (Glyma.20G113600 (SEQ ID NO:7) and Glyma.10G276100 (SEQ ID NO:6)).

[0188] A phylogenetic tree of the NAC domain-containing protein Nac094 was also constructed to identify orthologs and paralogs in legumes and non-legumes ( Figure 7 Nac094 (LotjaGi2g1v0259200; SEQ ID NO: 31) is orthologous to soybean Glyma.19G021900.1 (SEQ ID NO: 42) and Glyma.13G063300.1 (SEQ ID NO: 41). Example 6: Fun mutants in soybean and cowpea exhibit enhanced nitrogen fixation

[0189] The following examples describe the generation and characterization of fun mutants in soybean and cowpea. Specifically, experiments are described to evaluate the nitrogen fixation activity and yield performance of fun mutants in soybean and cowpea under different stress conditions. Materials and methods Plant materials and growth conditions

[0190] Glycine max (soybean) and Vigna unguiculata (cowpea) lines were used for Agrobacterium transformation and regeneration of CRISPR fun knockout mutants. Nitrogen fixation activity

[0191] Nitrogen fixation was assessed using the method of Example 1. Generation of plant expression vectors

[0192] Expression constructs were generated to express CRISPR / Cas and multiple guide RNAs targeting the Fun gene coding sequence in G. max (soybean) and V. unguiculata (cowpea). Plant transformation and regeneration

[0193] Plant transformation and regeneration were performed using standard methods for soybean and cowpea. Production performance

[0194] Standard methods were used to evaluate the yield performance of soybean and cowpea under conditions ranging from low to high nitrogen application. Yield performance was also evaluated in environments with companion crops. result Improved nitrogen fixation in soybeans and cowpeas increases yield

[0195] To determine whether the function of the Fun gene in Lotus is conserved in legumes, a CRISPR knockout soybean ( Figure 6A) and cowpea. Multiple knockout lines will be selected and propagated to generate homozygous fun mutants. Once homozygous lines are generated, the nitrogen fixation activity of the fun mutants in soybean and cowpea will be evaluated under limiting nitrate conditions. The nodulation phenotype and nitrogen fixation activity will be determined in a manner similar to that of the fun mutants in the genus Nelumbo. If the function of the Nelumbo Fun gene is conserved in soybean and cowpea, then the fun mutants in both species will exhibit a pink (active) nodulation phenotype and increased nitrogen fixation activity under limiting nitrate conditions. In addition, to determine whether the loss of the functional Fun gene enhances nitrogen fixation activity under other stress environments, the nitrogen fixation rate of the fun mutants in Nelumbo, soybean, and cowpea will be evaluated under drought, heat stress, and waterlogging conditions.

[0196] Soybean and cowpea varieties with enhanced nitrogen fixation activity under stress conditions could sustainably enhance the yield of both crops. Promising soybean and cowpea fun mutant lines identified from the stress assays described above will be evaluated for field performance. The yield characteristics of the fun mutants in soybean and cowpea will be evaluated in the field to determine whether enhanced nitrogen fixation translates into increased yield. Example 7: Engineered FUN variants improve nitrogen fixation in legumes

[0197] The following examples describe the structure-function characterization of FUN for the engineering and characterization of FUN variants in Nelumbo nucifera and target crops. Materials and methods Plant materials and growth conditions

[0198] The fun mutants of Nelumbo nucifera described in Example 1 and fun mutants identified in soybean and cowpea in Example 6 will be used for Agrobacterium transformation and regeneration of engineered FUN variants. Growth conditions are as described in Examples 1 and 6. Experimental techniques

[0199] The experimental techniques are as described in any one of Examples 1-6. result Structure-based engineering of FUN variants

[0200] Structural characterization of the fun mutant protein and its alleles will identify regions of the protein that are critical for its function. Combined with computational modeling, this structural analysis will pinpoint key residues within the FUN protein for engineering novel variants with further enhanced nitrogen fixation properties. These engineered FUN protein variants will be introduced into Nelumbo nucifera plants and evaluated for their nitrogen fixation properties. The functions of engineered FUN variants are conserved in soybean and cowpea

[0201] To evaluate whether structure-based FUN engineering translates into improved crop yields, mutations corresponding to engineered FUN Lotus variants will be introduced into soybean and cowpea. The performance of engineered FUN soybean and cowpea lines will be evaluated under various stress environments and in the field. Example 8: The FUN sensor domain mechanism is conserved among TGA transcription factors

[0202] The following examples describe the structural characterization of TGA-type transcription factors that share a conserved domain structure with FUN. Furthermore, experiments are described to assess the regulation of TGA sensor domain conformation by metal ion ligands and the identification of nitrate-responsive metal ion transporters. Materials and methods

