Use of a PTXD / phosphite system to enhance plant or seed performance

Abstract

The present disclosure pertains to a method of treating a plant or seed by applying a composition to the plant or seed. The composition may include a phosphite (Phi), such as H2PO3, [HPO3]-2, salts thereof, or combinations thereof. The composition may also include an orthophosphate (Pi), such as [PO4]-3, [HPO4]-2, [H2PO4]-, H3PO4, salts thereof, or combinations thereof. The present disclosure also pertains to plants or seeds that have been treated with the compositions of the present disclosure. The plant and seeds of the present disclosure may express a phosphite oxidoreductase-encoding gene, such as ptxD. The methods of the present disclosure may also include a step of introducing a phosphite oxidoreductase-encoding gene into a plant or seed of the present disclosure.

Description

PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128TITLEUse of a ptxD / phosphite system to enhance plant or seed performanceCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 686,746, filed on August 24, 2024. The entirety of the aforementioned application is incorporated herein by reference.SEQUENCE DISCLOSURE STATEMENT

[0002] Pursuant to 37 C.F.R. § 1 .834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF13368.P078WO.xml”. The date of the creation of the Sequence Listing is August 25, 2025. The size of the Sequence Listing is 9,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing.BACKGROUND

[0003] A need exists for more efficient production of crops in various environments. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to a method of treating a plant or seed. In some embodiments, such methods include applying a composition to the plant or seed. In some embodiments, the composition includes a phosphite (Phi), such as H2PO3 , [HPO3]2, salts thereof, or combinations thereof. In some embodiments, the composition also includes an orthophosphate (Pi), such as [PO4]'3, [HPO4]'2, [H2PO4]', H3PO4, salts thereof, or combinations thereof.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0005] The compositions of the present disclosure can have various effects on plants and seeds. For instance, in some embodiments, the compositions of the present disclosure enhance the growth rate of a plant or seed relative to untreated plants or seeds. In some embodiments, the compositions of the present disclosure enhance a plant’s or seed’s efficiency in utilizing nitrogen and / or phosphorus relative to untreated plants or seeds. In some embodiments, the compositions of the present disclosure enhance a plant’s or seed’s resistance to one or more sources of environmental stress relative to untreated plants or seeds. In some embodiments, the compositions of the present disclosure reduce the growth of weeds along with a plant or seed relative to untreated plants or seeds.

[0006] Additional embodiments of the present disclosure pertain to plants or seeds that have been treated with the compositions of the present disclosure. In some embodiments, the plants and seeds of the present disclosure include soybean. In some embodiments, the plant and seeds of the present disclosure include a phosphite oxidoreductase-encoding gene, such as ptxD. In some embodiments, the methods of the present disclosure also include a step of introducing a phosphite oxidoreductase-encoding gene into a plant or seed of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] A more complete understanding of the subject matter of the present disclosure may be obtained by reference to the following Detailed Description when taken in conjunction with the accompanying Drawings In some embodiments:

[0008] FIG. 1 illustrates a method of treating a plant or seed in accordance with various embodiments of the present disclosure.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0009] FIGS. 2A-G illustrate the generation and selection of transgenic soybean plants expressing the ptxD gene, using phosphite (Phi) as the phosphorus source. FIG. 2A shows a map of the gene construct harboring the codon-optimized ptxD sequence under control of the CaMV35S promoter. FIG. 2B shows a T1 progeny screened in the greenhouse using foliar application of BASTA® (glufosinate ammonium, 1% v / v) to identify herbicide-resistant events. FIG. 2C shows a T1 herbicide-resistant plant showing normal growth after BASTA selection. FIGS. 2D-F shows null or wild-type (Wt) and T2 ptxD progeny seedlings grown using Phi as the only phosphorus source and selection. ptxD transgenic lines exhibit healthy leaf development, whereas non-transgenic controls present chlorosis and necrosis. Representative photographs of Wt and ptxD plants to show (FIG. 2E) impaired root development and (FIG. 2F) foliar damage of Wt due to Phi treatment. FIG. 2G shows quantitative real-time PCR of ptxD transgenic lines and the wild type to determine ptxD expression levels. The genes GmACT2 and G / nACTl l were used as internal controls. All samples were processed with three experimental replicas each. Results are expressed as 2A(-AA Cq).

[0010] FIGS. 2H-M show comparative growth and yield traits of ptxD and wild-type (Wt) soybean plants under phosphate fertilization (100 ppm). FIG. 2H shows representative ptxD plant (top panel) and harvested seeds (bottom panel) at maturity. FIG. 21 shows representative Wt plant (top panel) and harvested seeds (bottom panel) at maturity. Scale bar = 2 cm. A U.S. quarter coin (24.26 mm diameter) is included for reference. FIG. 2J shows total pods per plant. FIG. 2K shows total seed yield per plant. FIG. 2L shows weight of 100 seeds. FIG. 2M shows plant height before harvesting (cm), n = 3; no statistical differences were found.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0011] FIGS. 3A-D illustrate representative photographs of soybean under different phosphorus regimens. Plants were grown under controlled conditions and fertilized as follows: optimal phosphate (HPi, Pi 100 ppm), low phosphate (LPi, 0 added Pi), phosphate and phosphite (HPi + Phi, Pi 75 ppm + Phi 25 ppm), and only phosphite (LPi + HPhi, 0 Added Pi + Phi 100 ppm), and imaged using the PhenoAxpert LemnaTec HTC system once a week, during five consecutive weeks after treatment application. Representative photographs of the lateral view and canopy of (FIG. 3A) wild-type (Wt) and (FIG. 3B) ptxD expressing soybean plants are shown. The (FIG. 3C) height (cm) and (FIG. 3D) canopy area (cm2) for all plants were calculated using RGB imagery.

[0012] FIGS. 4A-C illustrate high-throughput phenotyping and hyperspectral imaging. FIG. 4C shows a flowchart of the steps followed to analyze the high-throughput phenotyping and hyperspectral data. Systematic comparison of all wavelength pairwise combinations to generate a hyperspectral landscape and highlight differences between treatments in (FIG. 4B) wild-type (Wt) and (FIG. 4C) ptxD soybean plants after 5 weeks of the treatments.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0013] FIGS. 5A-B illustrate that Phi treatment modulates the expression of key components in the putative STOP1 and PHR1 pathways in G. max. Schematic representation of gene expression changes in response to phosphite (Phi) treatment, highlighting the regulation of key genes associated with phosphate starvation responses (PSRs) in the STOP1 and PHR1 pathways. Heatmaps represent expression changes (Z-scores) for individual genes under different phosphate and Phi treatment conditions. FIG. 5A shows a STOP putative regulatory pathway based on Arabidopsis pathway. FIG. 5B shows a PHR putative regulatory pathway based on Arabidopsis pathway. The homologous Arabidopsis genes displayed are: SAP AND MIZ DOMAIN 1 (SIZ1), REGULATION OF ATALMT1 EXPRESSION 1 (RAE1), MEDIATOR SUBUNIT16 (MED16), SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1), ALUMINUM- ACTIVATED MALATE TRANSPORTER 1 (ALMT1), LOW PHOSPHATE ROOT 1 (LPR1), LOW PHOSPHATE ROOT 2 (LPR2), CLAVATA3 / ESR-related genes (CLEs), VIP HOMOLOG 1 (VIH1), VIP HOMOLOG 2 (VIH2), INOSITOL- TETRAKISPHOSPHATE 1-KINASE 1 (ITPK1), SYG1 / Pho81 / XPR1 (SPX1 SPX2 SPX3, and SPX4), Phosphate 2 (PHO2), PHOSPHATE RESPONSE 1 (PHR1), and PHRl-like (PHL1).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0014] FIGS. 6A-F illustrate phosphite treatment differentially modulates canonical phosphate starvation responses (PSRs) depending on phosphate levels. FIG. 6A provides a heatmap showing the upregulated genes in plants in low phosphate ( LPi) relative to optimal phosphate (HPi) that are attenuated in the LPi + Phi relative to the HPi treatment. Counts were normalized with Z-score. FIG. 6B shows a Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) attenuated by LPi + Phi treatment, with the percentage of significant genes contributing to each category. Determination of (FIG. 6C) Glutathione peroxidase (GPX) activity, and (FIG. 6D) H2O2 content on leaves of wild-type (Wt) and and ptxD soybean plants grown under the different treatments after 5 weeks under stress. All samples and blanks were analyzed in triplicate and averaged to get a single measurement per sample; four biological replicates were processed per group. FIG. 6E shows a Venn diagram of DEGs across treatments (LPi vs. HPi, LPi + Phi vs. HPi, and HPi + Phi vs. HPi) in Wt plants. FIG. 6F shows a GO enrichment analysis of Phi- activated DEGs in HPi (intersection LPi vs. HPi & HPi + Phi), with the number of significant genes contributing to each category.

[0015] FIGS. 7A-C illustrate effects of phosphite (Phi) treatment on the biosynthesis and signaling of abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA)-related genes. In all cases, the left panel shows the Z-score distribution of genes involved in (FIG. 7A) ABA, (FIG. 7B) JA, and (FIG. 7C) SA signaling across different treatments. The middle panel represents the expression patterns of genes associated biosynthesis, and the right panel presents a heatmap of Z-score- normalized expression values for key enzymes involved in the biosynthetic pathway for the specific plant hormones.

[0016] FIG. 7D shows relative abundance of phosphonic acid negative ionic species [M-H]' =80.9746 in the samples of wild type (WT) and ptxD transgenic soybean plants under the following treatments: optimal phosphate (HPi, Pi 100 ppm), low phosphate (LPi, 0 added Pi), phosphate and phosphite (HPi + Phi, Pi 75 ppm + Phi 25 ppm), and only phosphite (LPi + HPhi, 0 Added Pi + Phi 100 ppm).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0017] FIGS. 8A-I illustrate correlation between phosphite (Phi) accumulation in leaves and defense-related metabolites under different phosphate (Pi) and Phi treatments in wild-type (Wt) plants. Scatter plots display the correlation (Pearson’s correlation coefficient, r) between Phi accumulation and the normalized abundance of various defense-related metabolites across different treatment. Shown are (FIG. 8A) mannitol- 1 -phosphate, (FIG. 8B) sorbitol, (FIG. 8C) a- trehalose, (FIG. 8D) oxo-proline, (FIG. 8E) 1-proline, (FIG. 8F) 1-lysine, (FIG. 8G) D- phenylalanine, (FIG. 8H) coumaroyl, (FIG. 81) coumaroyl quinic acid, (FIG. 8J) phenethyl-2- primeveroside, (FIG. 8K) 2-phenylethanol, and (FIG. 8L) salicyolyl aspartate. The correlation coefficient (r) and corresponding p-value for each metabolite are indicated in the top left comer of each panel.

[0018] FIGS. 9A-D show the effect of phosphite (Phi) treatment and water limitation on plant height and canopy area in wild-type (Wt) and ptxD soybean plants. Shown are representative photographs of Wt (FIG. 9A) and ptxD plants (FIG. 9B) under the different stresses. Plant height (mm) and canopy area (cm2) of Wt (FIG. 9C) and ptxD plants (FIG. 9D) are also shown. Soybean lines grown under high phosphate (HPi) or low phosphate (LPi) conditions, with (+Phi) or without (-Phi) phosphite supplementation, under well-watered (WC80, cyan) and water-limited conditions (WC30, yellow). Bars represent mean ± SE (n = 4). Letters indicate statistically significant differences between treatments according to Tukey’s HSD test (p < 0.05).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0019] FIGS. 10A-E show transcriptomic responses to water deficit and phosphite (Phi) supplementation in wild-type soybean. FIGS. 10A-C show Gene ontology (GO) enrichment analysis of biological processes among differentially expressed genes (DEGs) between water treatments (80 or 30% water content, WC) and Phi treatment comparisons. Shown are (FIG. 10A) HPi_WC30 vs. HPi_WC80; (FIG. 10B) HPi + Phi_WC30 vs. HPi_WC80; and (FIG. 10C) HPi + Phi_WC80 vs. HPi_WC80. Dot size represents the number of DEGs, and color intensity indicates the proportion of significant genes per term. FIG. 10D shows Venn diagrams showing overlap of DEGs under water treatment and Phi conditions. The Venn diagram compares drought (HPi_WC30), Phi-supplemented / water-limited (HPi + HPhi_WC30), and Phi- supplemented well- watered (HPi + HPhi_WC80) conditions against HPi_WC80. Color indicates Z score of gene expression. FIG. 10E shows GO enrichment analysis of shared DEGs between the three conditions.

[0020] FIGS. 11A-D show a Venn diagram showing overlap of DEGs under water treatment and Phi conditions (FIG. 11A), which compares LPi vs. HPi and HPi_WC30 vs. HPi_WC80 (FIG. 11B). FIGS. 11C-D show heatmaps showing expression profiles of DEGs in (FIG. 11C) LPi_WC80 vs. HPi_WC80, and (FIG. 11D) HPi_WC30 vs. HPi_WC80. Color indicates Z score of gene expression.

[0021] FIGS. 12A-D show the effect of phosphite (Phi) treatment and high temperature on plant height and canopy area in wild-type (Wt) and ptxD soybean plants. FIGS. 12A-B show representative photographs of Wt (FIG. 12A) and ptxD plants (FIG. 12B) under the different stresses. FIGS. 12C-12D show plant height (mm) and canopy area (cm2) of Wt (FIG. 12C) and ptxD plants (FIG. 12D). Soybean lines grown under high phosphate (HPi) or low phosphate (LPi) conditions, with (+Phi) or without (-Phi) phosphite supplementation, under optimal temperature (25°C, green) and high temperature (40 °C, red) conditions. Bars represent mean ± SE (n = 4). Letters indicate statistically significant differences between treatments according to Tukey’s HSD test (p < 0.05).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0022] FIGS. 13A-G show tran scrip tomic responses to high temperature stress and phosphite (Phi) supplementation in soybean. FIGS. 13A-C show Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in wild-type (Wt) soybean plants under various treatments. Shown are (FIG. 13A) HPi_40C vs. HPi 25C; (FIG. 13B) HPi + Phi_40C vs. HPi_25C; and (FIG. 13C) HPi + Phi_25C vs. Hpi_25C. Bubble size indicates the number of significant genes per GO term, while color represents the proportion of significant genes in each category. Also shown are Venn diagrams showing the overlap of DEGs between (FIG. 13D) high temperature stress (40C), Phi at 25 °C (Phi_25C), and their combination under HPi conditions (HPi + Phi_40C) in Wt plants; and (FIG. 13F) high temperature stress with optimal phosphate (Pi) fertilization (HPi_40C), Pi starvation treated ith Phi (LPi + Phi) at 25°C and LPi treated with Phi under high temperature stress (LPi + Phi_40C) in ptxD plants. FIGS. 13E and 13G show GO enrichment analysis of shared DEGs between the three conditions in FIGS. 13D and 13F. Dot size represents the number of DEGs, and color intensity indicates the proportion of significant genes per term. Color scale represents Z-score expression values.

[0023] FIGS. 14A-F show differentially expressed transcription factors (TFs) under low phosphorus and water-limited conditions in wild-type (Wt) soybean. FIG. 14A shows Venn diagrams showing the number and overlap of TFs downregulated under HPi + Phi_WC80, LPi_WC80, and HPi + Phi_WC30. FIG. 14B shows Venn diagrams showing the number and overlap of TFs upregulated under HPi + Phi_ WC80, Lpi_WC80, and HPi + Phi_WC30. FIGS. 14C and 14D show Gene Ontology (GO) enrichment analysis of differentially expressed TFs from the shared genes in all treatments in FIG. 14A and FIG. 14B. Bubble size indicates number of genes; bubble color represents the percentage of significant genes per term. FIGS. 14E-F show classification of up and downregulated TFs based on TF family.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0024] FIGS. 15A-F show differentially expressed transcription factors (TFs) under low phosphorus and high temperature conditions in wild-type (Wt) soybean. FIG. 15A shows Venn diagrams showing the number and overlap of transcription factor genes downregulated under LPi_25C, HPi_40C, and HPi + Phi_25C. FIG. 15B shows Venn diagrams showing the number and overlap of TFs upregulated under LPi_25C, HPi_40C, and HPi + Phi_25C. FIGS. 15C-D show Gene ontology (GO) enrichment analysis of differentially expressed TFs from the shared genes in the intersection of LPi_25C, HPi_40C, and HPi + Phi_25C. Bubble size indicates number of genes; bubble color represents the percentage of significant genes per term. FIGS. 15E-F show classification of up and downregulated TFs based on TF family.

[0025] FIGS. 15G-J show best-ranked transcription factors (TFs) in stress experiments. Shown are best-ranked TFs using the robust rank aggregation method in multiple network parameters and the number of interactor genes that are upregulated under stress in the low irrigation experiments (FIG. 15G), and in the high temperature experiments (FIG. 15H). FIG. 151 shows a bubble plot of the enrichment analysis of the GO terms of the interactor genes of the top 10 TFs showed in the low irrigation experiment (FIG. 15G). FIG. 15 J is the same as FIG. 151 but from the high heat experiment (FIG. 15H).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0026] FIGS. 16A-F show shared and unique transcriptomic responses to phosphite (Phi) under high temperature and water limitation in wild-type (Wt) and ptxD soybean plants. FIGS. 16A-B show Venn diagrams showing the overlap of upregulated differentially expressed genes (DEGs) in response to phosphite (Phi) under water limitation (FIG. 16A) and high temperature (FIG. 16B) stress. Comparisons were made between Phi-treated in HPi conditions Wt vs. ptxD plants under Phi only fertilization. FIGS. 16C-D show Gene Ontology (GO) enrichment of shared DEGs under water limitation (FIG. 16C) and high temperature (FIG. 16D) stress. Bubble color represents the proportion of significant genes, and bubble size indicates the number of DEGs per GO category. FIG. 16E shows a rain plot and heatmap of the expression values of the overlap of upregulated differentially expressed genes (DEGs) in response to phosphite (Phi) under water limitation. FIG. 16F shows Rain plot and heatmap of the expression values of the overlap of upregulated differentially expressed genes (DEGs) in response to phosphite (Phi).

[0027] FIGS. 17A-F show metabolomic enrichment analysis under drought, high-temperature, and phosphite (Phi) treatment in Wt soybean plants. Shown are scatterplots displaying enriched metabolite main classes according to the “Chemical structure” metabolites set in Metaboanalyst. FIGS. 17A-B show metabolic changes under the low irrigation experiments, including HPi (FIG. 17A) and (FIG. 17B) under HPi + Phi_WC30 conditions. FIG. 17C shows a comparison between HPi + Phi_WC80 and HPi_WC80. FIGS. 17D-F show metabolic changes in response to high- temperature stress (FIG. 17D) in HPi, exposure to high temperature and Phi treatment (FIG. 17E), and in (FIG. 17F) Phi supplementation at 25 °C.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128DETAILED DESCRIPTION[00281 It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0029] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0030] Crop production is essential to national and global food security. In particular, crop production provides the essential nutrients and calories that support human life and livestock feed.

[0031] For instance, soybean holds immense economic significance both in the United States and globally, serving as a cornerstone of agricultural production and trade. In the U.S., soybeans are one of the most valuable crops, with the U.S. being the largest global producer and exporter, accounting for over 34% of the world's soybean production.

[0032] However, a need exists for more efficient production of crops in various environments, such as environments that are vulnerable to heat, drought, and other sources of environmental stress. Numerous embodiments of the present disclosure aim to address the aforementioned need.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0033] In some embodiments, the present disclosure pertains to methods of treating a plant or seed. In some embodiments illustrated in FIG. 1, such methods include applying a composition to the plant or seed (step 10). In some embodiments, the composition includes a phosphite (Phi). In some embodiments, the composition can have various effects on the plant or seed, such as enhanced growth rate (step 12), enhanced resistance to environmental stress (step 14), and / or enhanced nitrogen and / or phosphorous utilization (step 16). Additional embodiments of the present disclosure pertain to plants and seeds that have been treated with the compositions of the present disclosure.

[0034] As set forth in more detail herein, various methods and compositions may be utilized to treat various plants and seeds. Moreover, the plants and seeds of the present disclosure can have various advantageous properties.

[0035] Compositions

[0036] The plants and seeds of the present disclosure may be exposed to various compositions. For instance, in some embodiments, the compositions of the present disclosure include a phosphite (Phi). In some embodiments, the phosphite includes, without limitation, H2PO3 , [ HPC ]’2, salts thereof, or combinations thereof. In some embodiments, the phosphite formulations can also include formulations based on potassium, sodium, calcium, ammonium, sodium, and aluminum. In some embodiments, the formulations can be made from phosphorous acid and other derivatives. In some embodiments, the phosphite includes without limitation, KH2PO3, NH4H2PO3, Na2HPC>3, CaHPCh, or combinations thereof. In some embodiments, the phosphite includes KH2PO3.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0037] The compositions of the present disclosure can include various concentrations of phosphites. For instance in some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 50 pM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 100 pM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 200 pM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 500 pM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 750 pM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 1 mM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 2.5 mM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 4 mM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 7.5 mM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 10 mM. In some embodiments, the compositions of the present disclosure include phosphite concentrations of at least 20 mM. In some embodiments, the compositions of the present disclosure include phosphite concentrations ranging from 50 pM to 1 mM._In some embodiments, the compositions of the present disclosure include phosphite concentrations ranging from 50 pM to 4.7 mM. In some embodiments, the compositions of the present disclosure include phosphite concentrations ranging from 10 to 150 parts per million when applied directly to soil.