[0203] The experimental techniques are as described in any one of Examples 1-7. result Sensor domain filamentation is a conserved feature of TGA transcription factors

[0204] FUN from the genus Nelumbo is a member of the TGA family of transcriptional regulators that play a wide range of roles in plant development, immunity, and nitrate signaling. TGA family members all share a conserved domain structure, a DNA binding domain and a sensor domain. To determine whether sensor domain filamentation is conserved in the TGA transcription factor family, the sensor domain structure of representative TGA family members will be determined. The ability of metal ion ligands to induce sensor domain multimerization will also be studied. If metal ion ligands can regulate sensor domain complex formation and activity similar to FUN, then the entire family can be regulated by metal ion treatment to target a variety of pathways related to crop improvement. Identification of nitrate-responsive metal ion transporters to regulate nitrogen fixation

[0205] To identify metal ion transporter genes regulated by nitrate, differential gene expression in Nelumbo plants was determined under three sets of conditions: nitrate treatment versus mock, zinc treatment versus mock, and nitrate and zinc co-treatment versus mock.

[0206] The differentially expressed candidate genes will be further evaluated for expression, activity, and zinc concentration in nodulation in response to nitrate conditions. Nelumbo nucifera knockout lines for the identified metal ion transporter genes will be obtained or constructed and evaluated for improved nitrogen fixation properties. Identification of these genes will provide mechanistic insights into zinc regulation by nitrate and offer additional means for regulating nitrogen fixation. Example 9: Effect of FUN on heat and drought tolerance

[0207] The following examples describe the generation and characterization of fun mutants in Medicago and Nelumbo, as well as experiments to evaluate nitrogen fixation activity under heat and drought conditions, and the yield performance of fun mutants in Medicago and Nelumbo. Materials and methods

[0208] The experimental techniques are as described in any one of Examples 1-8. result Manipulating FUN to improve drought tolerance

[0209] RNAseq revealed that NAC094 and HO1 were both robustly upregulated after 4 days of drought in both Medicago and Nelumbo ( Figure 8A To determine whether FUN regulation of NAC094 and / or HO1 plays a role in drought tolerance, fun mutants were generated in Medicago and Nelumbo and tested under drought conditions. Drought tolerance of the plants was assessed. Manipulating FUN to improve heat resistance

[0210] Fun mutant plants showed increased nitrogen fixation activity after 7 days of heat stress, as quantified using the acetylene reduction assay (ARA) ( Figure 8C To determine whether manipulation of FUN could improve heat tolerance, fun mutants were tested under hot conditions. The heat tolerance of the plants was assessed.

Claims

1. A genetically modified plant or part thereof comprising one or more genetic alterations which result in reduced activity or expression of a FUN protein in the genetically modified plant or part thereof as compared to the activity or expression of the FUN protein in a control plant grown under the same conditions, wherein the FUN protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 The proteins of the group consisting of SEQ ID NO:30, SEQ ID NO:80, SEQ ID NO:81 and SEQ ID NO:82 have polypeptides that are at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical or at least 99% identical.

2. The genetically modified plant or part thereof of claim 1 , wherein the FUN protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, or SEQ ID NO:

82.

3. A genetically modified plant or part thereof comprising one or more genetic alterations that result in a decrease in the activity or expression of an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, an NRT2.1 protein, or an AS1 protein in the genetically modified plant or part thereof as compared to the activity of the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein, or the AS1 protein in a control plant grown under the same conditions, wherein the protein is an NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:74; wherein the protein is a bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:75; wherein the protein is a NAC domain-containing protein, and wherein the NAC domain-containing protein comprises a polypeptide selected from the group consisting of SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73 group of proteins having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a polypeptide; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 78; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:

79.

4. The genetically modified plant or part thereof of claim 3, wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:

79.

5. A genetically modified plant or part thereof comprising one or more genetic alterations which result in a decrease in the activity or expression of one or more of a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein or an AS1 protein in the genetically modified plant or part thereof as compared to the activity or expression of a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein or an AS1 protein in a control plant grown under the same conditions, wherein the FUN protein, the FUN-like protein, the NRT3.1 protein, the bZIP28 protein, the NAC domain-containing protein, the HO1 protein, the NRT2.1 protein or the AS1 protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 or SEQ ID NO:

84. A group of polypeptides that are at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical, and wherein the FUN protein, FUN-like protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein has enhanced expression in nodules in the absence of one or more genetic alterations.

6. The genetically modified plant or part thereof according to any one of claims 1 to 5, wherein the reduction is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%, optionally wherein the reduction is due to knocking out a gene for a protein, introducing a premature stop codon in the coding sequence of a gene for a protein, RNAi silencing, knocking out a domain of a protein, introducing a binding site for a transcription repressor protein, or knocking out a binding site in the promoter region of a gene and / or the genetic alteration comprises knocking out a gene for a protein, introducing a premature stop codon in the coding sequence of a gene for a protein, RNAi silencing, knocking out a domain of a protein, introducing a binding site for a transcription repressor protein, or knocking out a binding site in the promoter region of a gene, preferably the binding site is a transcription activator protein binding site or a TATA box.

7. The genetically modified plant or part thereof of any one of claims 1-6, wherein the growing conditions include moderate nitrate levels, high nitrate levels, or nitrate levels around the plant that reduce or suppress nitrogen fixation, optionally wherein the nitrate level is between about 10 mM and about 250 mM nitrate, or includes at least about 10 mM nitrate, at least about 20 mM nitrate, at least about 30 mM nitrate, at least about 40 mM nitrate, at least about 50 mM nitrate, at least about 100 mM nitrate, at least about 150 mM nitrate, at least about 200 mM nitrate, or at least about 250 mM nitrate.

8. The genetically modified plant or part thereof of claim 7, wherein the genetically modified plant has increased nitrogen fixation compared to a control plant grown under the same growth conditions, optionally wherein the nitrogen fixation is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%.

9. The genetically modified plant or part thereof according to any one of claims 1 to 8, wherein the plant forms nodules and wherein the number of nodules is increased, the hemoglobin content is increased, or the acetylene reduction assay (ARA) activity is increased compared to control plants when grown under the same conditions.

10. A method of growing a genetically altered plant having enhanced nitrogen fixation under conditions including nitrate levels around the plant's roots that inhibit nitrogen fixation, the method comprising: a) providing a genetically modified plant, wherein the plant or part thereof comprises one or more genetic alterations which result in a decrease in the activity or expression of a FUN protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein or an AS1 protein, or any combination thereof, in the genetically modified plant or part thereof as compared to the activity or expression of a FUN protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein or an AS1 protein in a control plant grown under the same conditions, and wherein one or more of the genetic alterations reduces nitrate level repression of nitrogen fixation; and b) growing the genetically modified plant in the presence of nitrate levels surrounding the roots of the plant, wherein the genetically modified plant has increased nitrogen fixation compared to control plants grown under the same conditions.

11. A genetically modified plant or part thereof produced by the method of claim 10, wherein the number of nodules is increased or the hemoglobin content is increased compared to control plants when grown under the same growth conditions, and wherein the increased nitrogen fixation is measured using a method selected from the group consisting of measuring the number of pink nodules per plant compared to control plants, measuring the amount of acetylene (C2H2) reduced to ethylene (C2H4) per hour (Acetylene Reduction Assay (ARA)) compared to control plants, and measuring micrograms of hemoglobin per plant compared to control plants.

12. A method of growing a genetically modified plant capable of fixing nitrogen when grown under nitrogen fertilizer conditions, the method comprising: a) providing a genetically altered plant, wherein the plant or part thereof comprises one or more genetic alterations which result in a decrease in the activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein, or any combination thereof, in the genetically altered plant or part thereof as compared to the activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions, and wherein the one or more genetic alterations reduce nitrate level repression of nitrogen fixation; b) growing the plants under conditions that include standard nitrate levels around the plant roots; and c) applying nitrogen fertilizer, thereby generating conditions including nitrate levels around the roots of the plant that inhibit nitrogen fixation, wherein the genetically modified plant has increased nitrogen fixation compared to control plants grown under the same conditions.

13. The method of claim 12, wherein the genetically modified plant is grown in an intercropping system with a non-nitrogen fixing plant, or in a sequential system after a non-nitrogen fixing plant.

14. A method for delaying nodule senescence, comprising: a) providing a genetically altered plant, wherein the plant or part thereof comprises one or more genetic alterations that result in reduced activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein, or any combination thereof, in the genetically altered plant or part thereof, as compared to the activity or expression of a FUN protein, NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein in a control plant grown under the same conditions, and wherein the one or more genetic alterations delay nodulation senescence; and b) Growing the genetically modified plant under stress conditions, wherein the genetically modified plant has delayed nodulation senescence compared to control plants grown under the same conditions.