[0038] In some embodiments, the compositions of the present disclosure also include an orthophosphate (Pi). In some embodiments, the compositions of the present disclosure include a mixture of phosphate and orthophosphate. In some embodiments, the orthophosphate includes, without limitation, [PCh]'3, [HPC ]'2, [FfoPC ’, H3PO4, salts thereof, or combinations thereof. In some embodiments, the orthophosphate includes KH2PO4. In some embodiments, the orthophosphate includes, without limitation, KH2PO4, NH4H2PO4, Na2HPO4, CaHPC , or combinations thereof.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0039] The compositions of the present disclosure can include various concentrations of orthophosphates. For instance in some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 10 pM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 50 pM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 100 pM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 150 pM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 200 pM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 500 pM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 750 pM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 1 mM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 2.5 mM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 4 mM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 7.5 mM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 10 mM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations of at least 20 mM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations ranging from 10 pM to 1 mM. In some embodiments, the compositions of the present disclosure include orthophosphate concentrations ranging from 10 pM to 4.7 mM.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0040] The compositions of the present disclosure can be in various forms. For instance, in some embodiments, the compositions of the present disclosure are in the form of a fertilizer composition. In some embodiments, the fertilizer compositions also include nitrogen (N), phosphorus (P), and potassium (K). In some embodiments, the fertilizer compositions also include calcium, magnesium, sulfur, boron, copper, iron, manganese, zinc, chlorine, cobalt, or combinations thereof.

[0041] The compositions of the present disclosure can have various properties. For instance, in some embodiments, the compositions of the present disclosure have at least one of pesticidal activities, herbicidal activities, anti-microbial activities, nematocidal activities, fungicidal activities, bactericidal activities, algicidal activities, anti-weed activities, or combinations thereof.

[0042] Application of compositions to plants and seeds

[0043] The compositions of the present disclosure may be applied to plants and seeds in various manners. For instance, in some embodiments, the application includes spraying the composition onto a plant or seed. In some embodiments, the application includes incubating the plant or seed with the composition. In some embodiments, the application includes applying the composition onto a field, soil or substrate that includes the plant or seed. In some embodiments, the application includes liquid or granular formulations based on phosphites, and can be applied pre- or postsowing seed and pre- or post-transplanting seedlings. In some embodiments, the applying includes applying the composition onto a plant as a foliar application, or onto a seed as a seed coat or soaking treatment.

[0044] Effects of compositions on plants and seedsPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0045] The compositions of the present disclosure can have various effects on plants and seeds. For instance, in some embodiments, the compositions of the present disclosure enhance the growth rate of a plant or seed relative to untreated plants or seeds. In some embodiments, the plants and seeds of the present disclosure demonstrate such enhanced growth rates relative to untreated plants and seeds. In some embodiments, the enhanced growth rate of a plant or seed of the present disclosure relative to untreated plants or seeds is at least 25%. In some embodiments, the enhanced growth rate of a plant or seed of the present disclosure relative to untreated plants or seeds is at least 50%. In some embodiments, the enhanced growth rate of a plant or seed of the present disclosure relative to untreated plants or seeds is at least 100%.

[0046] In some embodiments, the compositions of the present disclosure enhance a plant’s or seed’s efficiency in utilizing nitrogen and / or phosphorous relative to untreated plants or seeds. In some embodiments, the compositions of the present disclosure enhance a plant’s or seed’s efficiency in utilizing nitrogen relative to untreated plants or seeds. In some embodiments, the plants and seeds of the present disclosure demonstrate such enhanced nitrogen utilization relative to untreated plants or seeds. In some embodiments, the nitrogen uptake of a plant or seed of the present disclosure is at least 25% higher than the nitrogen uptake of untreated plants or seeds. In some embodiments, the nitrogen uptake of a plant or seed of the present disclosure is at least 50% higher than the nitrogen uptake of untreated plants or seeds. In some embodiments, the nitrogen uptake of a plant or seed of the present disclosure is at least 75% higher than the nitrogen uptake of untreated plants or seeds.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0047] In some embodiments, the compositions of the present disclosure enhance a plant’s or seed’s efficiency in utilizing phosphorous relative to untreated plants or seeds. In some embodiments, the plants and seeds of the present disclosure demonstrate such enhanced phosphorous utilization relative to untreated plants or seeds. In some embodiments, the phosphorous uptake of a plant or seed of the present disclosure is at least 25% higher than the phosphorous uptake of untreated plants or seeds. In some embodiments, the phosphorous uptake of a plant or seed of the present disclosure is at least 50% higher than the phosphorous uptake of untreated plants or seeds. In some embodiments, the phosphorous uptake of a plant or seed of the present disclosure is at least 75% higher than the phosphorous uptake of untreated plants or seeds.

[0048] In some embodiments, the compositions of the present disclosure enhance a plant’s or seed’s resistance to one or more sources of environmental stress relative to untreated plants or seeds. In some embodiments, the plants and seeds of the present disclosure demonstrate such enhanced resistance to one or more sources of environmental stress relative to untreated plants and seeds. In some embodiments, the one or more sources of environmental stress includes, without limitation, abiotic stress, biotic stress, drought, heat, or combinations thereof.

[0049] In some embodiments, the one or more sources of environmental stress includes biotic stress. In some embodiments, the biotic stress includes, without limitation, microbial infections, viral infections, fungal infections, bacterial infections, or combinations thereof.

[0050] In some embodiments, the one or more sources of environmental stress includes abiotic stress. In some embodiments, the abiotic stress includes, without limitation, drought, heat, or combinations thereof.

[0051] In some embodiments, the one or more sources of environmental stress includes drought. In some embodiments, drought is represented by lack of water or rainfall for at least 3 days. In some embodiments, drought is represented by lack of water or rainfall for at least 5 days. In some embodiments, drought is represented by lack of water or rainfall for at least 7 days. In some embodiments, drought is represented by lack of water or rainfall for at least 10 days.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0052] In some embodiments, the one or more sources of environmental stress includes heat. In some embodiments, the heat includes heat at temperatures of at least 40°C for at least 2 days. In some embodiments, the heat includes heat at temperatures of at least 50°C for at least 2 days. In some embodiments, the heat includes heat at temperatures of at least 60°C for at least 2 days. In some embodiments, the heat includes heat at temperatures of at least 70°C for at least 2 days.

[0053] In some embodiments, the compositions of the present disclosure reduce the growth of weeds along with a plant or seed relative to untreated plants or seeds. In some embodiments, such weeds can include, without limitation, Palmer amaranth ( Amamnlhus palmerip Morning glory (Ipomoea sppAp Johnson grass (Sorghum halepense), Green foxtail (Setaria viridis), Barnyard grass (Echinochloa spp.), or combinations thereof.

[0054] Plants or seeds

[0055] The methods of the present disclosure can be utilized to treat various plants and seeds. Additionally, the plants and seeds of the present disclosure can include various species.

[0056] For instance, in some embodiments, the plants and seeds of the present disclosure include, without limitation, crops, weeds, a legume, maize, rice, soybean, cotton, wheat, N. benthamiana, Arabidopsis, Amaranthus palmeri, tobacco, tomato, lettuce, common beans, potato, grapes, sorghum, onion, strawberry, banana, spinach, mustard, varieties thereof, or combinations thereof. In some embodiments, the plants and seeds of the present disclosure includes a legume. In some embodiments, the plants and seeds of the present disclosure include soybean.

[0057] In some embodiments, the plants and seeds of the present disclosure include various parts. In some embodiments, the parts include tubers.

[0058] The plants and seeds of the present disclosure can have various applications. For instance, in some embodiments, the plants and seeds of the present disclosure (including plant parts) can be used for food, feed, biomolecule farming, bioactive molecule production, or combinations thereof.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0059] In some embodiments, the plants and seeds of the present disclosure include a phosphite oxidoreductase-encoding gene. In some embodiments, the phosphite oxidoreductase-encoding gene expresses a phosphite oxidoreductase. In some embodiments, the phosphite oxidoreductase- encoding gene is expressed exogenously in the plant or seed. In some embodiments, the phosphite oxidoreductase-encoding gene is expressed heterologously in the plant or seed. In some embodiments, the phosphite oxidoreductase-encoding gene is overexpressed in the plant or seed. For instance, in some embodiments, the phosphite oxidoreductase-encoding gene is overexpressed in an expression vector that is introduced into the plant or seed.

[0060] In some embodiments, the plants and seeds of the present disclosure include a transgenic crop that expresses a phosphite oxidoreductase-encoding gene. In some embodiments, the transgenic crop includes, without limitation, maize, a legume, soybean, tobacco, cotton, tomato, sorghum, potato, pepper, rice, wheat, lettuce, common beans, grapes, onion, strawberry, banana, spinach, mustard, varieties thereof, or combinations thereof. In some embodiments, the transgenic crop includes a legume. In some embodiments, the transgenic crop includes soybean.

[0061] In some embodiments, the phosphite oxidoreductase-encoding gene includes a ptxD gene. In some embodiments, the ptxD gene is a codon-optimized ptxD gene. In some embodiments, the ptxD gene includes SEQ ID NO: 1. In some embodiments, the ptxD gene includes a sequence with at least 65% sequence identity to SEQ ID NO: 1. In some embodiments, the ptxD gene includes a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the ptxD gene includes a sequence with at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, the ptxD gene includes a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the ptxD gene includes a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the ptxD gene includes a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the ptxD gene includes a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the ptxD gene includes a sequence with at least 99% sequence identity to SEQ ID NO: 1.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0062] In some embodiments, the ptxD gene includes SEQ ID NO: 2. In some embodiments, the ptxD gene includes a sequence with at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the ptxD gene includes a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the ptxD gene includes a sequence with at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, the ptxD gene includes a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the ptxD gene includes a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the ptxD gene includes a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the ptxD gene includes a sequence with at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the ptxD gene includes a sequence with at least 99% sequence identity to SEQ ID NO: 2.

[0063] In some embodiments, the ptxD gene includes SEQ ID NO: 3. In some embodiments, the ptxD gene includes a sequence with at least 65% sequence identity to SEQ ID NO: 3. In some embodiments, the ptxD gene includes a sequence with at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the ptxD gene includes a sequence with at least 75% sequence identity to SEQ ID NO: 3. In some embodiments, the ptxD gene includes a sequence with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the ptxD gene includes a sequence with at least 85% sequence identity to SEQ ID NO: 3. In some embodiments, the ptxD gene includes a sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the ptxD gene includes a sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the ptxD gene includes a sequence with at least 99% sequence identity to SEQ ID NO: 3.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0064] The phosphite oxidoreductase-encoding genes of the present disclosure may express various phosphite oxidoreductases. For instance, in some embodiments, the expressed phosphite oxidoreductase includes one or more mutations that enable more efficient use of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as cofactors. In some embodiments, the expressed phosphite oxidoreductase includes SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 50% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 55% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 60% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 65% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 75% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 85% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 99% sequence identity to SEQ ID NO: 4.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0065] In some embodiments, the expressed phosphite oxidoreductase includes SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 50% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 55% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 60% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 65% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 75% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 85% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the expressed phosphite oxidoreductase includes a sequence with at least 99% sequence identity to SEQ ID NO: 5.

[0066] In some embodiments, the expressed phosphite oxidoreductase (e.g., the phosphite oxidoreductase in SEQ ID NOS: 4-5) is a mutant phosphite oxidoreductase with two amino acid substitutions (E175A / A176R). In some embodiments, the amino acid substitutions allow the enzyme to use both NAD and NADP as cofactors efficiently.

[0067] Introduction of phosphite oxidoreductase-encoding genes into plants or seedsPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0068] In some embodiments, the methods of the present disclosure also include a step of introducing a phosphite oxidoreductase-encoding gene into a plant or seed of the present disclosure. Various methods may be utilized to introduce phosphite oxidoreductase-encoding genes into plants and seeds. For instance, in some embodiments, the introduction occurs at a seedling stage of a plant. In some embodiments, the introduction occurs within somatic embryos of a plant cell. In some embodiments, the introduction occurs in tissue culture prior to somatic embryo development and subsequent germination into seedlings. In some embodiments, the introduction occurs at an adult stage of a plant.

[0069] In some embodiments, the introduction of a phosphite oxidoreductase-encoding gene into a plant or seed includes transformation. In some embodiments, the transformation includes Agrobacterium-mediated transformation.

[0070] In some embodiments, the introduction of a phosphite oxidoreductase-encoding gene into a plant or seed includes introduction of a gene editing system into the plant or seed. In some embodiments, the gene editing system includes a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system). In some embodiments, the CRISPR / Cas system includes at least one Cas nuclease and at least one guide RNA. In some embodiments, the Cas nuclease includes, without limitation, class 2 of Cas nucleases, Cas 9, Cas . Cas 2, Cpfl, or combinations thereof. In some embodiments, the CRISPR / Cas system includes a CRISPR / Cas9 system.

[0071] Additional Embodiments

[0072] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0073] Example 1. Expression of the ptxD gene confers soybean effective metabolism of phosphite, which modulates growth, phosphate signaling, and defense mechanisms in a phosphate-dependent manner

[0074] Optimizing phosphorus fertilizer usage while effectively controlling weeds are two major needs in agriculture. The Phi- / v system, based on the expression of the ptxD bacterial gene, which encodes a phosphite oxidoreductase that enables plants to utilize phosphite (Phi) as the sole phosphorus (P) source, has provided promising outcomes in both avenues in model plants and crops. Additionally, Phi has gained popularity as a plant biostimulant when used in well-fertilized plants. However, the mechanisms through which Phi promotes plant growth and activates defense responses are just beginning to be unraveled.

[0075] In this Example, Applicant demonstrated that the expression of a codon-optimized ptxD sequence in soybean confers plants the capacity to effectively metabolize Phi as the only P source, which becomes highly important in low-P environments. Applicant conducted a comprehensive characterization of the transgenic plants and the non-transformed counterpart using transcriptomics, metabolomics, and high-throughput plant phenotyping technologies to understand in detail the effects of Phi when used as fertilizer in the context of optimal and deficient P fertilization. The findings demonstrate that Phi significantly enhances the growth, metabolism, and stress responses of soybean, with its effects been influenced by orthophosphate (Pi) availability.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0076] In wildtype (Wt) plants, for instance, Phi enhances growth under optimal P-fertilization conditions, suppresses the responses to Pi starvation at short time frames, and induces phytotoxicity in the long term. Phi triggered the upregulation of genes related to ABA and JA signaling, suggesting that the Phi treatment enhances plant defense and stress adaptation, which correlated with the metabolomic analysis data that shows increased levels of osmoprotectants such as proline, sorbitol, and mannitol, as well as phenylpropanoids and coumarins associated with antioxidant defenses and biotic stress responses. Hyperspectral imaging provided additional understandings, revealing distinct spectral shifts in response to Phi and Pi treatments, suggesting changes in pigment and chemical composition, and stress responses.

[0077] This Example shows the multiple interactions between Phi and Pi signaling and stress responses, providing data that can be used in further research to inform a possible mechanistic explanation of how Phi acts as a beneficial molecule and growth enhancer. The ability of ptxD- expressing plants to activate defense responses highlights its use not only as it enables the plants to utilize Phi effectively, but also as a stress-resilience tool in agricultural systems.

[0078] Example 1 , 1. Introduction

[0079] Phosphorus (P) is an essential macronutrient required for plant growth and development. Orthophosphate (Pi), its primary bioavailable form, is often one of the most limiting nutrients in agricultural soils. In highly weathered or low-input systems, such as tropical oxisols and ultisols, Pi becomes immobilized by iron and aluminum compounds, reducing its availability to crops. As a result, plants require higher and more frequent Pi fertilization to compensate for this. The integration of the Phi-pIxD system into crops represents a promising solution to these challenges. The system is based on the expression of a phosphite oxidoreductase that enables plants to utilize phosphite (Phi) as the only P source. This enzyme can be encoded by the ptxD gene from Pseudomonas stutzeri WM88. Phi is a reduced form of P, more mobile in the soil solution than Pi and less prone to fixation with soil components, potentially improving P availability in the rhizosphere.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0080] Expression of the ptxD gene in plants, in combination with Phi-based fertilization, has been successfully implemented in Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), rice (Oryza sativa), cotton (Gossypium hirsutum), and rapeseed (Brassica napus) as proof of concept. Transgenic plants of all plant species tested to date used Phi as the only P source effectively, and produced more biomass than their non-transformed counterpails, which often died under Phi treatment. Transgenic N. tabacum plants that express the ptxD gene use Phi more efficiently, as they produced double the amount of total biomass per plant when fertilized with 30 mg kg'1in the form of Phi compared to 60 mg kg'1Pi fertilization. Tobacco and rice transgenic plants expressing the ptxD gene exhibited normal and vigorous growth of aboveground and belowground parts when fertilized with Phi. Furthermore, Phi has been shown to effectively control the growth of weeds, which cannot utilize this form of P, particularly in low-Pi soils.

[0081] When ptxD plants are grown in competition with weeds and fertilized with Phi, Phi fertilization reduces weed growth while selectively improving ptxD plant growth. This effect has been successfully demonstrated for tobacco, cotton, and rice growing in competition with both broadleaf and grass-type weeds, including Brachypodium distachyon, Ipomoea purpurea, Chloris barbata, Euphorbia hirta, Portulaca oleracea, Phyllanthes niruri, Brachiaria plantaginea, Amaranthus hybridus, Malachium aquaticum, Alopecurus aequalis, and Rumex acetosa. In the case of cotton, the technology was also effective at controlling a glyphosate-resistant ecotype of Amaranthus palmeri, which threatens numerous agricultural lands globally, offering an effective and more sustainable non-herbicide weed control strategy. The Phi-ptxD system has also been utilized as an effective selectable marker during the genetic transformation process of economically important crops, including cotton, maize (Zea mays'), and sorghum (Sorghum bicolor).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0082] Phi is a structural analog of orthophosphate. Therefore, it can be taken up by the plant through its roots and leaves using the same protein transport system as Pi and can be mobilized through the xylem and phloem. Both root and foliar Phi fertilization have been successfully tested with ptxD transgenic plants. Crops expressing the ptxD gene and capable of utilizing Phi as the sole P source represent a sustainable alternative to conventional agricultural practices and hold immense potential for integration into sustainable farming systems. It can help optimize the use of P resources, whose reserves are limited, mitigate constraints associated with low P availability in extensive areas with agricultural potential, reduce the use of herbicides and other pesticides, and combat the evolution of herbicide-resistant weeds, and lessen the environmental impact associated with phosphate overuse, such as eutrophication.

[0083] Given its immense economic significance both in the U.S. and globally, soybean Glycine max) represents an excellent crop for implementing the Phi-ptxD system. Soybeans are one of the most valuable crops in the world. From 2019 to 2022, the soybean industry generated an average annual economic impact of $124 billion, supporting sectors like animal feed, biodiesel, and food manufacturing in the U.S. alone. Globally, soybeans serve as a vital protein source for livestock and an essential food staple, particularly in Asia.

[0084] The various uses and high yields make soybean a valuable crop for meeting global food demand. Despite its capacity for biological nitrogen fixation, soybean still remains heavily dependent on external fertilization to support key physiological processes, including root growth, nodule development, and seed formation, which depend on Pi. Soybean fertilization rates vary significantly, but it is generally accepted that soybeans require more than 30 kg P2Os / ha, with maximum values up to 90-120 kg P2O5 / ha, depending on soil fertility, crop system, and environmental conditions. This dependence of external P application contributes to increased input costs and environmental risks through runoff, underscoring the need for more efficient P management strategies in soybean cultivation. The ptxD gene in combination with Phi fertilization could help overcome the inherent downsides of Pi fertilization by allowing access to a more bioavailable P pool, thereby enhancing plant performance in low-P environments.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0085] Besides its role as an alternative P source, Phi has other beneficial effects on plants, such as contributing to enhanced growth and resilience. One key advantage of Phi over standard Pi fertilization is its ability to stimulate plant defense, enabling plants to withstand a variety of pathogens. Research has shown that Phi induces plants' systemic acquired resistance (SAR), helping them defend themselves against fungal pathogens, such as Phytophthora, which is the causal agent of root rot and blight in several crops. Phi also enhances root growth, which could improve nutrient uptake and plant health, making it a valuable tool in nutrient-poor environments, where an enhanced root system could allow plants to access more nutrients. Phi applications also improved tolerance to abiotic stresses, such as drought and salinity, but the underlying mechanisms arc unknown. The multiple benefits of Phi make it a valuable asset in sustainable agriculture, opening new opportunities for biotic and abiotic stress resistance while enhancing plant growth in fields.

[0086] In this Example, Applicant implemented the Phi- / . D system in soybean and investigated its effectiveness in enabling transgenic soybeans to metabolize Phi as the sole P source, as well as the feasibility of controlling weeds that affect soybean fields. To better understand how the Phi treatments act over soybean plants under different Pi fertilization levels, Applicant performed an in-depth characterization of soybean responses using a combination of platforms, including RNA- sequencing, metabolomics, and hyperspectral imaging to evaluate plant responses. The results revealed that the p / xD-expressing plants effectively metabolized Phi as the only source of P, exhibiting vigorous growth and no negative phenotypic effects. The results also suggest that Phi partially attenuates the systemic and local phosphate starvation responses (PSR) and activates the biosynthesis and signaling of plant hormones, like abscisic acid (ABA) and jasmonic acid (JA) pathways in both plxD and Wt plants. These findings provide new understanding into the mechanisms through which Phi enhances growth, attenuates phosphate signaling, and enhances defense responses, showing Phi potential applications in crop management and stress resilience.

[0087] Example 1 ,2. Materials and MethodsPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0088] Biological material and plant growth'. Glycine max cultivar Williams 82 was used for this Example. For all experiments, unless stated otherwise, seeds were sown directly in a substrate composed of organic matter and mineral substrates that better resemble natural soil with less than 10 ppm of P. This P concentration is considered below the critical level for soybeans; to achieve profitable returns, farmers must add P fertilization to the soil, as it is a common P deficiency that soybeans face in agricultural environments. Applicant used monopotassium phosphate (KH2PO4, Pi) (Sigma Aldrich, St. Louis, MO, USA, CAS. No. 7778-77-0, 99%) or monopotassium Phi (KH2PO3, Phi) (Wanjie International Company Ltd., Hangzhou, China, CAS. No. 13977-65-6, 98%) as required for the Pi and Phi treatments. Murashige and Skoog (MS) media, full strength, were used to provide the other nutrients. Soybean plants were grown in a Conviron chamber at 25°C and a 16-h light / 8-h dark photoperiod with a PAR intensity of -300 pmol-m-2s-1.

[0089] Analysis ofptxD gene expression by real-time qPCR'. Frozen samples were ground to a fine powder in liquid nitrogen with a high torque stirrer Caframo™. The total RNA extractions were performed from 50 mg of each macerate plant tissue in liquid nitrogen, using PurcLink® Plant RNA Reagent (Invitrogen) according to the protocol of the manufacturer. RNA concentration and purity were determined using a NanoDrop™ Spectrophotometer ND- 1000 (Thermo Scientific), and the integrity of RNA was also assessed by 1.2% agarose gel electrophoresis and SYBR® Safe DNA gel staining. The presence of contaminant DNA in the RNA samples was verified by PCR using ptxD and GmACT genes, and gel electrophoresis analysis. To ensure high-quality RNA samples, the RNeasy MinElute Cleanup kit (Qiagen) was used following the manufacturer’s instructions.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0090] Gene-specific primers for ptxD, GmACT2, and GmACTl 1 were designed using the NCBI Primer-BLAST tool. GmACT2 and GmACTl 1 were selected to use as internal controls for cDNA content normalization. cDNAs were synthesized by adding 2pM of gene-specific reverse primer from ptxD, GmACT2 and GmACTl 1 and 500pM of dNTPs mix to 500 ng of total RNA. This mixture was incubated at 65°C for five min, and briefly chilled on ice. First Strand Buffer, 20 mM of dithiothreitol (DTT) and 200 units of Superscript III (Invitrogen) were added to the prior mixture, and the total volume (20 pL) was incubated at 55°C for 1 h following manufacturer’s instructions. Inactivation of the reverse transcriptase was performed by incubating the mixture at 70°C for 15 min, and the cDNA solution was stored at -20°C.

[0091] Polymerase chain reactions were performed with the SensiFAST SYBR No-ROX Kit (BIOLINE) in MIC qPCR Magnetic Induction Cycler (BIOLINE) system. Reaction mixtures contained Ipl cDNA, 400nM of each primer and SensiFAST SYBR® No-ROX Mix, in a total volume of 10 pL. Reaction mixtures were incubated for two minutes at 95 °C, followed by 40 amplification cycles of 5 s at 94°C, 10 s at 60°C and 20 s at 72°C. Results were analyzed using the MIC qPCR Cycler on-board software (BIOLINE).

[0092] Effect of Phi on weed growth'. Seeds of six common weed species, Palmer amaranth (Amaranthus palmeri). Morning glory (Ipomoea spp.f Johnson grass (Sorghum halepense). Green foxtail (Setaria viridis), and Barnyard grass (Echinochloa spp.) (Azlin Seed Service, MS, US), were sown directly in germination trays containing 1.6 kg of a 1:1 (v / v) sand: vermiculite substrate mixture. One hundred seeds of each species were sown separately per pot. Plants were fertilized with MS nutrient solution lacking P. P was applied as required to get 80, 100, or 120 ppm using KH2PO4 or KH2PO3. When foliar treatment was performed, 0.05% Silwet L-77 was added to the solution as a wetting agent to ensure the contact of the solution with the plant leaves. The aboveground biomass was harvested and measured 1.5 months after germination. Six replicates were performed for each weed species.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0093] Experiments for RNA-seq Studies: To avoid any interference from organic P, Applicant decided to use a mineral-only substrate to better control P levels that can affect the RNA-seq results. Applicant used MS media as fertilizer at full strength. Plants were germinated directly in the fertilized substrate with the required Pi and Phi levels. Three biological replicates per treatment were generated and named as follows: HPi (100 ppm added Pi), LPi (0 added Pi), HPi + Phi (75 ppm added Pi + 25 ppm added Phi), and LPi + Phi (100 ppm added Phi only). Two trifoliate leaves v3 (21 days after germination, dag) per treatment were collected and pooled to form a biological replicate, then flash-frozen in liquid nitrogen and homogenized to isolate total RNA using TRIzol (Invitrogen, Carlsbad, CA, USA). mRNA-seq libraries were generated using the TruSeq Illumina protocol for the pool of 2 plants from each of the different treatments. Libraries were sequenced using a NovaSeq 6000 platform with paired-end 150 bp reads at Novogene Corporation Inc. (California, USA).

[0094] Data Checks and RNA-seq Data Analysis: Quality checks of the reads from the RNA-seq were performed using FastQC v0.11.9 to remove low-quality reads. The read estimation was performed using the pseudo-alignment program Kallisto vO.46.2. The official release of the Glycine max genome, Wm82.a4.vl, was used to build the index file for pseudoalignment. To integrate the counts from Kallisto into downstream analysis, the R package tximport v 1.20.0 was used. Differential expression analysis was performed with R using the edgeR package v3.34.0. The pipeline was similar to a standard edgeR pipeline but using the between- sample normalization method and model correction. After that Applicant used LRT-tests to determine differentially expressed genes (DEGs) between treatments, and Applicant used t-tests relative to a threshold to control the false discovery rate (FDR). The threshold for selecting differentially expressed genes with this method was a log fold change of 0.263, an increase or decrease in expression of 20%. Metabolic pathway genes were downloaded from the SoyCyc database vl l using the Plant Metabolic Network. The Arabidopsis thaliana homologs were retrieved from the Phytozome Database using the biomaRt R package.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0095] Functional Annotation and GO Enrichment Analysis'. Functional annotation of proteincoding genes was performed using a modified version of the MAIZE-gamer pipeline. Briefly, annotations were primarily assigned based on Gene Ontology (GO) terms derived from reciprocal blast hits (BLASTP) versus Arabidopsis thaliana (Araportl l) and UniProt Swiss-Prot proteins (including Brachypodiutn distachyon, Chlamydotnonas reinhardtii, Glycine max, Oryza saliva subsp. Japonica, Physcomitrium patens, Populus trichocarpa, Solanum lycopersicum, Sorghum bicolor, and Vitis vini / era). Additional GO annotations were retrieved from significant hits (bit score > 80) in the mentioned Araportl l and UniProt datasets. Protein domains were identified using InterProScan v5.57-90.0. The annotation server PANNZER2 was used, and results were filtered by a PPV value > 0.5. Finally, the results were combined into a non-redundant file including all GO annotations.

[0096] Gene Ontology (GO) enrichment analysis was carried out using a custom annotation, and the R package topGO v2.44.0, using the “elim” algorithm. Relations between sets of DEGs were analyzed using the R package dplyr v 1.0.7 and visualized with the R packages ggvenn vO.1.9 and ggplot2 v3.3.5.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0097] Determination of GPX, SOD, H2O2, and total Protein Levels'. The fully developed top trifoliated leaf from each treatment was harvested and immediately flash-frozen in liquid nitrogen (four biological replicates) and stored at -80°C until use. All samples were processed between 1 and 4 weeks after collection. For glutathione peroxidase (GPX) activity, samples were homogenized in a buffer containing 50 mM Tris-HCl and 5 mM EDTA (pH 7.6), and GPX activity was assayed using cumene hydroperoxide and reduced glutathione (GSH) as substrates. The oxidation of GSH was measured spectrophotometrically following the oxidation of NADPH at 340 monitored at 412 nm, and one unit of GPX activity was defined as the amount of enzyme required to oxidize 1 pmol of GSH per minute. For H2O2 quantification, 150 mg of frozen ground leaf powder was homogenized in 1 mL of a solution containing 0.25 mL of 0.1% (w / v) trichloroacetic acid (TCA), 0.5 mL of 1 M potassium iodide (KI), and 0.25 mL of 10 mM potassium phosphate buffer (pH adapted to tissue), all maintained at 4°C. An additional control using H2O instead of KI was included for background correction. After 10 min of incubation on ice and protection from light, the samples were centrifuged at 12,000 x g for 15 min at 4°C. Two hundred microliters of the supernatant were transferred to UV-compatible microplate wells and incubated at room temperature (20-22°C) for 20 min before absorbance measurement. All samples and blanks were analyzed in triplicate, and averaged to get a single measurement per sample; four biological replicates were processed per group.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0098] Metabolomic analysis: To investigate the metabolic responses induced by Phi fertilization in soybean, Applicant performed a metabolite extraction protocol that ensured comprehensive coverage of both polar and non-polar metabolites in / vD-cxprcssing and Wt plants at 5 weeks after treatment. Leaf samples were collected from plants grown under controlled conditions, immediately frozen in liquid nitrogen, and stored at -80°C until further processing to prevent degradation. Before extraction, the samples were ground using a homogenizer with liquid nitrogen to maintain metabolic integrity. Metabolites were extracted using a two-step solvent-based approach to maximize the recovery of a diverse range of compounds. Identified compounds were submitted to the Enrichment Analysis module of MetaboAnalyst 6.0 online platform for classification into the Super-class categories (39 super chemical class metabolite sets) using the compound list obtained from Compound Discoverer as input and specifying the feature type as Metabolites. Name / ID standardization was then performed by the platform and submitted for metabolite classification in the enrichment analysis. A detailed view of the results table lists the metabolites belonging to each set. Compounds not found on the platform were categorized as “other”.

[0099] High-throughput phenotyping and hyperspectral imaging: The High-Throughput Plant Phenomics Platform used in this Example is located within the IGCAST facility at Texas Tech University and is based on a PhenoAxpert LemnaTec HTC system (hereafter HTC system). The system is equipped with RGB and VNIR cameras. Seven dag, germinated seedlings were transplanted into pots containing a mix of 90% perlite and 10% BM7 substrate (Berger, Saint- Modeste, QC, Canada). After acclimation for one week before applying the HPi, LPi + Phi, and HPi + Phi treatments, all pots were incubated in a reach-in growth chamber (Conviron model PGR15) at 25°C and a 16-h light / 8-h dark photoperiod with a PAR intensity of -300 pmol m-2- s-1and manually loaded into the system when needed. Plants were scanned once a week for five consecutive weeks, starting one week after the treatment was applied.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0100] After data acquisition, RGB images were processed using LemnaGrid v22.9.0.863 to segment plant pixels from the background. To isolate plant pixels, Applicant first split the RGB images into three individual color channels (R, G, and B) and applied a transformation using LemnaTec’s recommended formula:R G g - 255 + - . 5

[0101] This transformation assigns values of around 255 or lower to the green pixels. Then, Applicant filtered out pixels with values lower than 246. Independently, Applicant filtered the RGB images in the HSV (Hue, Saturation, Value) color space, selecting pixels within the following thresholds: H values between 60 and 150, S values between 61 and 200, and V values between 20 and 256. After filtering, Applicant applied a series of morphological operations consisting of four erosion and dilation steps using values of -1, +1, -7, and +7. The segmented pixels from both processes were then merged into a single object for further analysis.

[0102] For hyperspectral data, images were segmented using Lemna 3D v24.4.0.5463. The Normalized Difference Vegetation Index (ND VI) was calculated for each pixel. Any pixel with an ND VI value below 0.5 was filtered out, ensuring that only relevant vegetation signals were considered. After this, Applicant averaged the value of all plant pixels for each single band captured by the VNIR camera to get the average value for each band, totaling 448 individual bands ranging from 397.62 nm to 1003 nm.

[0103] After segmentation, both RGB and hyperspectral data were integrated and processed using LemnaExperiment v23.11.0.5449, which enabled automated trait extraction: height and canopy from RGB data, and individual band values from Hyperspectral images. Data was exported to .csv format for downstream analysis. ND VI, CRI1, CRI2, and ANT 1 indices were implemented in R and calculated from the individual bands from the hyperspectral data based on the following formulas:PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0104] For the systematic pairwise comparisons of all the bands, Applicant used the formula:1 1 pairwise index = — — —RxRy

[0105] This formula follows the same mathematical structure as established indices such as CRI1, CRI2, and ANTI. While these published indices have been widely used to assess specific physiological traits, they are often designed for targeted spectral responses and may not fully capture the complexity of Phi-induced changes in soybean. By implementing a broader, band-by- band comparative approach, Applicant aimed to uncover spectral differences that traditional indices might overlook, providing a more comprehensive analysis of plant responses under varying P and Phi conditions. This method enables the detection of subtle spectral shifts that may be associated with metabolic, structural, or pigment-related changes, providing a more nuanced understanding of how various treatments affect canopy reflectance.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0106] The approach, therefore, serves as an exploratory tool to complement existing indices, identifying novel spectral markers associated with Pi and Phi metabolism and stress adaptation. This approach of pairwise comparison of the entire hyperspectral narrow bands is similar to others previously reported. The entire matrix of values was plotted as a heatmap. The heatmap was divided into color areas based on the color ranges suggested by the CRC Handbook of Fundamental Spectroscopic Correlation Charts and the detection ranges of the HTC system, as follows: violet, 449 nm and any lower value; blue, 450 nm-499 nm; green, 500 nm-569 nm; yellow / orange, 570 nm-619 nm; red, 620 nm-749 nm; NIR (near- infrared reflectance), 750 and any higher value. Due to the narrow range of yellow and orange colors, these two ranges were treated as one. This docs not affect any analysis or interpretation and was made only for visualization and data description purposes.

[0107] Example 1,3. ResultsPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0108] Soybean plants can use Phi as the only P source by expressing a codon-optimized ptxD sequence'. To implement Phi metabolism in soybean, the ptxD gene coding sequence from P. stutzeri WM88 was codon-optimized for soybean and chemically synthesized. A gene construct with this new sequence under the control of the CaMV35S promoter was generated using the Gateway technology (FIG. 2A). Soybean transformation was performed using the Agro / wcterrnm-mediated transformation protocol and cotyledonary tissue. The first progeny of transgenic plants was screened under greenhouse conditions using the herbicide BASTA® (glufosinate ammonium, 1% v / v) as a foliar application (FIGS. 2B-C). After recovering the seed from independent resistant events, the homozygous progenies were grown directly in an artificial substrate (sand: vermiculite, 1:1) using Phi as the only P source in the greenhouse, to verify the plant’s capacity to metabolize Phi from early developmental stages (FIGS. 2D-F). Applicant observed that the herbicide-resistant plants effectively utilized Phi as the P source with no apparent phenotypic changes, whereas the null and Wt controls exhibited severe symptoms of foliar damage and reduced growth of the root system due to Phi toxicity. The expression of the ptxD gene was verified by quantitative real-time PCR, which revealed different gene expression levels in the transgenic lines, and the absence of expression in the Wt control (FIG. 2G). In preliminary experiments for seed increase and optimization of treatment conditions, seed yield of the line G / nptxD-35 A was evaluated and compared to that of the Wt plants (FIGS. 2H-2M). No significant statistical differences in pods per plant, total seeds per plant, weight of 100 seeds, and plant height at the end of the growth cycle were found between GmptxD-35A and Wt plants (FIGS. 2H-2M). Based on these results, Applicant decided to perform a more detailed characterization of one of the lines with higher ptxD expression levels, referred to as GmptxD-35A plants.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0109] ptxD-expressing plants grow normally and produce seed utilizing Phi as the P -fertilizer: To investigate the capacity of the pZxD-expressing plants to metabolize Phi and complete the growth cycle using this P source, Applicant performed a comprehensive characterization using the PhenoAlxpert HTC LemnaTec platform. This system allows morphological and physiological phenotyping using RGB and hyperspectral cameras, thus facilitating studies on growth, development, stress responses, and biochemical properties. Using the LemnaGrid (v22.9.0.863) and Lemna3D (v24.4.0.5463) software, RGB and hyperspectral imagery were analyzed to investigate how soybean plants respond to P limitation, and how Phi affects these responses (FIGS. 3A-D and FIGS. 4A-C).

[0110] Seeds from the G7?zp(xD-35A transgenic line and the Wt control were germinated and transplanted 7 days after germination to their final pots. After one week of acclimation, plants were treated with the following fertilization regimes: HPi, only Pi (100 ppm) as control; LPi, no added P; LPi + Phi, with Phi (100 ppm) only and no added Pi; and HPi + Phi, with a combination of Pi (75 ppm) and Phi (25 ppm). Both Wt and ptxD plants exhibited robust and comparable growth in the HPi treatment, which was reduced under Pi starvation (LPi) in both genotypes (FIGS. 3A-D). The supplementation of Phi to both LPi and HPi treatments had dramatic effects on plant growth.

[0111] Under low phosphate conditions supplemented with Phi (LPi + Phi), Wt plants displayed marked height and canopy reductions. These plants exhibited signs of stunted growth and reduced foliage spread, which were generally lower than those grown under LPi alone. This suggests that Wt plants cannot utilize Phi as a P source, and that Phi reduces plant growth compared to untreated plants. On the contrary, under LPi + Phi conditions, ptxD plants exhibited a clear growth advantage over Wt, with taller plants and more developed canopies observable in photographic and quantitative data comparable to those of the plants in HPi (FIGS. 3B and 3D). This genotype exhibited a progressive increase in height and canopy size across the time course experiment, indicating sustained growth when Phi was the sole P source. The addition of Phi in combination with HPi, resulted in an enhancement in growth, as reflected in both lateral and top-view images, showing increased plant height and canopy area (FIGS. 3A-D).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0112] Phi controls the growth of soybean-threatening weeds'. Phi has been shown to control the growth of several weeds that affect economically important crops. Given the negative effect of Phi on the development of non-Phi metabolizing controls, Applicant decided to test whether different levels of Phi suppress the growth of weeds that commonly affect soybean fields. Applicant examined six representative weed species, including Palmer Amaranth (Amaranthus palmeri), Velvet Leaf (Abutilon theophrasli). Morning Glory (Ipomoea spp.), Johnson Grass (Sorghum halepense). Green Foxtail (Setaria viridis), and Barnyard Grass (Echinochloa spp). Weeds were planted in an artificial substrate (sand: vermiculite, 1:1) and grown under three treatments, HPi, HPi + Phi, and LPhi + Phi. Biomass of all weeds significantly decreased in all Phi treatments (HPi + Phi, LPhi + Phi), regardless of the presence of Pi fertilization. In some species, such as Ipomoea spp., Setaria viridis, and Echinochloa spp., LPiPhi-treated plants exhibited biomass reductions exceeding 75%, indicating their inability to metabolize Phi. Foliar spray of Phi combined with soil application improved weed control, even using higher weed densities (five-fold). These results demonstrate that Phi application selectively suppresses the growth of plants without the ptxD gene, showing the Phi-p / xD system's utility as a dual-function strategy for fertilization and weed management, with the potential to enhance growth.