15. The method of claim 14, wherein the stress condition is selected from the group consisting of moderate nitrate levels, high nitrate levels, nitrate levels around the plant that promote nodulation and senescence, moderate heat levels, high heat levels, heat levels around the plant that promote nodulation and senescence, moderate water deficit levels, high water deficit levels, water deficit levels around the plant that promote nodulation and senescence, moderate waterlogging levels, high waterlogging levels, or waterlogging levels around the plant that promote nodulation and senescence.

16. A method for inducing filamentation of a FUN protein, the method comprising: a) providing FUN protein; as well as b) increasing the amount of zinc or manganese in the environment of the FUN protein, wherein the increased amount of zinc or manganese induces filament formation compared to a control FUN protein in an environment without the increased amount of zinc or manganese, optionally wherein filament formation is induced under high nitrate conditions, and / or the method is performed in vitro.

17. A method for inducing filamentation, the method comprising: a) providing a plant comprising a FUN protein; as well as b) growing a plant under conditions of increased zinc or manganese, wherein filament formation of a FUN protein in said plant is induced compared to the FUN protein in a control plant grown under conditions without increased zinc or manganese, optionally wherein said plant comprises a genetic alteration, and / or wherein said filament formation is induced under conditions of high nitrate.

18. The method of claim 16 or 17, wherein the genetic alteration reduces the activity of the FUN protein without eliminating the activity of the FUN protein, and / or wherein the induction of filamentation results in increased nitrogen fixation in the genetically altered plant compared to control plants grown under the same conditions, or the induction of filamentation reduces the activity of the FUN protein or inactivates the FUN protein.

19. A method for regulating nodulation function according to the amount of available nitrogen in soil, the method comprising: a) providing a genetically modified plant comprising a FUN protein having altered activation by nitrate; as well as b) growing the genetically modified plant under conditions of nitrate concentration, wherein the genetically modified plant has reduced FUN activity or expression and / or reduced FUN active form compared to a WT plant grown under the same nitrate conditions, optionally wherein (i) altering the activity of the FUN protein by nitrate comprises downregulating FUN, reducing the activity of FUN, knocking out FUN by mutation, knocking down the expression of FUN, knocking out or disrupting the promoter element of FUN, or a combination thereof; (ii) altering FUN protein activity by nitrate involves manipulating environmental or cellular zinc or manganese levels, wherein said manipulation results in the maintenance of the FUN protein in an inactive filamentous form; or (iii) Altering the activity of the FUN protein by nitrate involves genetically modifying the FUN protein sequence to alter its sensitivity to zinc or manganese.

20. A method of making a genetically altered plant having enhanced nitrogen fixation under conditions comprising nitrate levels around the plant's roots that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic alterations that reduce the activity or expression of a FUN protein as compared to the activity or expression of the FUN protein in a control plant grown under the same conditions.

21. A method for making a genetically altered plant having enhanced nitrogen fixation under conditions including nitrate levels around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or part thereof one or more genetic alterations that reduce the activity or expression of one or more of the NRT3.1 protein, bZIP28 protein, NAC domain-containing protein, HO1 protein, NRT2.1 protein, or AS1 protein compared to a control plant grown under the same conditions.

22. A method of making a genetically altered plant having increased nitrogen fixation under conditions comprising nitrate levels around the roots of the plant that inhibit nitrogen fixation, the method comprising introducing into the plant or a part thereof one or more genetic alterations that reduce the activity or expression of one or more of a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein, as compared to the activity or expression of the FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein in a control plant grown under the same conditions.

23. A method for preparing a genetically modified plant or part thereof according to any one of claims 1 to 9, the method comprising: A genetic alteration is introduced into a plant cell that reduces or knocks out the activity or expression of a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein, wherein the genetic alteration comprises a first nucleic acid sequence capable of reducing or knocking out a second nucleic acid sequence encoding a FUN protein, a FUN-like protein, a NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, a NRT2.1 protein, or an AS1 protein operably linked to a promoter, and wherein the genetically altered plant is selected from one or more of the group consisting of alfalfa, Bambara peanut, beans (e.g., kidney beans, black beans, etc.), blackcurrants, chickpeas, clover, cowpeas, forage legumes, legume trees, lentils, lotus plants, lupins, Medicago spp., peas, peanuts, pigeon peas, soybeans, Sophora japonica, alder, and elm.