[0113] Spectral vegetation indices suggest ptxD soybean plants are healthy when using Phi as the only P source: Spectral vegetation indices are valuable tools to evaluate the plant physiological status in a non-destructive manner, providing insights into pigment composition, stress responses, and plant health. By analyzing hyperspectral data, four conventional indices, the Normalized Difference Vegetation Index (ND VI), Carotenoid-related indices 1 (CRIl) and 2 (CRI2), and Anthocyanin Reflectance Index (ANTI), were calculated for Wt and p / xD-cxprcssing soybean plants grown under the four established treatments (FIGS. 4A-C).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0114] The ND VI vegetation index is widely used to estimate chlorophyll content and canopy greenness, serving as a measurement of photosynthesis and biomass accumulation. High ND VI values reflect healthy plants, while lower values indicate stress, senescence, or nutrient deficiency. Under HPi conditions, NDVI values for both Wt (x = 0.77) and ptxD (x = 0.76) plants were consistent, indicating healthy plants with adequate chlorophyll content and canopy greenness, which is indicative of robust photosynthetic activity and biomass accumulation. The NDVI values for both Wt (x = 0.77) and ptxD (x = 0.76) under LPi were similar to those under HPi, suggesting that P deficiency does not have a significant impact on NDVI at this developmental stage. The addition of Phi to the HPi treatment decreased the NDVI values of ptxD plants (x = 0.75) compared to those of the Wt (x = 0.77), with no noticeable negative effects on plant growth. NDVI values substantially decreased in Wt plants under LPiPhi treatment, with the lowest NDVI values recorded (x = 0.74), likely due to Phi toxicity, as Wt plants lack the metabolism to use Phi as a P source, and accumulate it until toxic levels are reached and plants die. Importantly, in LPi + Phi treatments, ptxD plants maintained high NDVI values (x = 0.77), similar to those of the Wt and ptxD plants in HPi conditions, suggesting optimal plant growth and minimal stress levels in the transgenic plants using Phi as the only P source.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0115] The CRI1 and CRI2 indices are used to show changes in carotenoids, which play crucial roles in photoprotection and stress mitigation. These indices are helpful in detecting shifts in pigment composition under abiotic stress conditions, including nutrient limitation. CRIi and CRI2 values in Wt plants under HPi conditions were xCRIl = 38.55 and xCRI2 = 41.33, respectively, which increased to xCRIl = 39.39 and xCRI2 = 42.6 under LPi, indicating increased carotenoid content. CRII and CRI2 values in ptxD plants were higher than in the Wt in HPi, with xCRIl = 40.45 and xCRI2 = 43.83, which decreased in LPi to xCRIl = 39.16 and xCRI2 = 42.85. In HPi + Phi-treated Wt soybeans, the carotenoid-related index values were higher, with xCRIl = 42.54 and xCRI2 = 45.80, suggesting a stress response even when phosphate availability was adequate in response to Phi. On the contrary, ptxD plants displayed values of xCRIl = 36.71 and xCRI2 = 40.38, indicating overall less carotenoid pigments. LPi + Phi-treated Wt plants displayed xCRIl = 37.04 and xCRI2 = 41.77, whereas ptxD plants’ values were consistently lower than in the HPi treatment, xCRIl = 39.34 and xCRI2 = 42.71.

[0116] The ANTI index is used to measure changes in anthocyanin accumulation, induced under stress to scavenge reactive oxygen species and differentially accumulated in response to Pi starvation. In Wt plants under HPi conditions, the ANTI value was x ANTI = 2.78, which increased to xANTl = 3.22 in LPi, indicating higher anthocyanin levels under phosphate limitation. Baseline ANTI value in ptxD plants under HPi conditions was xANTl = 3.38, higher than in the Wt, which slightly increased to xANTl = 3.69 under LPi, reflecting anthocyanin accumulation due to limited phosphate availability. The addition of Phi to both LPi and HPi caused an increase in the ANTI values in the Wt and had a minor effect on the ptxD plants. For instance, the Wt displayed xANT 1 = 3.26 in HPi + Phi and xANTl = 4.72 in LPi + Phi conditions, whereas the ptxD plants have xANTl =3.36 in LPi + Phi and xANTl = 3.67 in HPi + Phi treatment.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0117] Hyperspectral Landscape Reveals Spectral Shifts in Response to Phi and Phosphate Levels'. To better understand the narrowband changes induced by Pi and Phi availability, Applicant employed a pairwise hyperspectral comparison approach, allowing Applicant to capture a broader range of reflectance variations beyond conventional indices. Applicant systematically compared all wavelength pairwise combinations to generate a hyperspectral landscape that displays spectral values for each treatment and wavelength combination. Here, “mountains” refer to increased value of R1 relative to R2 in treatment A relative to treatment B, or an increase of R1 in treatment A or a reduction of R2 in treatment B. “valleys” describe a reduction of R1 in treatment A or an increase in treatment B of R2.

[0118] In Wt plants, the comparison of LPi-treated plants to HPi revealed an increased value of the R1 proportion relative to R2 values ("mountains") in the violet region when contrasted with the middle of the green, yellow / orange, and red regions (FIG. 4B). These peaks extended horizontally into the blue area, though with decreasing intensity. Additionally, in the green, yellow / orange, and red regions relative to the infrared, distinct vertical valleys (a reduced value of the R1 proportion relative to R2) were observed when compared to the infrared region, indicating significant depressions of the infrared values or increased values in the visible spectrum. Another distinct valley was observed at the intersection of the yellow / orange and red regions, suggesting alterations in pigment composition or stress-induced changes in reflectance properties (FIG. 4B). The comparison of LPi + Phi vs. HPi revealed horizontal mountains when the violet region was compared to the green and red regions, as well as in the yellow and red regions when compared to red itself. These mountains are similar to the ones found in the LPi vs. HPi treatment, suggesting that Phi increases some stresses caused by LPi in the Wt plants.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0119] Furthermore, the vertical valleys that emerged due to LPi treatment in the green, yellow / orange, and red vs. infrared comparison are also more profound, indicating that the Phi further increases the stress of LPi (FIG. 4B). The addition of Phi to LPi conditions (LPi + Phi vs. LPi) in Wt plants resulted in a spectral pattern that was very different from that of LPi or LPi + Phi, both vs. HPi, with valleys appearing in the transition between blue and green and red light when compared to inferred light. Additionally, the mountains in the violet regions become apparent when comparing them to red and green, suggesting a complete change in the response to Phi depending on Pi levels (FIG. 4B). When comparing HPi + Phi vs. HPi in Wt plants, Applicant observed valleys in the violet region that were mountains in the LPi+Phi vs. LPi comparison, particularly when compared to the green, yellow / orange, and red regions. However, within the infrared relative to green, yellow / orange, and red regions, Applicant identified multiple mountains, that are not present in any other comparison. These peaks indicate that Phi treatment under HPi conditions leads to broader spectral changes, possibly linked to altered metabolic composition or pigment production, such as chlorophylls and carotenoids (FIG. 4B).

[0120] When comparing LPi vs. HPi, ptxD plants exhibited a highly distinct landscape. Unlike in Wt plants, where valleys appeared when comparing visible to infrared reflectance, in ptxD plants, these valleys were closer to zero, indicating minimal shifts in NIR reflectance under LPi. In addition, two prominent spectral peaks (mountains) emerged in the violet and blue spectral regions when compared to green, yellow / orange, and red bands. This suggests an increased proportion of violet / blue light reflectance relative to longer wavelengths, possibly due to modifications in pigment composition or stress-related physiological adjustments (FIG. 4C). When comparing LPi + Phi vs. HPi, the hyperspectral landscape in ptxD plants closely resembled that of HPi alone, but with mountains and valleys closer to zero. This suggests that Phi treatment attenuates the spectral shifts associated with phosphate deficiency in ptxD plants, reducing the magnitude of hyperspectral responses. This indicates that the conversion of Phi into Pi within the plant alleviates LPi stress, leading to a spectral landscape more similar to that of HPi conditions.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0121] The comparison LPi + Phi vs. LPi in ptxD plants revealed distinct horizontal valleys when the violet region was compared to the green, yellow / orange, and red regions. Mountains also appear in the green, yellow / orange, and red areas when compared to infrared light (FIG. 4C), similar to HPi + Phi in Wt plants. This suggests that Phi in LPi + Phi conditions is accumulating enough in plxD plants to give a similar response to HPi + Phi in Wt plants. When comparing HPi + Phi vs. HPi, ptxD plants exhibited mountains in the transition between blue and green, as well as in the middle of the red spectrum, when compared to infrared reflectance. The blue-to-green transition also exhibited a spectral peak when compared to green itself, suggesting that Phi supplementation in HPi conditions altered pigment composition, possibly affecting the dynamics of chlorophyll or carotenoids, given that the bluc-grccn transition is highly influenced by chlorophyll absorption. The spectral peaks in the red vs. NIR arc fewer but higher compared to the Wt plants, as Applicant only found two peaks, suggesting that the effect of Phi in ptxD plants is minor or insignificant.

[0122] Transcriptomic changes induced by levels of Pi and Phi treatment'. To gain more detailed information about the plant’s internal responses to the different fertilization regimes, Applicant conducted RNA-sequencing and metabolomics analysis of Wt and ptxD soybean plants at the end of the fifth week of treatment. During the initial phase of the RNA-seq analysis, the functional annotation publicly available for the soybean genome (version Wm82.a4.vl) was utilized. However, Applicant found that more than 50% of the annotated genes have only one or no functional annotation, limiting the depth of biological insights drawn from transcriptomic analyses. Therefore, to ensure a more comprehensive functional characterization of soybean genes, a new functional annotation was developed following an in-house pipeline, which significantly improved annotation coverage with over 50% of annotated genes assigned five or more functional terms.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0123] In Wt plants, the comparison of LPi relative to HPi found 4528 upregulated and 3432 downregulated differentially expressed genes (DEGs). The Gene Ontology (GO) enrichment analysis of DEGs showed that pathways associated with stress and the immune system were considerably downregulated. This includes how the plant fights off bacteria, fungi, oomycetes, and chemicals that come from bacteria under LPi. Under LPi, upregulated genes were primarily involved in photosynthetic processes, such as photosynthesis, photosynthetic electron transport, photosystem II assembly, chloroplast organization, and thylakoid membrane structure. Comparing LPi + Phi relative to HPi in Wt, 5836 DEGs were upregulated and 4490 downregulated. GO enrichment analysis showed that the upregulated DEGs were mostly involved in photosynthesis, chloroplasts organization, and thylakoid membrane organization, reflecting the physiological alterations induced by Phi in LPi treatment. In Wt plants, the LPi + Phi vs. LPi treatment resulted in 365 DEGs upregulated and 558 downregulated. Notably, the enriched GO categories in the upregulated DEGs included oxylipin biosynthesis, jasmonic acid biosynthesis, lipid oxidation, and phosphate starvation response. This indicates that Phi exposure in Pi-limited settings resulted in more stress and hormone signaling. The HPi + Phi vs. HPi comparison in the Wt revealed 5076 upregulated and 3138 downregulated DEGs; enriched GOs of upregulated DEGs predominantly associated with photosynthesis and chloroplast processes, such as photosynthetic electron transport and photosystem II assembly, suggesting Phi treatment targets these processes under optimal Pi conditions.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0124] In the case of ptxD plants, the LPi vs. HPi comparison identified 1275 upregulated and 2257 downregulated DEGs. Enriched GO categories in the upregulated and downregulated DEGs were similar to those enriched in the Wt plants. The LPi + Phi vs. HPi comparison in ptxD plants revealed 3029 upregulated and 4241 downregulated DEGs, with strong enrichment in photosynthesis-related processes, the NADH dehydrogenase complex, and chloroplast rRNA processing, indicating that efficient Phi metabolism mitigates Pi deficiency stress. In ptxD plants, the LPi + Phi vs. LPi treatment showed 58 upregulated and 356 downregulated DEGs. Enriched GO functional categories in the upregulated DEGs included auxin-mediated signaling, response to salt stress, xyloglucan metabolism, glucosinolate metabolism, and brassinosteroid signaling pathways. This suggests that Phi supplementation caused hormonal and stress-response changes. Finally, the HPi + Phi vs. HPi comparison in ptxD plants showed 53 upregulated and 19 downregulated DEGs. The enriched GO categories of upregulated DEGs were involved in photosynthesis, photosynthetic electron transport, photosystem II assembly, and thylakoid organization. This suggests minimal stress and efficient metabolic adjustments to Phi supplementation under optimal Pi conditions.

[0125] Phi differentially modulates Pi starvation responses depending on Pi levels: In basic research, Phi is recognized as an effective attenuator of the phosphate starvation responses (PSRs) in plants, which arc mainly controlled by the transcription factors PHR1 (Phosphate Starvation Response 1) / PHL1 (Phosphate Response 1-like) and STOP1 (Sensitive to Proton Rhizotoxicity 1). Based on the signaling pathways established in other model plant species like Arabidopsis, Applicant analyzed the STOP1- and PHR1 -related signaling transduction pathways in soybean in response to the different treatments. Based on the Arabidopsis molecular components of the STOP1 and PHR1 signaling pathways, orthologue genes were searched in soybean and used to analyze their expression patterns (FIGS. 5A-5B).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0126] In the case of the STOP1 signaling pathway, Applicant observed changes in expression in key PSR genes under LPi + Phi conditions. These changes were characterized by the attenuation of PSR gene expression. Specifically, SIZ1 (E3 ligase SAP AND MIZ 1) expression increases, which negatively regulates STOP1, whose expression is also attenuated. The STOP1 expression going down causes the low expression of its target genes, ALMT1 (Aluminum-Activated Malate Transporter 1). Also, MED16 (Mediator 16), a recognized positive regulator of STOP1, is downregulated under the LPi + Phi treatment, reinforcing the attenuated PSR response. In contrast, minimal transcriptional changes were observed in genes associated with PDR2 (Phosphate Deficiency 2), LPR1 / 2 (lipoprotein receptor-related protein 1), and CLEs (Clavata3 / ESR-related), which indicates that these components arc less influenced by the conditions tested (FIG. 5A). In the PHR1 regulatory pathway, both PHR1 and PHL1 exhibit downregulation in the LPi + Phi treatment. Interestingly, SIZ1, which activates PHR1 through SUMOylation in this pathway, is upregulated in LPi + Phi. Furthermore, homologs of PHO2 (Phosphate Starvation 2), which regulate SPX4 (SYG1 / Pho81 / XPR1), a negative regulator of PHR1 / PHL1, show reduced expression, suggesting a complex, coordinated attenuation of PSR gene activation mediated by Phi supplementation under LPi conditions (FIG. 5B).

[0127] To further explore the attenuation of other genes in LPi conditions in response to Phi in the Wt, Applicant analyzed the expression of genes that were upregulated in LPi conditions, and their expression was diminished in LPi + Phi (LPi > LPi + Phi). Applicant identified 1706 genes exhibiting this pattern (FIG. 6A), and GO enrichment analysis of these genes revealed associations with iron sequestration and transport, regulation of abscisic acid (ABA)-activated signaling, and responses to oxidative stress (FIG. 6B). These data correlated with a decrease in H2O2 content and glutathione peroxidase (GPX) activity in plants treated with LPi and Phi in both plant genotypes (FIGS. 6C-D). Increased GPX activity and H2O2 content are both present in response to Pi starvation, therefore, indicating that Phi interferes with these responses mainly in the LPi condition (FIGS. 6C-6D).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0128] The analysis also revealed that the HPi + Phi condition elicits a set of responses like those of the LPi condition (FIG. 6E). Applicant found 2779 upregulated LPi-responsive genes that are also activated by Phi in the HPi condition (HPi + Phi, 2596 + 183 genes). The enriched GO terms of this set of genes present a significant enrichment in photosynthesis-related processes, electron transport, NADH dehydrogenase complex activities, and chloroplast organization (FIG. 6F). These findings suggest that under high Pi availability, Phi actively stimulates processes related to energy metabolism and photosynthetic efficiency. To further investigate this, Applicant examined the expression profile of genes associated with tetrapyrrole biosynthesis, a critical pathway involved in chlorophyll formation and defense activation. A significantly low expression of these genes was observed under HPi compared to the other conditions.

[0129] Phi and Phosphate Deficiency Influence ABA and J A Signaling Pathways in Soybean'. The analysis of the enriched GO categories for Wt and ptxD plants under different conditions highlighted several GO terms related to ABA, SA, and, in particular, J A. Given that these three well-known plant hormones play central roles in stress responses and defense regulation, Applicant investigated whether the expression levels of genes involved in the biosynthesis and signaling of ABA, SA, and JA are affected. The expression patterns of the genes annotated under the terms "response to ABA," "response to JA," and "response to SA," as well as all their respective child terms, were analyzed. This ensured a broad representation of hormone-responsive transcripts. Genes implicated in ABA signaling were upregulated in all treatments compared to HPi control.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0130] This suggests that both Phi treatment and LPi therapy cause ABA-dependent stress responses (FIG. 7A). The strongest upregulation was observed in the LPi + Phi treatment, which supports the idea that LPi and Phi work together to make ABA-related signaling pathways stronger. However, despite this increase in signaling genes, the ABA biosynthesis pathway genes were not significantly upregulated in any treatment relative to the control, and the crucial biosynthesis step, Zeaxanthine to Violaxanthin, were not enhanced in any treatment. In fact, HPi + Phi-treated plants exhibited downregulation of some ABA biosynthetic genes, suggesting that while Phi triggers ABA-dependent responses, it does not necessarily enhance ABA production. Similar to ABA, genes that are part of JA signaling were turned on in all treatments except the HPi control. The LPi + Phi treatment had the most activation (FIG. 7B). This implies that both LPi treatment and Phi application can cause JA-dependent responses, which supports the idea that JA helps plants adapt to stress when LPi is present. However, in contrast to ABA, where biosynthesis remained unchanged or downregulated, JA biosynthetic genes exhibited a distinct pattern. Both HPi + Phi and LPi + Phi treatments showed the highest JA biosynthesis gene expression levels, indicating that Phi specifically promotes the production of JA precursors and intermediates.

[0131] In the case of the LPi treatment, JA biosynthesis genes are not significantly upregulated compared to the HPi treatment. However, the enhanced expression of the signaling components may amplify small changes and make them significant. These results suggest that Phi induces a higher activation of JA biosynthesis, possibly as part of a Phi-driven stress response that enhances defense priming and adaptive metabolic adjustments. Unlike ABA and JA, Applicant did not detect transcriptional changes in the SA pathway, with only two genes annotated under "Response to SA" and its child terms detected in a dataset (FIG. 7C).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0132] Phi-Induced Metabolic Reprogramming'. The metabolomic analysis of soybean plants indicated genotype and treatment- specific metabolite accumulation patterns. There were considerable alterations in metabolite accumulation in Wt plants grown under LPi treatment when compared to HPi conditions. One hundred sixteen molecules were considerably downregulated, and only 12 were strongly upregulated. Functional enrichment analysis revealed enrichment in phenylpropanoids and polyketides, homogeneous non-metal compounds, nucleosides and analogues, organic acids and derivatives, and organic oxygen compounds, indicating a broad metabolic effect in LPi. In Wt plants, metabolic reprogramming became more comprehensive under LPi + Phi settings compared to HPi conditions. This was shown by 142 metabolites being considerably downregulated and 195 being upregulated. Prominent metabolites showing increased accumulation included azelaoylglucuronide, methyl-P-mannopyranoside, and deacetyldiglucuronide. Functional enrichment analysis showed that the same classes were enriched as they were under LPi. This shows that metabolic stress responses were stronger when both LPi and Phi were supplemented together. In Wt plants under HPi + Phi vs. HPi conditions, metabolite shifts were comparatively modest, with only two metabolites downregulated and 25 significantly upregulated, suggesting minor metabolic changes in response to treatment. In the case of / D-cxprcssing plants, LPi conditions relative to HPi elicited a notably different response, with only 13 significantly downregulated metabolites and 104 significantly accumulated metabolites.

[0133] Under LPi + Phi conditions compared to HPi, ptxD plants showed a strong metabolic response with 124 metabolites significantly upregulated and none significantly downregulated. There weren't enough significant differential metabolites found under these two conditions, LPi + Phi and Pi, to do a relevant enrichment analysis. Under HPi + Phi conditions relative to HPi, ptxD plants showed the most substantial response, accumulating 285 metabolites significantly with no downregulated metabolite reductions. This wide metabolic adjustment demonstrates the impact of Phi metabolism and the ptxD gene, even in the presence of sufficient Pi. In Wt plants, Phi accumulation in leaves was strongly correlated with the upregulation of several defense-related metabolites, suggesting a potential link between Phi and stress adaptation.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0134] To better understand the impact of Phi fertilization on the metabolite accumulation in plants, Applicant analyzed whether Phi treatment or potential Phi accumulation correlated with stress responses in ptxD and Wt plants, respectively. For this purpose, Applicant performed systematic correlation analyses between the Phi concentrations and the abundances of all detected metabolites, which helped identify important molecules that respond to Phi treatment and possible changes that could come from the fact that ptxD plants have a distinct metabolic competence. Phi accumulation was significant in Wt plants and negligible in ptxD plants (FIG. 7D). Applicant found 11 metabolites that positively correlated with Phi concentration in Wt plants; mannitol, sorbitol, OxoProline, L-Proline, L-Serine, D-phenylalanine, coumaroyl, p-coumaroylquinic acid, phenethyl 2-primcvcrosidc, 2-phcnylcthanol, and salicyoyl aspartate (FIGS. 8A-8I). These molecules are known to help protect cells from osmotic stress, act as antioxidants, and fight off pathogens. This supports the hypothesis that Phi helps plants get ready to defend themselves (FIGS. 8A-8I). A more exhaustive correlation analysis identified 164 additional substances with correlations higher than 0.5 or lower than -0.5 to Phi levels. These compounds include a diverse array of flavonoids, amino acid derivatives, and secondary metabolites. Many of them are associated with reducing oxidative stress, adjusting metabolism when nutrients are low, and signaling molecules that help plants defend themselves.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0135] In ptxD plants, a positive correlation of Phi treatment with an increase in defense-related metabolites was also found, although to a lower degree and with some distinctions compared to the Wt plants. Of the 11 metabolites that strongly correlated with Phi content in leaves in Wt plants, seven remained significantly correlated in ptxD plants: sorbitol, oxoproline, serine, phenylalanine, phenethyl 2-primeveroside, 2-phenylethanol, and salicyoyl aspartate, even when the Phi concentrations were much lower than Wt plants. Beyond these metabolites, Phi concentration displayed high correlations with several secondary metabolites in ptxD plants (252 in total), which are associated with P metabolism, osmoprotection, and stress tolerance. This suggests that the molecular priming effects of Phi are long-lasting, leading to the activation of metabolic pathways and reflected in the accumulation of stress-related molecules. In addition, in the case of the ptxD plants, these exhibit additional metabolic adjustments due to their ability to metabolize Phi, which provides them with advantages to respond to environmental stresses.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0136] Example 1.4. Discussion[001371 This Example shows that the Phi- / x technology is a promising tool in agriculture for both fertilizing and controlling weeds in soybeans, much like they have been in other crops, including rice, cotton, and maize. Furthermore, Applicant also demonstrates that Phi has a significant effect on soybean physiology, influencing both growth and defense responses in a P level-dependent manner. In Wt plants, Phi enhances canopy growth and height in well Pi-fertilized conditions. However, under Pi deficiency, it results in toxicity. On the other hand, ptxD plants showed enhanced growth when Phi is supplemented as the only P source, demonstrating that ptxD plants can fully metabolize Phi and fully alleviate Pi starvation effects. At the molecular level, Phi treatments triggered the upregulation of defense-related pathways, particularly ABA and JA signaling, which are commonly linked with responses to both biotic and abiotic stresses. Interestingly, Phi also induced the upregulation of tetrapyrrole biosynthesis genes, which have a role in modulating both primary and secondary metabolism. Hyperspectral imaging also indicates spectral shifts linked to Phi and Pi treatments, showing underlying biochemical and physiological changes. Finally, Phi accumulation in leaves and Phi treatment correlated with defense metabolites, with ptxD plants exhibiting a distinct metabolic profile compared to Wt, suggesting that the Phi-ptxD technology modulates stress-related pathways. These findings provide new information about the roles of Phi in plant metabolism and stress tolerance, shedding light on its potential applications in sustainable agriculture.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0138] Phi enhances soybean growth and the expression of the ptxD gene allows plants to use Phi as the sole source ofP: Phi has been used on several crops to enhance their growth and improve nutrient uptake. In wheat (Triticum aestivum) and Arabidopsis, foliar application of Phi increases the root biomass, leading to improved nutrient and water use efficiency. Similarly, Phi treatments on oilseed rape (Brassica napus) led to enhanced root growth and increased biomass production. Using Phi on sugarcane (Saccharum officinarum) has been shown to help the plants grow bigger and produce more. However, it is crucial to note that the effectiveness of Phi appears to vary depending on the P status of the soil. For instance, in common bean (Phaseolus vulgaris), Fol iar application of Phi under Pi-deficient conditions led to reduced growth, whereas in turfgrass species such as ryegrass (Lolium multifloruni) and bentgrass (Agrostis stolonifera), Phi applications when soil Pi levels were adequate did not have negative effects on growth. In this Example, Applicant corroborated a similar behavior in soybean. Wt plants had enhanced growth when both Phi and Pi (HPi + Phi treatment) were applied, but were the worst-performing plants when they received Phi treatments under Pi starvation conditions (LPi + Phi) (FIGS. 3A-B).

[0139] Applicant demonstrated that soybean plants expressing the codon-optimized ptxD gene efficiently metabolize Phi as the only P source, as demonstrated previously for other plant species. The results show that / xD-cx rcssing soybean plants have all the beneficial effects on the growth of Phi, while avoiding any toxicity symptoms similar to those observed in the Wt plants. ptxD transgenic plants were phenotypically normal, fertile, and able to complete the growth cycle using Phi exclusively as the P source. Likewise, the use of Phi allowed the effective control of the six weeds tested, as shown before for other broadleaved and grass-type weeds.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0140] Phi modulates vegetation indices depending on Pi status: The hyperspectral indices demonstrate that soybeans respond differently to the Phi treatment according to the Pi levels. NDVI values showed difference between Wt and ptxD plants after 5 weeks of treatment. LPi treatments showed that ptxD plants had lower NDVI values than Wt plants (data not included), suggesting that ptxD plants either had less chlorophyll or were reacting to stress in a way that was unique to them (FIGS. 3A-3D, FIGS. 4A, B, C). Under LPi + Phi conditions, Wt plants had the lowest NDVI values, suggesting that Phi application to non-Phi metabolizing plants imposes a stress that negatively impacts canopy greenness. These data align with the observations on soybean growth in LPi, and previous reports that indicate Phi is phytotoxic in low Pi conditions and causes growth inhibition, metabolic imbalances, and plant death. In the case of ptxD plants, the NDVI values under LPi + Phi are similar to those in the Wt in HPi treatment, suggesting that Phi does not cause stress or health issues in ptxD plants.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0141] The carotenoid-related CRI2 and CRI1 indices gave Applicant more information about how limiting Phi and Pi affects leaf pigmentation. At week 5, no significant differences were observed between Wt and ptxD plants under HPi conditions. However, in both LPi and LPi + Phi treatments, Wt plants displayed higher CRI2 and CRI1 values, indicating an increase in carotenoid- associated reflectance. This data suggests that low Pi levels and Phi treatment increase the production carotenoid in Wt plants. This could be a way for the plants to defend themselves from oxidative stress caused by changes in P homeostasis. A similar trend was reported in soybean under drought stress treated with selenium nanoparticles, where treated plants accumulated more carotenoids and coped better with drought. This suggests that increasing the production of carotenoids helps the plant cope with drought and high ROS levels. In LPi conditions, both Wt and ptxD showed high anthocyanin accumulation, with ptxD plants exhibiting higher ANTI values. However, in LPi + Phi conditions, Wt plants displayed the highest ANTI values, suggesting increased anthocyanin accumulation, which is often associated with stress adaptation and Pi starvation responses. This suggests that Phi toxicity induces anthocyanin production in Wt plants as a response to long exposures to Phi (five weeks). The change in spectral patterns also supports the fact that ptxD plants can effectively utilize Phi as their only P source, and Wt plants experience metabolic stress when exposed to Phi in LPi conditions, leading to altered pigment dynamics and potential oxidative stress responses.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0142] Phi and Pi starvation influence the hyperspectral signals landscape in soybean'. Hyperspectral analysis showed that Pi availability and Phi supplementation caused significant and different spectral modifications. These changes in the hyperspectral landscape, could reflect alterations in the pigment composition, and leaf structure. In Wt plants, the hyperspectral landscape comparison of HPi + Phi vs. HPi (FIG. 4B) showed valleys in the violet region when compared to the green, yellow / orange, and red regions. These valleys, particularly when associated with green and yellow wavelengths, indicate shifts in pigment composition, which may be linked to the metabolism of chlorophyll and carotenoids. Notably, Applicant observed 1, 3, and 5 spectral peaks (mountains) in the green, yellow / orange, and red regions, respectively, relative to NIR, which could indicate increased chlorophyll or altered leaf structural properties. These findings align with previous reports suggesting that Phi application enhances chlorophyll content and photosynthetic efficiency.

[0143] In ptxD plants, Phi supplementation in HPi had no significant effects (FIG. 4C). The LPi + Phi vs. LPi comparison in Wt plants (FIG. 4B) revealed a spectral landscape vastly different from that observed under HPi + Phi conditions, with valleys appealing in the transition between blue-green and red light relative to NIR. Furthermore, the valleys observed in the violet spectrum now appear as mountains, indicating a fundamental shift in the way plants modulate pigments in response to the Phi treatment, depending on Pi availability.

[0144] These changes show that, under Pi starvation, Phi induces changes in the metabolic state, likely involving changes in pigment production or shifts in stress responses. This could also be attributed to the toxic effects of Phi in low Pi conditions, which impede fundamental physiological processes. In HPi conditions, Phi enhanced growth and also modified pigment composition, but under Pi starvation, Phi exacerbates stress responses rather than mitigating them. Phi toxicity is well-documented as it competes with Pi for uptake by plants. This interferes with Pi-dependent activities, such as ATP synthesis, nucleotide metabolism, and membrane phospholipid formation.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0145] The valleys in the blue-green and red light relative to NIR suggest pigment breakdown, oxidative stress, or structural damage, consistent with stress-related chlorophyll degradation and impaired photosynthesis. Additionally, the presence of mountains in the violet spectrum, which were valleys in LPi alone, indicates that Phi triggers a compensatory response that shifts metabolic priorities, possibly toward stress signaling and secondary metabolite production. When Applicant add Phi to Wt plants under LPi conditions (FIG. 4B), all the features shown in LPi condition seem exaggerated. While Phi has been used as a suppressor of the Pi starvation responses, Applicant observed that at 5 weeks of treatment, Phi aggravates LPi symptoms. However, at 2 weeks of treatment, Phi treatment actually attenuates the Pi starvation responses; Wt plants under LPi conditions have attenuated features when treated with Phi. This suggests that the attenuation of the PSR is temporal, revealing Phi toxicity after a couple of weeks due to the absence of P to sustain cellular functions in the Wt. These findings demonstrate that in low Pi environments, Phi is not metabolized by Wt plants, and is a stress-inducing compound, leading to spectral and physiological changes that indicate disrupted Pi homeostasis.

[0146] Interestingly, in ptxD plants, the LPi + Phi vs. LPi comparison (FIG. 4C) revealed a pattern similar to HPi + Phi in Wt plants, with valleys appearing in the violet region relative to the green, yellow / orange, and red regions. Additionally, Applicant observed mountains in green, yellow / orange, and red regions when compared to infrared, resembling the spectral features of HPi + Phi Wt plants. This suggests that ptxD plants present a dual response; they take-up enough Phi under LPi + Phi conditions to trigger similar spectral shifts as those observed in Phi-treated Wt plants under high Pi availability, and still metabolize enough Phi into Pi to support normal metabolism, growth, and reproduction. When comparing LPi to HPi in Wt plants (FIG. 4B), violet spectral mountains relative to green, yellow / orange, and red bands suggest that Pi starvation alters pigment ratios. These spectral changes extended into the blue spectrum, reinforcing Pi starvation- induced shifts in anthocyanin and carotenoid composition. Additionally, the valleys in green, yellow / orange, and red vs. NIR reflectance suggest a decrease in chlorophyll content, as the relative proportion of green reflectance compared to spongy mesophyll-related NIR decreased, indicating chlorophyll degradation under Pi starvation.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0147] In ptxD plants, the LPi vs. HPi comparison showed a drastically different spectral landscape from Wt plants under the same condition (FIG. 4C). The valleys in the visible vs NIR comparisons indicate a reduced chlorophyll degradation. The presence of two large mountains in the violet and blue regions relative to green, yellow / orange, and red bands further suggests an adaptation to Pi starvation, which could involve alternative photoprotective strategies or stress mitigation mechanisms. Unlike in Wt plants, where Phi supplementation under Pi starvation (LPi + Phi) exacerbated stress responses, in ptxD plants Phi mitigated spectral shifts associated with Pi deficiency, leading to a landscape closer to that of HPi conditions, supporting the fact that ptxD plants can metabolize Phi as the only source of P (FIG. 4C).

[0148] This hyperspectral data show that plants can modulate leaf reflectance properties in response to Phi in a Pi-dependent manner, modifying pigment composition and leaf structure. The observed spectral differences between Wt and ptxD plants show the ability of the Phi-ptxD system to alter the physiological impact of Phi, potentially helping plants cope with stress under Pi deprivation.

[0149] The Phi-ptxD system and activation of stress responses'. RNA-seq analysis revealed a substantial overlap in DEGs between LPi and HPi + Phi conditions (FIGS. 6A-6F), with 34.7% of the upregulated DEGs in LPi also upregulated in HPi + Phi. This suggests that Phi, despite being a non-metabolizable analog of Pi, triggers similar transcriptional responses to Pi deprivation. These findings align with previous studies, which have shown that Phi disrupts Pi homeostasis and induces stress-related gene expression. The upregulation of PSR genes in Phi-treated plants (HPi or LPi) supports the hypothesis that plants treated with Phi disrupt part of the Pi signaling pathway, likely due to its ability to compete with Pi for uptake and intracellular sensing mechanisms. However, some PSRs were diminished by Phi, highlighting the complexity of Pi-starvation stress signaling. Both repression and enhancement of PSR responses by Phi have been previously reported, suggesting that the mechanism is conserved across different plant species.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0150] An interesting transcriptomic response to Phi treatment was the upregulation of tetrapyrrole biosynthesis genes, under both HPi and LPi. Tetrapyrroles are essential for chlorophyll, heme, and phytochromobilin biosynthesis, playing a crucial role in photosynthetic efficiency, redox homeostasis, light perception, and signaling. The observed induction of tetrapyrrole -related genes suggests that Phi, despite being a non-metabolizable P source in Wt plants, strongly affects chlorophyll metabolism and light-harvesting pathways. Notably, the LPi treatment also upregulated tetrapyrrole biosynthesis genes, but Phi application in both high- and low-Pi conditions amplified this response. The upregulation of genes involved in chlorophyll biosynthesis under Phi treatment suggests that Phi may enhance chlorophyll retention or production, possibly as a compensatory mechanism for disrupted Pi homeostasis. This aligns with the hyperspectral data, where Phi-treated plants exhibited high chlorophyll-associated spectral reflectance in HPi conditions and LPi conditions, avoiding chlorophyll degradation (FIG. 4B). Similar findings have been reported in other crops, where Phi application led to changes in photosynthetic activity, chlorophyll fluorescence, and antioxidant responses. Given that tetrapyrroles are precursors for key signaling molecules involved in plant defense responses, this raises the possibility that Phi enhances specific branches of the tetrapyrrole pathway that influence stress perception and photosynthetic acclimation.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0151] RNA-seq data also revealed that Phi treatment and Pi deficiency induce complex hormonal responses, particularly in ABA and JA (FIGS. 7A-7C). These plant hormones are central to stress adaptation, defense priming, and metabolic reprogramming, and their transcriptional regulation under Phi treatment provides key insights into how plants perceive and respond to altered P availability. Genes associated with ABA signaling were significantly upregulated in all treatments, except for the HPi control, with the strongest activation occurring in LPi + Phi-treated plants. This suggests that Phi, particularly under low Pi conditions, triggers ABA-dependent stress responses. Interestingly, despite the upregulation of ABA-responsive genes, ABA biosynthesis genes were not significantly induced in any of the treatments. HPi + Phi- trcatcd plants showed a downregulation of some ABA biosynthetic genes. The apparent contradiction of upregulated ABA signaling but not ABA biosynthesis suggests possible explanations: (1) it could indicate an enhanced receptor sensitivity or interactions with other stress- related pathways rather than ABA synthesis, (2) Phi disrupts Pi homeostasis in a way that mimics drought or osmotic stress signals, leading to an ABA-like response, and (3) Phi primes ABA pathways, preparing plants for a faster response to future stressors. In support of the first case, a report has shown enhanced transcription of the auxin receptor, TIR1 (TRANSPORT INHIBITOR RESPONSE1), in response to Pi starvation, which leads to enhanced auxin sensitivity and transcriptional activation of auxin-related genes, with no significant increase in auxin levels. Similarly, studies have reported instances where Pi deprivation increased ABA-related responses without significantly altering ABA levels, reinforcing the notion that ABA-dependent stress pathways are tightly linked to Pi metabolism. It is also plausible that, like other beneficial molecules and chemical priming molecules, the activation of perception components prepares the plant to respond swiftly to stresses without compromising growth.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0152] In the case of JA, associated pathways exhibited transcriptional activation in both signaling and biosynthetic genes (FIGS. 7A-7C). Both HPi + Phi and LPi + Phi treatments showed the highest JA biosynthesis gene expression levels, suggesting that Phi specifically promotes the production of JA. Since JA is crucial in regulating plant responses to biotic and abiotic stress, its upregulation under Phi treatment may indicate a defense-related metabolic shift. The simultaneous activation of JA biosynthesis and signaling suggests Phi might contribute to a primed defense state, enhancing the plant’s ability to cope with potential stresses. This is particularly relevant given the established links between JA signaling and Pi starvation responses, as Pi-deficient plants often exhibit increased JA-dependent defense pathways.

[0153] In contrast to ABA and JA, Applicant found limited transcriptional activation of SA- related genes. While the low number of SA-related genes prevented a statistically significant conclusion, their expression patterns are similar to those observed in JA and ABA signaling genes, showing upregulation in all treatments except HPi, with the highest response in LPi + Phi. This suggests that Phi and LPi may contribute to SA-related defense signaling, although at a much lower magnitude than JA and ABA. Despite these transcriptional changes in SA signaling genes, Applicant did not observe any significant upregulation of SA biosynthetic genes in any of the treatments. This suggests that Phi does not strongly induce SA production at the transcriptional level in soybean, indicating that any SA-mediated effects may arise from post-transcriptional regulation or altered hormone sensitivity. The similarity in response patterns between ABA, JA, and SA may suggest a coordinated hormonal response to phosphite and phosphate stress, potentially reinforcing a stress-adaptive state in soybean.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0154] The simultaneous activation of ABA and JA signaling pathways suggests that Phi treatment may induce a complex stress-adaptive state, potentially mimicking Pi deficiency that, as a pleiotropic effect, also primes plants for enhanced defense responses. This hormonal regulation likely interacts with the observed hyperspectral and phenotypic changes, reinforcing the idea that Phi application alters physiological processes at multiple levels, ranging from transcriptomic shifts to pigment metabolism and canopy reflectance changes. Understanding how these hormonal pathways intersect with Phi-induced metabolic shifts will be crucial for optimizing Phi use in agricultural systems, particularly in P-limited soils where Phi could serve as a growth booster and as an alternative P source for ptxD plants.

[0155] PSR modulation in the Phi-ptxD system'. It has been previously described that Phi can be used as a structural analog of Pi that suppresses PSRs. Applicant’s findings reveal that Phi treatments in LPi conditions can modulate some PSRs in soybean. However, unlike hyperspectral data, the attenuation effect is not as pronounced at the transcriptomic level as in other plants. It is also known that Phi does not completely attenuate all genes induced by Pi starvation but enhances the expression of some of them. The observed changes in expression levels suggest that Phi modulates Pi starvation-induced responses in multiple ways (FIG. 6A).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0156] A key observation is that Phi activates the expression of 2779 genes in the HPi + Phi treatment that were also upregulated under LPi alone (FIG. 6E). These genes are primarily involved in photosynthesis, JA signaling, regulation of defense responses, and ROS-coping mechanisms. This aligns with previous reports, which demonstrate that Phi can activate ABA and JA signaling pathways, thereby enhancing stress adaptation and immune priming. The activation of JA and ABA signaling, in particular, suggests that Phi contributes to a plant defense-like state, potentially enhancing resilience to environmental stressors. Additionally, Phi enhances the expression of genes involved in chlorophyll biosynthesis and redox regulation, further supporting its role in modulating energy metabolism and oxidative stress responses. This was also observed in Arabidopsis, but it appears to be more critical in soybean. Phi attenuates the expression of only 19 genes under LPi + Phi compared to LPi alone, most of which are involved in cell wall modifications and lateral root development. This suggests that while Phi does not broadly suppress PSR in soybean, it still attenuates some of its more critical responses, such as lateral root formation.

[0157] To further explore the regulatory basis of these changes, Applicant analyzed the expression of putative STOP1 and PHR1 pathway components. Phi treatment altered the expression of key Pi-homeostasis regulators, reinforcing the idea that it interferes with Pi sensing. In the STOP1 pathway, Phi increased the expression of STOP1 homologs itself, as well as its downstream target ALMT1, which plays a role in rhizosphere acidification and nutrient mobilization. Additionally, Phi modulated SIZ1 and MED 16, known regulators of STOP1 stability and function. This suggests that Phi ion in fact interferes with this signaling pathway, although not in the same way as in Arabidopsis (FIG. 5A). In the PHR1 pathway, Applicant observed that SPX homologs, which are downregulated under LPi, exhibited less repression under LPi + Phi, suggesting that Phi disrupts the Pi-sensing mechanism controlled by SPX-initiated feedback loops. Conversely, PHR1 homologs were more strongly activated in LPi + Phi than in LPi alone, indicating that Phi reinforces some aspects of the Pi starvation response despite its inhibitory effects on SPX-mediated regulation. However, the upregulation of SIZ1, a SUMOylation enzyme known to modulate PHR1 activity, suggests the existence of a regulatory feedback mechanism that fine-tunes Phi-induced responses (FIG. 5B).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0158] Overall, these findings reinforce the idea that Phi is not merely a passive Pi analog but an active regulatory molecule that reshapes Pi starvation responses. Phi enhances stress-related gene expression, modulates key regulatory pathways, and selectively suppresses certain starvation- induced defense mechanisms. These effects appear to be context-dependent, varying based on Pi availability. Given its ability to modulate plant metabolism, Phi holds potential as a tool for improving nutrient management strategies in agriculture, particularly in low-Pi environments where its controlled use could optimize plant growth and stress resilience.