24. A method for preparing a genetically modified plant or part thereof according to any one of claims 1 to 17, the method comprising genetically modifying a plant cell by transforming the plant cell with one or more gene editing components, wherein the gene editing components target an endogenous nuclear genomic sequence encoding a FUN protein, a FUN-like protein, an NRT3.1 protein, a bZIP28 protein, a NAC domain-containing protein, a HO1 protein, an NRT2.1 protein or an AS1 protein, wherein the endogenous nuclear genomic sequence or a portion thereof is knocked out, wherein the one or more gene editing components comprise a ribonucleoprotein complex that targets the nuclear genomic sequence; a vector comprising a TALEN protein coding sequence, wherein the TALEN protein targets the nuclear genomic sequence; a vector comprising a ZFN protein coding sequence, wherein the ZFN protein targets the nuclear genomic sequence; an oligonucleotide donor (OND), wherein the OND targets the nuclear genomic sequence; or a vector CRISPR / Cas enzyme coding sequence and a targeting sequence, wherein the targeting sequence targets the nuclear genomic sequence.

25. An expression vector or isolated DNA molecule comprising (i) one or more nucleotide sequences encoding a FUN protein, a FUN-like protein, a HO1 protein, a protein containing an NAC domain, a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence, and (ii) a nucleotide sequence encoding a FUN protein, a FUN-like protein, a HO1 protein, a protein containing an NAC domain, a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence, and (ii) a nucleotide sequence encoding a FUN protein, a FUN-like protein, a HO1 protein, a protein containing an NAC domain, a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the nucleotide sequence is capable of reducing or knocking out the expression of the protein. or (iii) one or more nucleotide sequences comprising a nucleic acid sequence comprising a combination thereof, wherein the one or more nucleotide sequences are operably linked to at least one expression control sequence, or (iv) one or more nucleotide sequences comprising a mutation in a gene for a FUN protein, a FUN-like protein, a HO1 protein, a NAC domain-containing protein, a bZIP28 protein, a NRT2.1 protein, a NRT3.1 protein, an AS1 protein, or a combination thereof, wherein the mutation reduces or knocks out the activity or expression of the protein, and the one or more nucleotide sequences are operably linked to at least one homologous nucleic acid sequence that hybridizes adjacent to the mutation site in the gene.

26. The expression vector or isolated DNA molecule of claim 25, wherein the protein is a FUN protein, and wherein the FUN protein comprises a residue selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO:

82. NO:82, wherein the protein of the group has a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity; wherein the FUN protein includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 NO:81 and SEQ ID NO:82; or wherein the FUN protein comprises SEQ ID NO:1, SEQ ID NO:8 or SEQ ID NO:9; wherein the protein is a FUN-like protein, and wherein the FUN-like protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a protein selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 83, and SEQ ID NO: 84; wherein the FUN-like protein comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 83, or SEQ ID NO: 84; and / or wherein the protein is an NRT3.1 protein, and wherein the NRT3.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 74; wherein the protein is a bZIP28 protein, and wherein the bZIP28 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO: 75; wherein the protein is an NAC domain-containing protein, and wherein the NAC domain-containing protein comprises a polypeptide selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73 group of proteins having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to a polypeptide; wherein the protein is HO1 protein, and wherein the HO1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:77; wherein the protein is NRT2.1 protein, and wherein the NRT2.1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:78; or wherein the protein is an AS1 protein, and wherein the AS1 protein comprises a polypeptide having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO:79; wherein the NRT3.1 protein comprises SEQ ID NO:74; wherein the bZIP28 protein comprises SEQ ID NO:75; wherein the NAC domain-containing protein comprises SEQ ID NO:76, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:51 NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 or SEQ ID NO:73; wherein the HO1 protein comprises SEQ ID NO:77; wherein the NRT2.1 protein comprises SEQ ID NO:78, or wherein the AS1 protein comprises SEQ ID NO:

79. NO:79; or wherein the protein containing the NAC domain comprises SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:

42.

27. A bacterial cell or Agrobacterium cell comprising the expression vector or isolated DNA molecule according to claim 25 or claim 26.

28. A genetically modified plant, plant part, plant cell or seed comprising the expression vector or isolated DNA molecule of claim 25 or claim 26.

29. A kit comprising the expression vector or isolated DNA molecule according to claim 25 or claim 26 or the bacterial cell or Agrobacterium cell according to claim 27.

30. A method for increasing nitrogen fixation, delaying nodulation senescence, or inducing FUN filamentation in a plant, the method comprising: (a) introducing a genetic alteration by means of an expression vector or an isolated DNA molecule according to claim 25 or claim 26; and optionally (b) treating the plant with zinc or manganese or growing the plant under high zinc, high manganese or high nitrate conditions.

31. A genetically altered plant genome comprising: (i) one or more genetic alterations in the genetically modified plant or part thereof according to any one of claims 1-9, or (ii) one or more genetic alterations in the genetically modified plant or part thereof produced by the method according to any one of claims 20-24.

32. Non-regenerable parts or cells of the genetically modified plant or part thereof according to any one of claims 1 to 9.

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