[0159] Metabolomic data show correlation between defense molecules and Phi, suggesting activation of defense responses'. Many metabolites that correlated with Phi accumulation in soybean are well-documented for their roles in stress mitigation, particularly in responses to drought, heat, oxidative stress, and pathogen defense. The observed changes in osmolytes, antioxidants, and phenylpropanoid derivatives indicate that Phi may enhance plant resilience under environmental stress conditions (FIGS. 8A-8L). Several sugar and amino acids were identified in this Example, including mannitol, sorbitol, oxoproline, L-proline, and serine, which are classic osmoprotectants that play crucial roles in mitigating drought, cold, and salinity stresses. These compounds help maintain cellular turgor pressure by stabilizing proteins and membranes, reducing water loss, and preventing osmotic damage. Proline, in particular, is a well-known drought and salt stress protectant, accumulating in plants under dehydration conditions to scavenge reactive oxygen species (ROS), stabilize proteins, and maintain cellular redox homeostasis. The upregulation of proline metabolism in Phi-treated plants suggests that Phi may prime plants for enhanced drought tolerance by promoting osmoprotectant accumulation.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0160] Additionally, sorbitol and mannitol function as compatible solutes, which protect cellular structures against osmotic stress caused by drought and salinity. These molecules also facilitate ROS scavenging and enhance antioxidant defense mechanisms, further reinforcing their role in stress resilience. The presence of these metabolites in both Wt and ptxD plants treated with Phi indicates that Phi exposure, whether metabolized or not, influences osmotic balance and water retention strategies in soybean. Additionally, phenethyl 2-primeveroside and 2-phenylethanol are phenyl compounds that are produced under stress conditions that help plants cope against drought.

[0161] Heat stress is closely linked to oxidative stress, as elevated temperatures cause an overproduction of ROS, leading to cellular damage. Several metabolites correlated with Phi accumulation, including phenylalanine and coumaroyl derivatives, are involved in phenylpropanoid metabolism, a crucial pathway in heat stress tolerance and antioxidant defense. These compounds contribute to the synthesis of flavonoids, lignin, and other secondary metabolites that reinforce cell wall integrity and mitigate oxidative damage caused by temperature fluctuations. Phenylalanine, a precursor for multiple phenylpropanoids and flavonoids, plays a critical role in stress adaptation, as flavonoid compounds function as UV protectants and ROS scavengers, shielding plant tissues from oxidative damage. The increased accumulation of p- coumaroylquinic acid and phenethyl glycosides further suggests that Phi exposure enhances the phenylpropanoid pathway, potentially improving heat tolerance and light stress adaptation in soybean.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0162] Interestingly, the Phi-responsive metabolites such as salicyoyl aspartate are also linked to plant immunity. Salicyoyl aspartate is a derivative of SA, a key hormone in systemic acquired resistance (SAR) and pathogen defense. The correlation between Phi accumulation and salicyoyl aspartate suggests that Phi may prime plant immunity, potentially enhancing resistance against biotic stressors. Furthermore, phenylpropanoids and flavonoid derivatives play a crucial role in phytoalexin biosynthesis, which strengthens plant defense mechanisms against pathogens. The presence of multiple phenylpropanoid-associated compounds in Phi-treated plants suggests that Phi exposure may induce a mild defense priming effect, improving plant resilience against future pathogen attacks.

[0163] The correlation between Phi accumulation in the Wt and Phi treatment in the ptxD plants and multiple stress-related metabolites strongly suggests that Phi acts as more than just a P analog in plants. Instead, Phi appears to function as a modulator of stress responses, influencing osmoprotection, antioxidant defense, and metabolic priming. The observation that these effects are retained in the Phi-ptxD system suggests that Phi metabolism modulates its stress-related signaling effects, reinforcing the idea that Phi may act as both a stress signal and a metabolic substrate. Overall, the metabolomic shifts observed in this Example indicate that Phi influences a broad network of physiological responses, many of which align with known plant stress adaptation mechanisms . Whether these responses are a direct effect of Phi perception, an indirect consequence of metabolic disruptions, or a combination of both remains an open question. Further research should investigate how Phi-responsive pathways intersect with classical stress signaling networks and whether Phi applications could be leveraged to enhance crop resilience under abiotic stress conditions.

[0164] Example 2. The Phi- / .vD system provides protection against high temperature and water limitation stresses by potentiating multiple transcriptional and metabolic responses in Glycine maxPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0165] In this Example, Applicant shows that Phi supplementation activates multiple stress- responsive genes in soybean, which help plants cope with both drought and heat stress. Phi treatments significantly enhanced plant growth and physiological parameters by activating protective mechanisms, including enhanced chlorophyll biosynthesis, maintenance of photosynthetic processes, and antioxidant defense responses involving SOD and GPX enzymes in control and the tested stress conditions. At the molecular level, Phi triggered more than a hundred TFs that are also activated under heat and drought, from which we can highlight TFs like AtHB 13, NF-YA7, ERF74, WRKY40, WRKY42, and PHR1, previously identified to help plants cope with drought and heat stresses.

[0166] Additionally, Applicant showed that the transgenic ptxD soybean lines can also benefit from Phi treatments, especially when Phi was the only source of phosphorus and used it at higher rates. They exhibit molecular and physiological responses similar to those of the wild-type plants when given low doses of Phi. These transcriptional responses were potentiated in the ptxD plants, as demonstrated in the RNA-seq analysis, which provided ptxD plants with the capacity to utilize Phi as the sole P source and to enhance stress tolerance responses more effectively. This shows that Phi has great promise as an alternative phosphorus fertilizer in transgenic crops. These results show that Phi can both promote growth and prepare plants for stress at the molecular level. This is a promising way to make crops more resistant to drought and heat while also preventing weed development.

[0167] The frequency and severity of high temperatures and drought events have increased in various agroecological regions worldwide, directly affecting global agricultural productivity. In the US, approximately $27 billion in crop yields have been lost due to global warming from 1991 to 2017, while in 2024 alone, about $5.4 billion was lost due to droughts. Since 79.6% of other economic sectors are directly linked to agriculture, changes in crop yields can influence the overall cost of many products. Consequently, this has a significantly negative effect on the global economy.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0168] Drought and high temperatures are stresses that impact virtually all aspects of the plant’s life, from early seed germination to grain production. Both stresses trigger complex plant responses that involve oxidative stress, membrane destabilization, hormonal signaling, and transcriptional reprogramming, impacting whole development. These stresses, therefore, adversely affect nutrient uptake, photosynthesis, root system development and integrity, and disrupt flowering, making them particularly detrimental to all food and feedstock cops.

[0169] Such environmental stresses significantly threaten soybean (Glycine max [L.] Men-.) cultivation, which is the second most valuable crop in the US and the top oilseed worldwide according to the latest data. It is a crucial crop both economically and for food supply, grown across a broad geographic area that is increasingly challenged by climate change, thus threatening its global sustainable production.

[0170] Severe drought has been reported to significantly reduce the growth, yield, and seed quality of soybean. Additionally, drought stress during the flowering and early pod-filling stages (R1-R6) notably reduces pollen germination (17%), lowers seed number by about 45%, and reduces seed weight by around 35%. A similar effect occurs under acute high-temperature stress; short-duration heat waves during early pod development (in U.S. conditions) caused approximately 10% loss in end-of-season seed yield due to flower and pod abortion, despite physiological recovery post-stress, indicating damage during the reproductive phase.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0171] As sessile organisms, plants are naturally subjected to multiple stresses, and few studies have investigated the interaction between water limitation, high temperatures, and low nutrient availability. Being an essential nutrient, low phosphorus (P) supplies have been extensively reported to have severe effects on plant growth and reproduction. In soybean, for instance, low P availability stunts shoot and root growth, reduces the number of lateral roots and lowers total root surface area, ultimately reducing seed yield and dry biomass, especially when P is limited during or after the flowering stage. This scenario requires the development of new technologies to protect agriculture, food security, and other economic sectors that rely on agriculture, such as dairy, cattle, textiles, biofuels, bioplastics, pharmaceuticals, nutraceuticals, and other industries, from multiple environmental stresses, including water limitation, high temperatures, and low P availability.

[0172] Phosphite (Phi; H2PO3 ), a reduced form of P, has been used for over 30 years as an agrochemical with biostimulant properties that enhance yields and plant defenses against both biotic and abiotic stresses. This has been demonstrated in several species, such as Capsicum annum, Solatium lycopersicum, Vigna unguiculata, Arabidopsis thaliana, Glycine max, Triticum aestivum, and Solanum tuberosum.

[0173] In the case of soybean, Phi has been tested mainly to control diseases such as anthracnose, which is caused by a complex of fungal species (e.g., Colletotrichum truncatum) and soybean root rot caused by Phytophthora sojae. In these reports, Phi treatment exhibited dual activity, both in terms of fungal toxicity and the ability to induce the activation of plant defense-related enzymes, such as catalase, peroxidase, and superoxide dismutase, resulting in effective control of C. truncatum and P. sojae. Phi was described as being as effective as the fungicide carbendazim + thiram in treating soybean seeds infected by the fungus C. truncatum. This effective control can be attributed to Phi's capacity for translocation in soybean from root to shoot within 1 h, with concentrations rising in the leaves after 36 h of application. While not directly metabolized by plants, Phi induces a chemical priming that pre-activates plant defenses, allowing swifter activation of biotic and abiotic stress adaptation pathways, through mechanisms that are just beginning to be unraveled.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0174] Applicant’s work with Arabidopsis thaliana revealed that, under optimal Pi fertilization, Phi application enhances growth and pre-activates defense hormone pathways. Applicant also found that Phi upregulates genes involved in abscisic acid (ABA) biosynthesis and signaling, which are crucial for adaptation to abiotic stress. Phi also pre-activates salicylic acid (SA) and jasmonic acid (JA) pathways, enhancing immune-related gene expression even under both optimal P nutrition and Pi starvation. These hormonal responses may be responsible for plant-enhanced resilience to environmental challenges while improving nutrient and water-use efficiency.

[0175] Engineered plants with the capacity to metabolize Phi as the only P source have been generated. These plants express the ptxD gene of bacterial origin, which encodes for a phosphite oxidoreductase that converts Phi into phosphate (Pi) using NAD as a cofactor, and in combination with Phi provides an interesting alternative agricultural system. Because these plants can use Phi as the only P source, the system enables an effective control of weeds, which cannot metabolize Phi. Moreover, this system holds great potential to optimize P usage and help address P resource shortages and decrease fertilizer and pesticide requirements.

[0176] Field trials with transgenic tobacco plants expressing the ptxD gene in low-P soil demonstrated that the Phi-ptxD system is robust and the transgenic plants grew successfully using Phi as the sole P source and achieved weed control comparable to that with glyphosate in two agricultural locations. In Example 1, Applicant demonstrated that ptxD soybean plants can metabolize Phi, and they are able to grow using it as the only source of P and show enhanced growth. Applicant’s analyses revealed that Phi treatment pre-activates stress-related hormone signaling pathways, particularly those involving ABA and JA, leading to increased accumulation of osmoprotectants (proline, sorbitol, mannitol) and defense-associated metabolites (phenylpropanoids, coumarins). These metabolic shifts suggest that Phi enhances the tolerance of soybean plants to abiotic stresses, such as high temperatures and drought, showing the potential of the Phi-ptxD system as a valuable strategy for improving P fertilization and resilience of ptxD plants in agricultural systems, while stunting weeds.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0177] The Phi-ptxD technology presents the opportunity to investigate the effects of Phi fertilization on enhancing the plant’s capacity to respond and thrive to stress conditions, adding to the proven benefits of weed control and P fertilization. Moreover, it provides an excellent system to investigate the effect of combined stresses on plant growth and yield performance.

[0178] Given that Phi-metabolizing soybean plants have been generated recently, Applicant aimed to evaluate the performance of ptxD plants using Phi as the sole P source, while subjected to limited water and high-temperature conditions, and determine the potential of Phi to mitigate deleterious effects on soybean plants and the molecular mechanisms underlying these responses. By integrating metabolomics, transcriptomics, and high-throughput plant phenotyping, Applicant found that Phi enhances growth of soybean under optimal conditions, as well as under both water limitation and high-temperature stress conditions.

[0179] Importantly, transgenic ptxD plants consistently showed higher growth than the wild-type (Wt) using Phi fertilization as the only P source under both stresses, demonstrating that they can metabolize Phi while improving stress responses. Applicant’s transcriptomic analysis also revealed that Phi induced stress tolerance at the molecular level. Biochemical profiling also indicated that Phi-treated plants exhibited enzymatic responses similar to those of low-water plants, suggesting a chemical priming effect.

[0180] This Example enhances Applicant’s understanding of how Phi influences defense responses in plants at the molecular level and presents the Phi- / .v soybean system as a highly promising alternative by tackling multiple abiotic stresses and offering a sustainable method to boost crop resilience against climate stresses.

[0181] Example 2,1, Water limitation and P starvation experimentsPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0182] Water limitation experiments were designed based on the gravitometric method. Sand was used as the substrate, and was dried for two days in an oven at 70°C. The weight was constantly monitored until the soil no longer lost any weight; this point was established as 0% of water content (WCO). A known volume of WCO soil was weighed and watered to its maximum capacity. The soil was monitored until the pots stopped dripping water, and afterward, the pot was weighed again. The difference in weight between the watered soil and the dry soil was 100% WC (WC100).

[0183] Wt and ptxD soybeans were sown in 72-well trays for germination, and after 1 week, seedlings with similar height were transplanted to pots at WC80 and maintained at this water level for one week. Then, plants were subjected to the respective treatments. In terms of P level and source, the following treatments were set up: HPi (100 ppm added Pi), LPi (0 added Pi), HPi + Phi (75 ppm added Pi + 25 ppm added Phi), and LPi + Phi (100 ppm added Phi only). In terms of watering level, Applicant set up 80% WC (WC80) as the control group and the 30% WC (WC30) as the water limitation group. This was because in previous experiments, plants at 20% WC died, and this prevented Applicant from evaluating plants and collecting tissues for the following experiments.

[0184] The weight of the pots was monitored twice per week to calculate their WC. Water was added as needed to maintain the WC80 level for the control groups. For the low irrigation treatment, pots were allowed to dry gradually, and water was adjusted to match the weight of the heaviest pot for this group at each time point. This process continued until all low water-stressed pots stabilized at WC30. Measurements were performed weekly until the WC30 level was reached, and a final evaluation was taken one week after plants were maintained at WC30. The duration of the experiment was seven weeks. Each experiment was performed with four replicates per treatment per genotype.

[0185] Example 2.2. High-temperature and P starvation experimentsPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0186] High-temperature experiments were set up similarly to the low-water experiments. The sand substrate was prepared, and Wt and ptxD soybeans pre-germinated in well trays as mentioned above. After transplanting, seedlings were subjected to the same P treatments, HPi, LPi, HPi + Phi, or LPi + Phi, and allowed to grow for another week. After that, plants were separated into two groups: group 1 (40C), to be grown at 40 °C for 16 h [day] and 35°C for 8 h [night]), and group 2 (25C), to be grown at 25°C for 16 h [day] and 25°C for 8 h [night]). Plants were maintained under temperature stress for one week. Measurements were made before the start of the high-temperature stress and after one week of treatment. The duration of each experiment was four weeks. The experiment was performed with four replicates per treatment per genotype.

[0187] Example 2,3. Effects of Phosphite on the growth of soybean under low irrigation and P deficiency

[0188] To investigate the impact of Phi on soybean growth under low irrigation conditions in the context of the Phi-pt D system, the previously generated and characterized transgenic line G7np(xD-35A (Example 1), was selected for these experiments. Applicant evaluated plant height and canopy area in both Wt and transgenic ptxD plants grown under two phosphate levels: optimal Pi (HPi) and low Pi (LPi), with or without Phi supplementation (+ Phi), and exposed to low irrigation (WC30) or well-watered conditions (WC80), for a total of 16 independent groups. Plants were manually evaluated weekly until the WC30 level was achieved, and a final evaluation was conducted one week after the plants had been stably maintained at WC30.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0189] In the HPi condition at WC80, Wt plants reached an average height of 156.02 mm, which increased to 182.36 mm when treated with Phi (FIGS. 9A and 9C). Water limitation (WC30) substantially reduced plant height to 99.26 mm in HPi, whereas the Phi treatment had a positive impact by increasing plant height to 138.79 mm even under WC30 (FIGS. 9A and 9C). LPi treatment negatively impacted the height of Wt plants in both WC80 (107.83 mm) and WC30 (90.28 mm) growing conditions, with the impact being more severe under water limitation. Under LPi conditions, Phi supplementation to Wt plants under both watering regimes, WC80 and WC30%, resulted in extremely deleterious effects, leading to plant death, which is indicative of Phi phytotoxicity, as Wt plants cannot metabolize Phi and accumulate it to toxic levels.

[0190] Well-watered Wt soybean plants had a canopy area of 152 cm2under HPi treatment, which reached 173.02 cm2with Phi treatment. Water limitation (WC30) caused a marked reduction in canopy area in Wt plants with 35.85 cm2, consistent with drought-induced growth inhibition (FIGS. 9A and 9C). However, supplementation with Phi under these conditions (HPi + Phi_WC30) resulted in a significant increase in canopy size (51.27 cm2). LPi conditions had detrimental effects on Wt plant growth; canopy expansion was severely reduced, with Wt plants reaching only 29.86 cm2under WC80 and 11.76 cm2under WC30.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0191] Well-watered (WC80) transgenic ptxD plants under HPi exhibited similar behavior to Wt plants under the same treatment. Their growth potential was reduced under water limitation (WC30), with an average plant height of 121.75 mm. When supplemented with Phi (HPi + Phi_WC30), a statistically significant increase in plant height was not observed, as in the case of Wt plants. However, ptxD plants reached the highest plant height values when using exclusively Phi as the only P source (LPi + Phi_WC80) with 192.94 mm, confirming that ptxD plants benefit most from Phi when both water and Phi availability are high. Water limitation under exclusive Phi fertilization increased plant height (129.08 mm) compared to the control with no Phi supplementation (FIGS. 9B and 9D). Canopy area of ptxD plants under well-watered conditions and HPi (153.3 cm2), was comparable to that of the Wt plants under the same treatment. Water limitation under optimal P fertilization resulted in a decrease in canopy area (34.42 cm2), as expected, with no significant improvement upon Phi treatment. Under exclusive Phi fertilization, ptxD plants exhibited a canopy area of 168.96 cm2when well-watered, which was higher than that of the ptxD and Wt plants under the HPi control treatment (FIGS. 9B and 9D). Plants with low irrigation showed a reduced area of 22.97 cm2, which was increased to 52.43 cm2when treated with Phi even under water stress, confirming that Phi has a beneficial effect when used as the sole P source in ptxD soybeans.

[0192] Example 2,4, The Phi-ptrD system displays better performance under water imitation stressPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0193] To gain a deeper understanding of the physiological changes induced by stress conditions, Applicant determined total chlorophyll and anthocyanin content and the activity of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPX), one week after the low irrigation group reached 30% WC. In Wt plants grown under HPi and well-watered conditions (HPi_WC80), chlorophyll content was moderately high (9.27 mg / g FW), whereas drought stress (HPi_WC30) caused a reduction of chlorophyll (6.03 mg / g FW). Phi supplementation under both water levels and HPi, significantly increased chlorophyll content, with 9.54 mg / g FW under WC30 and 12.2 mg / g FW under WC80. Pi starvation (LPi) caused a decrease in chlorophyll levels regardless of the watering level, with values of 4.72 mg / g FW in WC80 and 5.52 mg / g FW in WC30. In ptxD plants, overall chlorophyll content under the HPi and WC80 treatment was 9.36 mg / g FW, while low watering led to a reduction (6.92 mg / g FW).

[0194] This was similar to the Wt plants. Phi supplementation to plants grown under both watering levels and HPi, slightly increased chlorophyll content; 8.30 mg / g FW in WC30 and 10.19 mg / g FW in WC80. However, this increase was not statistically significant. Under LPi conditions, chlorophyll levels decreased in both well-watered (5.38 mg / g FW) and low-water-stressed plants (4.79 mg / g FW) compared to the HPi conditions. However, Phi supplementation as the only P source under drought (LPi + Phi_WC30) significantly increased chlorophyll accumulation (12.76 mg / g FW), compared to the control in WC80 (8.70 mg / g FW).

[0195] Anthocyanin content was also measured to assess potential pigment accumulation under stress and Phi treatments. However, Applicant did not detect any statistically significant differences among treatments in either Wt or ptxD genotypes. While anthocyanin content varied across all groups, none of these changes reached statistical significance.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0196] SOD activity is a marker of oxidative stress response in plants and varied across treatments in both Wt and ptxD plants. In Wt plants under the HPi_WC80, the observed SOD activity was 1.66 U / mg, which increased in response to water limitation (1.73 U / mg). Phi supplementation under HPi and WC80 slightly increased SOD activity to 1.99 U / mg, compared to the control with no Phi, whereas under water limitation, SOD showed a substantially higher activity of 2.38 U / mg. In contrast, under LPi conditions, SOD activity was low in both WC80 (1.51 U / mg) and WC30 (1.94 U / mg).

[0197] In ptxD plants, SOD activity under well-watered and HPi conditions was 1.41 U / mg, and water limitation caused a significant increase to 2.65 U / mg. Phi treatment caused minimal changes compared to the control with no Phi. Plants growing under LPi, in both well-watered and waterlimited conditions, showed comparable SOD levels, 2.30 and 2.11 U / mg, respectively. While ptxD plants under Phi fertilization only maintained high SOD activity (2.24 U / mg) when water was limited, compared to the well-watered controls (1.6 U / mg).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0198] GPX activity, another key indicator of antioxidant response, showed treatment- and genotype- specific trends in both Wt and ptxD plants. In Wt plants grown under HPi and WC80, GPX activity was 1.60 U / mg, which increased in response to water stress to 2.50 U / mg. Phi treatment of plants grown under WC80 caused no significant changes (2 U / mg). However, Phi supplementation under water limitation (HPi + Phi_WC30) had a more pronounced effect, elevating GPX activity to 3.52 U / mg. In contrast, Wt plants under LPi showed GPX levels of 2.13 U / mg under WC80 and 4.31 U / mg under WC30. In ptxD plants, GPX activity in the HPi treatment and WC80 was 2.22 U / mg, while water stress significantly decreased GPX activity to 0.39 U / mg. Phi supplementation to plants under both watering levels did not cause significant changes compared to the control; GPX activity was 0.74 U / mg under WC30 and 2.10 U / mg under WC80. In LPi conditions with optimal irrigation, GPX activity was higher (3.41 0.57 U / mg) than in HPi conditions, but the difference was not statistically significant. Water limitation severely decreased GPX activity to 0.57 U / mg. Surprisingly, even with Phi supplementation (LPi + Phi_WC30), GPX activity remained low (0.75 U / mg), and reached levels comparable to the control group (2.80 U / mg) in WC80.

[0199] Applicant also evaluated other photosynthetic parameters such as net photosynthetic rate (A), stomatai conductance (gsw), yield of Photosystem II (<|)PSII), and efficiency of PSII under light-adapted conditions (Fv' / Fm') in Wt and ptxD plants. Overall, Phi application improved all parameters in Wt plants under optimal Pi fertilization under water stress. However, the ptxD plants with Phi as the exclusive P-fertilizer, showed substantially improved parameters mainly under water limitation, suggesting the advantages of using the Phi-ptxD system.

[0200] Example 2,5. Global Gene Expression Changes Reveal Shared and Unique unregulated gene sets between water limitation and phosphorus starvationPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0201] To assess the overall transcriptomic responses of plants under different stress conditions, Applicant performed RNA sequencing (RNA-seq) analysis of most samples collected at the end of the experiment, after the low irrigation groups were exposed for one week to WC30. All samples were collected except the ones under LPi + Phi. Those samples were collected when plants reached WC30, as longer periods of exposure under both stresses cause most plants to die. A Multidimensional Scaling (MDS) analysis was performed for all RNA-seq libraries separated by genotype. Within each genotype, samples were further grouped based on P fertilization levels and Phi treatment rather than water availability, except in the case of LPi + Phi treatments in the Wt plants. Then, differentially expressed genes (DEGs) were quantified by comparing treatment conditions to the HPi _WC80 control reference.

[0202] In the Wt plants, water limitation alone (HPi_WC30 vs. HPi_WC80) resulted in 6,380 DEGs, with 3,766 upregulated and 2,614 downregulated genes, indicating a substantial transcriptional shift in response to the stress. When Phi was applied under water limitation (HPi + Phi _WC30 vs. HPi_WC80), the number of DEGs increased slightly to 8,431 (4,868 upregulated, 3563 downregulated). Phi supplementation under well-watered conditions (HPi + Phi_WC80 vs. HPi_WC80) induced more DEGs (8,209 total, and 3,538 upregulated, and 3,552 downregulated), indicating a smaller, Phi-specific transcriptional signature when stress is absent. Shifting to LPi, Wt plants showed extensive transcriptional reprogramming. The LPi + Phi_WC30 vs. LPi_WC80 contrast yielded 17,433 DEGs (8,320 upregulated, 9,113 downregulated), representing the highest DEG count among all Wt comparisons. This substantial response suggests a strong transcriptional perturbation caused by Phi toxicity in Pi-starved Wt plants and water limitation.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0203] In response to water stress alone (HPi_WC30 vs. HPi_WC80) (FIG. 10A), highly enriched Gene Ontology (GO) terms in the Wt plants were associated with cell wall organization, photosynthesis, ROS detoxification, and response to auxin, aligning with structural remodeling and stress adaptation. Several GO categories associated with oxidative stress, including glutathione metabolic processes and cellular oxidant detoxification, were significantly enriched, highlighting ROS scavenging as a fundamental response to water limitation. In the combined Phi and water deficit treatment (HPi + Phi_WC30 vs. HPi_WC80) (FIG. 10B), GO enrichment analysis revealed significantly increased categories, including photosynthetic light harvesting, high light response, chloroplast organization, and heat sensitivity, indicating that both water limitation and Phi affect photosynthetic capability while modifying cell wall organization. In Phi-trcatcd well-watered plants (HPi + Phi_WC80 vs. HPi_WC80) (FIG. 10C), the enriched GO terms were predominantly associated with photosynthesis, light harvesting, chlorophyll biosynthesis, and some terms related to ROS coping, indicating that Phi enhances energy metabolism and protein synthesis in non-stress conditions.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0204] In the case of the ptxD plant under water stress alone (HPi_WC30 vs. HPi_WC80), they exhibited a transcriptomic response composed of 1,636 upregulated and 2,522 downregulated DEGs. Interestingly, Phi supplementation under water limitation (HPi + Phi_WC30 vs. HPi_WC80) resulted in a reduction in DEGs, with only 247 upregulated and 545 downregulated DEGs, suggesting a potential stabilizing or dampening effect of Phi on the ptxD plants’ response to water stress. In contrast, Phi addition under well-watered conditions (HPi + Phi_WC80 vs. HPi_WC80) yielded 2,299 upregulated and 1,228 downregulated genes, suggesting that ptxD responds to Phi even in HPi conditions. The strongest DEG responses were observed under LPi conditions. When comparing LPi_WC30 vs. HPi_WC80, ptxD plants showed 4,702 upregulated and 4,179 downregulated DEGs, indicating a robust transcriptional reprogramming under combined water stress and P limitation. Interestingly, Phi supplementation as P source under LPi and water stress (LPi + Phi_WC30 vs. LPi_WC80) led to 2904 upregulated and 2139 downregulated DEGs, while Phi addition to the same well-watered conditions (LPi + Phi_WC80 vs. LPi_WC80) induced 3919 upregulated and 2,370 downregulated DEGs. These results show that the transcriptional response of ptxD plants to Phi fertilization, particularly when Phi serves as the only P source.

[0205] The enriched GO terms in response to water limitation alone (HPi_WC30 vs. HPi_WC80) in ptxD plants were NADH dehydrogenase complex, chaperone-mediated protein folding, cellular response to hypoxia, and non-photochemical quenching, as well as response to water deprivation and response to heat. In the combined Phi and water limitation treatment (LPi + Phi_WC30 vs. HPi_WC80), the enriched categories were mainly related to photosynthesis. These included photosystem II assembly, photosynthetic light harvesting, and chlorophyll biosynthesis. Similar to the HPi_WC30 vs. HPi_WC80 contrast, NADH dehydrogenase complex and chaperon-mediated protein folding were upregulated in this treatment. When Phi was used as the only source of P but under no water stress (LPi + Phi_WC80 vs. HPi_WC80), GO enrichment showed a significant increase in photosynthetic activities, light harvesting, chloroplast growth, and protein refolding, as well as response to heat and response to high light intensity.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0206] To evaluate the extent of shared and unique transcriptional responses between stresses, Applicant compared DEGs using Venn diagrams (FIG. 10D). The first comparison performed was between the following conditions: water stress alone (HPi_WC30 vs. HPi_WC80), Phi treatment to well-watered plants (HPi + Phi_WC80 vs. HPi_WC80), and Phi treatment to water- stressed plants (HPi + Phi_WC30 vs. HPi_WC80) in the Wt plants. Water limitation and Phi under well- watered conditions shared 1,320 DEGs (15.5%), while 1,290 DEGs (15.1%) were unique to Phi under well-watered conditions, and 819 DEGs (9.6%) were unique to low irrigation alone. Notably, 1,293 DEGs (14.9%) were shared across all three conditions, suggesting a core set of genes responsive to both low water stress and Phi regardless of water status. A GO enrichment analysis of this set of shared DEGS showed enriched categories related to multiple stresses (heat, abscisic acid, cold, among others), and multiple categories associated with ROS coping mechanisms (FIG. 10E).

[0207] In the case of ptxD plants, a comparison of DEGs under water limitation (HPi_WC30), exclusive Phi fertilization under well-watered conditions (LPi + Phi_WC80), and under water stress (LPi + Phi_WC30), revealed 1,131 DEGs (22.8%), 763 DEGs (15.4%), and 1,394 DEGs (28.1%), respectively, exclusively to each condition. In contrast, only 231 DEGs (4.7%) were consistently regulated across all three treatments, indicating a core group of genes influenced by the combination of water and Pi stress, along with Phi supplementation. This same intersection for the ptxD plants showed the most enriched terms primarily linked to photosynthesis, carbon utilization, redox homeostasis, and chaperone-mediated protein folding. Additional categories, such as glycerol catabolic process, triglyceride mobilization, and response to light intensity, were also found.

[0208] Example 2.6. A subset of LPi-responsive genes is co-activated by Phi application and water limitationPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0209] It has been reported that plants transcriptionally induce common responses to both Pi deficiency and drought. To investigate this, Applicant examined the overlap of DEGs between Pi deficiency (LPi_WC80 vs. HPi_WC80) and low irrigation (HPi_WC30 vs. HPi_WC80) only, in the Wt and ptxD samples. In the Wt, a total of 3,785 DEGs (50.1%) were unique to the LPi_WC80 condition, while 1,251 DEGs (16.6%) were specific to the low irrigation treatment. Importantly, 2,515 DEGs (33.3%) were shared between both treatments, suggesting a substantial core response to Pi deficiency that is also modulated by low irrigation stress. In the case of the ptxD plants, a significant proportion of DEGs (5,914 genes, 78.3%) were exclusive to Pi starvation, while 1,223 DEGs (16.2%) were particular to water constraint. There were only 413 DEGs (5.5%) shared by both treatments. This suggests that there is a small but important overlap between the plant responses to Pi shortage and drought stress. A GO enrichment analysis of DEGs from this intersection between both stresses revealed that multiple categories associated with photosynthesis and cell wall organization were enriched in both genotypes. In contrast, those related to carbon metabolism and NADH dehydrogenase complex were enriched only in the ptxD plants (FIGS.11A-B).

[0210] Applicant further explored the expression patterns of genes differentially expressed in response to Pi starvation (LPi_WC80 vs. HPi_WC80) and low irrigation (HPi_WC30 vs. HPi_WC80) across selected contrasts (FIGS. 11C-D). In FIG. 11C, Applicant visualized the expression of genes upregulated under phosphate deficiency across all other treatments. Many of these genes (6300 DEGs in total) were also upregulated in the low irrigation condition (HPi_WC30 vs. HPi_WC80, 2515 DEGs shared). This, and the fact that Phi treatment also shares genes with drought (FIG. 11D) indicates that a subset of LPi-induced genes is co-activated by Phi and low irrigation.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0211] Applicant then examined genes differentially expressed under water limitation (FIG. 11D), and tracked their expression across LPi and Phi conditions. Applicant observed that many drought-responsive genes showed similar induction patterns under both LPi and Phi treatments, and when Phi was applied under low water availability sharing 1627 (388 + 1239 DEGs) and 2515 DEGs when compared (FIGS. 10D and 11A). This suggests that drought stress may trigger part of the same transcriptional machinery activated by Pi deficiency and Phi exposure. In the case of ptxD plants, the hierarchical heatmaps of DEGs significantly regulated under Pi deficiency (LPi_WC80 vs. HPi_WC80) showed a clear clustering pattern, revealing that many of these genes were also induced under LPi and LPi + Phi in both conditions, but just a few in low irrigation stress. DEGs identified under water limitation (HPi_WC30 vs. HPi_WC80) also showed activation in LPi_WC80.

[0212] Example 2,7, Enriched metabolites under low irrigation

[0213] Applicant analyzed how water limitation and Phi influenced the metabolite profile of soybean plants. Water limitation alone caused the upregulation of 58 metabolites in Wt plants, from which isoflavonoids, flavonoids, fatty Acyls, in addition to Pyrans, linear 1 ,3- diarylpropanoids, octadecanoids, and tetrapyrroles were the most enriched chemical classes. These are all known to be involved in stress signaling, pigmentation, and antioxidant properties.

[0214] Phi treatment (HPi + Phi_WC30 vs HPi WC80), slightly changed the metabolite profile of water-limited Wt plants, inducing the upregulation of 77 metabolites, among which organooxygen compounds, pteridines and derivatives, isoprenoids, in addition to flavonoid and isoflavonoids, were upregulated. Other groups of metabolites significantly enriched were Lignan glycosides, Pyrans, Carboxylic acids and derivatives, Pyrimidine nucleotides, Benzimidazoles, Octadecanoids, Pteridines and derivatives, Tetrapyrroles and derivatives, and Keto acids and derivatives.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0215] The effects of Phi when water was in optimal quantity over the metabolome of Wt plants (HPi + Phi_WC80 vs. HPi_WC80) revealed 44 upregulated metabolites. Flavonoids and Isoflavonoids were again the two most significant groups with 11 and 4 hits, respectively. Other significantly enriched metabolite groups were Pteridines and derivatives, Organooxygen compounds, Pyrimidine nucleotides, Linear 1,3-diarylpropanoids, Keto acids and derivatives, Prenol lipids, and Isoprenoids.

[0216] To understand the impact of the low irrigation regime in ptD-transgenic soybean, Applicant compared the same treatments as in Wt, but for the ptxD plants. In the case of the ptxD- transgenic plants, water limitation triggered enrichment of 30 metabolic groups from 386 upregulated metabolites. Metabolite classification changed slightly compared to the Wt plants. Among the most enriched compound classes were Carboxylic acids and derivatives, Organooxygen compounds, Isoflavonoids, Keto acids and derivatives, and Flavonoids. Cinnamic acids and derivatives and Coumarins and derivatives, also showed significant enrichment ratios.

[0217] Under water-limited conditions, supplementation with Phi (LPi + Phi_WC30 vs. HPi_WC80) upregulated 51 metabolites. The metabolic profile shifted to Isoflavonoids and Flavonoids with 5 and 6 hits, respectively. Notably, octadecanoids, linked to jasmonate biosynthesis and lipid signaling, were also elevated. Additional responses included pteridines and derivatives, cinnamic acids, and carboxylic acid derivatives, organooxygen compounds and fatty acyls. In total 11 metabolic groups were significantly enriched.

[0218] Under the Phi treatment with no stress (LPi + Phi_WC80 vs. HPi_WC80), ptxD plants also showed 115 upregulated metabolites. Isoflavonoids and Flavonoids with 18 and 24 hits, respectively, were enriched. Alongside these metabolites, linear 1,3-diarylpropanoids, Linear 1,3- diarylpropanoids, Steroids and steroid derivatives, Cinnamic acids and derivatives, Pteridines and derivatives, Organooxygen compounds, Lignan glycosides, Pyrans, Pyrimidine nucleotides, Benzimidazoles, Octadecanoids, Keto acids and derivatives, and Peptidomimetics were groups significantly enriched.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0219] Example 2.8. Effects of Phosphite on the growth of soybean under high temperature

[0220] Independent experiments were set up to evaluate the effect of Phi supplementation in Wt and transgenic ptxD plants grown under similar P regimes, HPi and LPi, and subjected to high temperature stress. These experiments provided Applicant information about the Phi-ptxD system performance in response to those three environmental factors. Plant growth and canopy development were monitored once a week during the experiment to evaluate plant performance when grown under 25°C (25C), the optimal condition, or 40°C (40C), a high temperature stress, resulting in a total of 16 independent groups. All samples were collected after one week of exposure to the 40°C treatment, except the ones under LPi + Phi. These samples were collected after 3 days.

[0221] Under high temperature, Wt plants showed a significant reduction in height across all treatments when compared to the control temperature (FIGS. 12A and 12C). In the HPi_25C control condition, Wt plants reached an average height of 145.51 mm. However, under 40°C, the average height was 116.51 mm. The Phi-treated plants under high temperature (HPi + Phi_40C) cope better with the stress, growing to an average height of 133.29 mm. Additionally, in control conditions and treated with Phi (HPi + Phi_25C), the height of Wt plants was 186.36 mm on average, indicating a beneficial effect when plants are treated with Phi (FIGS. 12A and 12C).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0222] Under LPi at 25°C, height dropped to 111.08 mm, and it decreased further to 82.78 mm at 40°C, showing the compounding adverse effects of Pi deficiency and high temperature. As in the low irrigation experiments, Wt plants grown under LPi + Phi at both temperatures did not survive due to Phi toxicity and are therefore not included in the figures (FIGS. 13A and 13C). In plxD plants (FIGS. 13B and 13D), the response to heat stress differed. Under HPi_25C, plxD plants reached 190.56 mm, and high temperature reduced height to 119.25 mm (HPi_40C), even under optimal P fertilization. Phi supplementation under temperature stress (HPi + Phi_40C) did not significantly improve height (120.63 mm). However, under optimal temperature (HPi + Phi_25C), ptxD plants grew slightly better, reaching a height of 201.95 mm, which was comparable to that of the control treatment. In the LPi condition, plant height was reduced at both temperature conditions, with 107.29 mm at 25°C and 98.99 mm at 40°C. When Phi was provided as the only P fertilizer under temperature stress (LPi + Phi_40C), ptxD plants grew an average of 155.58 mm, indicating that ptxD plants had enhanced growth under temperature stress when supplied with Phi. The highest growth was observed in the LPi + Phi_25C condition (199.02 mm), indicating that the beneficial effect of Phi was potentiated when used as the sole P source by ptxD plants.

[0223] Canopy area was also affected by temperature, P availability, and Phi supplementation in both genotypes (FIGS. 13C and 13D). In Wt plants under HPi at 25°C, canopy expansion was robust, reaching 100.77 cm2(FIG. 13C). Exposure to 40°C (HPi_40C) led to a reduction in canopy size (79.52 cm2), while Phi supplementation under high temperature (HPi + Phi_40C) did not significantly improve it (80.68 cm2). Under HPi + Phi at 25°C, the canopy area increased to 119.88 cm2. In LPi treatments, canopy size was consistently lower in Wt plants, showing heights of 77.83 cm2at 25°C and 52.78 cm2at 40°C. In the case of ptxD plants (FIG. 13D) grown under HPi at 25°C, the canopy area reached 101.04 cm2, which was reduced in response to high temperature to 63.23 cm2.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0224] Interestingly, Phi supplementation under temperature stress (HPi + Phi_40C) had little impact (50.36 cm2), while the combination of HPi + Phi_25C did not improve canopy size as in Wt plants (76.27 cm2). In LPi conditions at 25°C, canopy size reached 73.08 cm2and was further reduced under 40°C (56.35 cm2). Notably, Phi supplementation as the only P source in the absence of Pi fertilizer, under temperature stress (LPi + Phi_40C), preserved canopy area to similar values as the HPi 40°C treatment (78.91 cm2), while at 25 °C it had also similar values as the control treatment (106.47 cm2).

[0225] Example 2.9. The Phi-p / system improves plant performance under high temperature stress

[0226] Chlorophyll content was strongly influenced by temperature, phosphate availability, and Phi supplementation in both genotypes. In Wt plants under HPi at 25°C, chlorophyll content was 9.46 mg / g FW, which decreased to 5.85 mg / g FW under high temperature (HPi_40C). When plants grow under optimal Pi and temperature stress (HPi + Phi_40C), the determined chlorophyll levels were similar to control conditions (8.98 mg / g FW), indicating that Phi alleviates chlorophyll loss under stress in Wt plants. In contrast, Phi treatment under optimal conditions (HPi + Phi_25C) further increased the chlorophyll content to 12.16 mg / g FW. Under LPi, Wt plants showed significantly reduced chlorophyll levels at both 25 °C (3.3 mg / g FW) and 40°C (5.48 mg / g FW). In ptxD plants, chlorophyll levels under HPi_25C were moderately high (9.27 mg / g FW), and decreased slightly under temperature stress (HPi_40C, 6.71 mg / g FW). Phi supplementation under high temperature (HPi + Phi_40C) yielded not significantly different values (7.77 mg / g FW). Under HPi + Phi at 25°C, chlorophyll content was maintained at 7.776 mg / g FW. Under FPi conditions, ptxD plants exhibited lower chlorophyll levels at 25°C (5.1 mg / g FW) and 40°C (4.97 mg / g FW). Notably, chlorophyll content significantly increased at both temperatures when Phi was used as the P source. Values were higher than those of the controls, with 10.51 mg / g FW and 11.76 mg / g FW at 25 °C and 40°C, respectively.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0227] In the case of anthocyanin accumulation, Wt plants under HPi at 25°C accumulated an average of 2.19 Abs / g FW, while at 40°C, it decreased to 1.59 Abs / g FW. When Phi was added to plants under temperature stress (HPi + Phi_40C), anthocyanin accumulation increased to 2.80 Abs / g FW. Under HPi + Phi_25C, the mean was 2.41 Abs / g FW. Under LPi, Wt plants showed reduced anthocyanin accumulation with means of 0.93 Abs / g FW at 25 °C and 1.49 Abs / g FW at 40°C. In contrast, all Wt plants exposed to LPi + Phi (at either temperature) died prematurely and were excluded from the analysis. In ptxD plants under HPi at 25°C, anthocyanin content averaged 1.57 Abs / g FW, while under temperature stress (HPi_40C), the mean increased to 2.05 Abs / g FW, indicating a more robust stress response. With Phi supplementation under 25°C (HPi + Phi_25C), it remained at 1.84 Abs / g FW but increased to 2.23 Abs / g FW in response to temperature stress (HPi + Phi_40C). Under LPi, ptxD plants maintained moderate levels of anthocyanins: 1.84 Abs / g FW at 25°C and 1.69 Abs / g FW at 40°C. Under LPi + Phi 40°C, anthocyanin content was 1.71 Abs / g F.W.

[0228] Wt plants grown under optimal Pi at 25°C showed a mean SOD activity of 1.50 U / mg, and increased to 1.70 U / mg as temperature increased to 40°C. Under HPi + Phi_40C treatment, SOD activity was further increased to 2.26 U / mg. In contrast, plants grown with HPi + Phi at optimal temperature had slightly higher activity than the HPi-only group, averaging 1.90 U / mg. In Wt plants under LPi at 25°C, the average SOD activity was 1.46 U / mg, which rose to 1.80 U / mg at 40°C, indicating some stress adaptation. In ptxD plants under HPi at 25°C, the mean SOD activity was 1.51 U / mg, which increased to 2.32 U / mg in response to high temperature. The treatment with Phi resulted in a slight increase in SOD activity at both temperatures: 1.64 U / mg at 25°C and 1.81 U / mg at 40°C, both of which were elevated compared to HPi alone. Under LPi at 25°C, ptxD plants displayed a mean SOD activity of 2.72 U / mg, while at high temperature stress, they showed similar levels of 2.21 U / mg. Under Phi fertilization at 40°C, the average was 1.68 U / mg.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0229] Wt soybean plants displayed distinct GPX activity profiles in response to the stresses. Under HPi at 25°C, the average GPX activity was 1.61 U / mg, which increased slightly to2.51 U / mg at 40°C, reflecting a mild oxidative response. When treated with Phi and grown at 25 °C, Wt plants had an intermediate mean activity of 2.00 U / mg, which increased to an average of3.52 U / mg at 40°C. Wt plants grown under LPi at 25°C displayed higher GPX activity with an average of 2.14 U / mg, while those at 40°C with the same Pi level showed an even stronger response (4.81 U / mg), indicating enhanced oxidative pressure. In ptxD plants, HPi at 25°C resulted in a mean GPX activity of 2.22 U / mg, which dramatically decreased to 0.39 U / mg in response to 40°C, indicating reduced oxidative pressure or altered regulation under this stress. Phi treatment under optimal Pi fertilization and 40°C similarly resulted in low GPX activity (0.72 U / mg), while the same condition at 25 °C had elevated activity (2.11 U / mg). This suggests that Phi enhances GPX activity mainly at moderate temperatures. ptxD plants under LPi growing at optimal temperature exhibited the highest GPX activity (83.41 U / mg) among all groups, followed by those under exclusive Phi fertilization. ptxD plants under temperature stress showed moderate GPX activity regardless Phi treatment.

[0230] Photosynthetic parameters were severely affected by both LPi and high temperature stresses. Interestingly, parameters such A, gsw, yield of Photosystem II (<|)PSII), and efficiency of PS11 under light-adapted conditions (FvVFm') in the ptxD plants when Phi was used as the only P source, were better than those of the Wt plants fertilized with Pi. ptxD plants also showed better performance under high temperature stress.

[0231] For instance, in Wt plants, A under the HPi_25C treatment was 3.2 pmol m2s *, but high temperature stress (HPi_40C) severely reduced it to 0.7 pmol m2s Adding Phi to both optimal (HPi + Phi_25C) and stressed (HPi_40C + Phi) growth conditions substantially increased A to 5.9 pmol m2s1and 2.1 pmol m2s ', respectively.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0232] LPi had severe effects on A, causing a drastic decrease to 0.07 pmol m2s1even at optimal temperature (25C), which decreased further (-0.61 pmol m2s ') under temperature stress (40C). Adding Phi caused most Wt plats to die. ptxD plants behaved similarly to Wt plants under optimal conditions and high temperature stress when using Pi fertilization. However, when Phi was the only source of P, A increased substantially under both control (7.3 pmol m2s *) and temperature stress (2.06 pmol m2s1). compared to the Wt plants, suggesting better plant performance.

[0233] Example 2,10, Global gene expression changes reveal shared and unique gene sets controlled by the stresses

[0234] RNA-seq analysis was conducted for samples collected from plants grown under these three stresses. An MDS analysis of all RNA-seq libraries separated by genotype, suggested additional grouping of samples along both dimensions based on temperature (25 °C vs. 40°C) and treatment (HPi, HPi + Phi, LPi, LPi + Phi). In Wt plants, samples from plants grown under 25°C were tightly grouped into clusters distinct from those at 40°C. Similarly, in ptxD, high- temperature-stressed samples diverged from their counterparts at 25°C.

[0235] To evaluate the transcriptional responses of soybean plants to high temperature and P treatments, Applicant quantified DEGs using HPi 25 °C as the control condition, and then to gain insights into relevant transcriptional mechanisms activated in the Wt and ptxD plants under high temperature stress and how Phi modulates these responses, Applicant performed GO enrichment analysis of DEGs. Under high temperature stress alone in the Wt (HPi_40C vs. HPi_25C), Applicant found 6,207 upregulated and 6,751 downregulated genes, totaling 12,958 DEGs.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0236] Phi supplementation under temperature stress (HPi + Phi_40C vs. HPi_25C) led to a comparable response, with 6,628 upregulated and 7,074 downregulated genes (13,702 total DEGs). In contrast, Phi application under non-stress conditions (HPi + Phi_25C vs. HPi_25C) resulted in fewer DEGs; 1,450 upregulated and 2218 downregulated genes. Pi deficiency induced stronger transcriptional changes. The LPi_40C vs. HPi_25C comparison revealed 5,981 upregulated and 7,036 downregulated genes (13,017 total). When Phi was applied under LPi and temperature stress (LPi + Phi_40C vs. LPi_25C), DEGs increased further to 17,225 (7,850 upregulated and 9,375 downregulated), highlighting a robust transcriptomic shift potentially associated with Phi-induced toxicity or signaling in the absence of Pi. In response to high temperature (HPi_40C vs. HPi_25C) (FIG. 13A), enriched GO terms were largely related to protein folding, chaperone activity, ROS, and response to heat. In Phi-treated plants under temperature stress (HPi + Phi_40C vs. HPi_25C) (FIG. 13B), similar stress-associated categories remained enriched, but additional emphasis was observed in translation, ribosomal assembly, and cytoplasmic protein localization.

[0237] In contrast, Phi application under optimal temperature (HPi + Phi_25C vs. HPi_25C) (FIG. 13C) resulted in enrichment of distinct GO categories, including maltose biosynthesis, chloroplast rRNA processing, and starch catabolism, as well as innate immune response and ammonia assimilation. Together, these GO enrichments suggest that while high temperature induces a strong proteostasis response in Wt plants, Phi enhances this response under temperature stress and promotes broader metabolic activity under optimal temperatures.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0238] In the case of ptxD soybean plants, temperature stress (HPi_40C vs. HPi_25C) triggered a transcriptional response with 6,286 upregulated and 6,667 downregulated genes (12,953 total DEGs). When plants were treated with Phi and subjected to temperature stress (HPi + Phi_40C vs. HPi_25C), the number of DEGs slightly increased to 16,627, with 8,531 genes upregulated and 8,096 downregulated. Phi supplementation under optimal temperature (HPi + Phi_25C vs. HPi_25C) had a moderate effect, with 6,200 upregulated and 5,449 downregulated genes. Under phosphate-deficient conditions, ptxD plants showed strong responses. The LPi_25C vs. HPi_25C contrast revealed 8,053 upregulated and 9,869 downregulated genes, consistent with the known activation of Pi starvation pathways. The magnitude of the response was similar under heat stress (LPi_40C vs. HPi_25C), with 8,057 upregulated and 9,869 downregulated DEGs. In the LPi + Phi_25C vs. LPi_25C comparison, ptxD plants showed a mild transcriptional response with 2,293 upregulated and 3,380 downregulated DEGs. Enriched GO terms in plants subjected to high temperature stress were related to heat response, protein folding, response to hydrogen peroxide, and chaperone cofactor activity. Terms related to salt and osmotic stress, photosynthetic light harvesting, and circadian control were also enriched. When Phi was the only source of P under heat stress (LPi + Phi_40C vs. HPi_25C), the enriched GO terms were response to heat, protein folding and refolding, response to fungus and bacteria, response to ABA, and processes linked to ERAD (Endoplasmic-reticulum-associated protein degradation) and autophagy. When the temperature was optimal and Phi was the only source of P (LPi + Phi_25C vs. HPi_25C), the GO terms enriched were defense responses to fungi, bacteria, and wounding, as well as jasmonic acid signaling, fatty acid -oxidation, and ubiquitin-mediated regulation.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0239] To further dissect the transcriptional overlap between treatments, Applicant compared DEGs from temperature stress and Phi-treated Wt plants using Venn diagrams (FIG. 13D). The top Venn diagram shows that 566 DEGs (6.6%) were shared among the three comparisons: heat stress (HPi_40C vs. HPi_25C), Phi application under optimal temperature (HPi + Phi_25C vs. HPi_25C), and the combined Phi and temperature stress condition (HPi + Phi_40C vs. HPi_25C). Many DEGs were unique to each condition, particularly 1,195 genes (13.9%) in the high temperature-only treatment, 648 genes (7.6%) in the Phi treatment, and 1,598 genes in the combined treatment. This suggests distinct transcriptional responses to individual and combined stresses.

[0240] Functional enrichment analysis of the shared upregulated DEGs between the treatments revealed enrichment of Gene Ontology (GO) terms related to ROS coping mechanisms, photosynthesis-related categories, including photosynthesis light harvesting, chloroplast organization, and photosystem 11 assembly (FIG. 13E). Additionally, responses associated with redox regulation, protein folding, and water transport were also overrepresented, suggesting a broad activation of abiotic stress and recovery pathways. The overlap between low phosphate (LPi_25C vs. HPi_25C) and high temperature (HPi_40C vs. HPi_25C) conditions revealed 532 genes (6.7%) overlapped between these two comparisons, suggesting some similarity in the transcriptional reprogramming induced by high temperature and Pi deficiency, but not as high as observed under water limitation. GO enrichment analysis showed terms related to response to water deprivation, response to oxidative stress, response to heat, and abscisic acid signaling.

[0241] The expression pattern of DEGs in LPi and high temperature was investigated in the other conditions. Applicant found that Phi treatment under Pi starvation attenuates the PSR response. But interestingly, the temperature stress appears to enhance the expression of multiple genes responsive to Pi starvation even with Phi application (LPi + Phi_40C). However, very few genes activated in LPi_25C, appear to be activated in HPi_40C. Similarly, a strong transcriptional response of these genes was found in response to Phi treatment under both HPi and LPi, under temperature stress.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0242] Similar analyses were performed for the ptxD plants (FIGS. 13F-G). The three-way Venn diagram (FIG. 13F) showed that a core set of 2765 genes (18.6%) was commonly regulated across temperature stress (HPi_40C), Phi treatment (LPi + Phi_25C), and their combination (LPi + Phi_40C). Applicant found 2147 genes (14.5%) were exclusively shared between temperature stress and the combined treatment, while 1990 genes (13.4%) were shared between the combined treatment and LPi + Phi_25C; this latter alone showed the highest number of unique DEGs (5260, 35.5%).

[0243] GO enrichment analysis of the shared upregulated DEGs indicated enrichment of GO terms overrepresented by processes such as response to heat, response to hypoxia, protein phosphorylation, and defense responses to fungi and bacteria. Key elements of post-translational regulation, including ubiquitination, protein targeting to vacuoles, and SCF-dependent proteasomal degradation, are also enriched (FIG. 13G).

[0244] When Applicant compared DEGs of ptxD plants under temperature stress (HPi_40C vs. HPi_25C) and exclusive Phi fertilization (LPi + Phi_25C vs. HPi_25C) at optimal temperature, 2580 genes (20.6%) were commonly regulated. The number of unique DEGs in each treatment was 6263 genes (49.9%) specific to Phi fertilization, and 3706 genes (29.5%) specific to high temperature. Enriched GO terms from the shared DEGs between both conditions were related to response to water deprivation, response to oxidative stress, response to heat, and abscisic acid signaling, in addition to GO terms associated with ROS coping mechanisms. Hierarchical clustering heatmaps of DEGs in the Phi-only treatment (LPi + Phi_25C) revealed that several LPi- related genes were also activated despite the sufficiency of P. The expression of a set of genes responsive to LPi at 25°C was also enhanced in response to high temperature. Interestingly, a cluster of upregulated genes under Phi fertilization at optimal temperature was downregulated in response to high temperature stress. This gene cluster was also downregulated in other treatments, including HPi_40C and HPi + Phi_40C, suggesting they are responsive to the temperature stress.

[0245] Example 2 ■ I L Enriched metabolites under high temperature stressPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0246] Metabolite profiling of Wt soybean plants under high temperature revealed 19 upregulated metabolites in response to the stress (FIG. 17D). The most significantly enriched metabolite classes were pteridines and derivatives, hydroxy acids and derivatives, steroids and steroid derivatives, organonitrogen compounds, tetrapyrroles and derivatives, and keto acids and derivatives.

[0247] In contrast, Wt soybean plants treated with Phi under temperature stress (HPi + Phi_40C vs. HPi_25C) led to a decreased number of metabolites with only 16 molecules upregulated. Significantly enriched classes were carboxylic acids and derivatives, phenols, pteridines and derivatives, cinnamic acids and derivatives (FIG. 17E).

[0248] When Wt soybean plants were grown at the optimal temperature of 25°C and treated with Phi, they showed 19 upregulated metabolites. The chemical classes with most hits were carboxylic acids and derivatives with 9 hits, fatty Acyls with 4 hits. Other enriched classes were imidazopyrimidines, cinnamic acids and derivatives, diazines, isoflavonoids, and azoles (FIG.17F).

[0249] In general, ptxD plants accumulated metabolites belonging to more diverse chemical classes than the Wt in these experiments. In response to high temperature stress, ptxD plants showed 80 up regulated metabolites, with organooxygen compounds, and carboxylic acids and derivatives, and keto acids and derivatives among the most enriched. Other significantly enriched classes were keto acids and derivatives, hydroxy acids and derivatives, lactones, phenols, organic carbonic acids and derivatives, pteridines and derivatives, purine nucleosides, cinnamic acids and derivatives, naphthalenes, diazines, pyridines and derivatives, azoles, and fatty acyls.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0250] The ptxD plants treated with Phi and subjected to 40°C (LPi + Phi_40C vs. HPi_25C) had 204 upregulated metabolites. Isoflavonoids, flavonoids, and organooxygen compounds were the three most enriched categories with 16, 22, and 24 hits, respectively. Carboxylic acids and derivatives and keto acids and derivatives were also highly enriched with 25 and 12 hits each. Other enriched compounds included keto acids and derivatives, cinnamic acids and derivatives, Hydroxy acids and derivatives, prenol lipids, fatty acyls, pteridines and derivatives, tetrapyrroles and derivatives, benzene and substituted derivatives, linear 1,3-diarylpropanoids, peptidomimetics, flavin nucleotides, imidazole ribonucleosides and ribonucleotides, imidazopyrimidines, furofurans, coumarins and derivatives, benzoxazines, pyrans, pyridines and derivatives, and phenols.

[0251] At 25 °C, Phi treatment alone caused a clear change in the metabolism of ptxD soybean plants compared to control (LPi + Phi_25C vs. HPi_25C). The most enriched chemical categories were isoflavonoids and flavonoids. Other 23 groups were significantly enriched, from which Applicant can highlight: keto acids and derivatives, hydroxy acids and derivatives, fatty acyls, phenols, urine nucleotides, pyrimidine nucleotides y purine nucleosides, cinnamic acids and derivatives.

[0252] Example 2,12, Transcriptional responses of soybean to Pi starvation, water limitation, and high temperature are coordinated by a common set of TFs that regulate photosynthesisPCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0253] To better understand the regulatory landscape underlying the transcriptional responses of soybean to Pi starvation, high temperature, and water limitation in the context of Phi application, Applicant analyzed differentially expressed transcription factors (TFs) that might be orchestrating these responses (FIGS. 14A-D). The Venn diagrams illustrate the overlap of downregulated (FIG. 14A) and upregulated (FIG. 14B) TFs between the following conditions: LPi_WC80, HPi_WC30, and HPi + Phi_WC80 in the Wt. A significant number of TFs were commonly regulated by all treatments, with 103 and 125 TFs downregulated and upregulated, respectively, indicating a core set of regulatory genes involved in both Pi starvation, low irrigation, and Phi treatment responses. Most downregulated TFs belong to four families, C2H2, bHLH, NF-YA, and G2-like, all of which play critical roles in regulating plant development, hormonal signaling, and responses to abiotic stress. Additionally, WRKY, ERF, NAC GRAS, SBP, MYB, and ARF TF families appear prominently among the downregulated genes, indicating a likely shift in transcriptional priorities away from growth and toward survival (FIG. 14E).

[0254] In the case of upregulated TFs, the most represented families include bHLH, ERFs, and MYB, all known for their roles in mediating abiotic stress responses. Families like ERF, GRAS, and NF-YA are also strongly represented, as are heat shock-related TFs such as HSF, as well as others like ZAT and C2H2 (FIG. 14F). A GO enrichment analysis suggests enriched terms of downregulated TFs relate to auxin-activated signaling pathways, ethylene, gibberellic acid signaling, and salicylic acid responses, and regulation of secondary metabolism (FIG. 14C). In the upregulated TFs, the enriched GO terms included regulators of cell wall and ROS coping mechanisms (FIG. 14D), suggesting a shared transcriptional program modulating both stress resilience and growth-related processes.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0255] Similar analyses were conducted for the downregulated and upregulated TFs for the high temperature experiments (LPi_25C, HPi_40C, and HPi + Phi_25C). In the downregulated DEGs, Applicant observed that about 60% of the TFs are uniquely downregulated by high-temperature stress (415 TFs), while 15.7% (115 TFs) are shared between high-temperature and Phi-treatment conditions, with only 9 (1.2%) of these TFs being shared with LPi (FIG. 15A). A similar trend is observed in the upregulated TFs, where 48% (269 TFs) are specifically regulated in response to high temperature, 6.1% (34 TFs) are shared between Phi treatment and high temperature stress, and only 9 TFs (1.6%) are shared with Pi starvation (FIG. 15B). Downregulated TFs belong to three TF families, bHLH, WRKY, and HD-ZIP, each comprising over ten genes. Families such as ERF, MYB, and LBD arc also substantially represented, suggesting the repression of genes typically involved in stress adaptation, cell differentiation, and hormonal crosstalk (FIG. 15E).

[0256] For the upregulated genes, Applicant found that SBP stands out with the highest number of TFs induced; TFs belonging to ZF-HD, NAC, HD-ZIP, ERF, Dof, bZIP, and bHLH families were also consistently upregulated, indicating a broad activation of stress-adaptive and hormone- regulatory networks. The presence of families like TCP, MYB, HSF, and MYB related underscores a coordinated response involving transcriptional regulators of heat shock, cell cycle, and signaling pathways (FIG. 15F). Because the low number of shared TFs with Pi starvation prevents GO enrichment analysis, this type of analysis was performed only for TFs shared between high temperature and Phi treatments (FIGS. 15C-D), which highlights distinct regulatory themes. The analysis revealed GO terms associated with heat acclimatization, gibberellin responses, nutrient transport, defense responses, and phosphate starvation responses (PSRs) (FIG. 15C), whereas for the upregulated TFs enriched GO categories related to auxin responses and regulation, response to heat and salt, abscisic acid (ABA) and gibberellic acid (GA), as well as the regulation of secondary shoots (FIG. 15D).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0257] Applicant conducted a more structured analysis of the transcriptional regulatory networks (TRNs) underlying soybean responses to water limitation and high temperature using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) algorithm. Due to their high degree of modularity, TRNs can help identify the central nodes of regulation, and determine whether TFs and TF-controlled genes with similar transcriptional profiles belong to the same gene community (grouped in the network) and conserve the same transcriptional interactions across treatments. The robust rank aggregation (RRA) algorithm was then applied to integrate the most influential nodes based on five network centrality measures (degree, closeness, betweenness, eigenvector, and PageRank). These analyses revealed the top-ranked TFs in the low irrigation experiments were AFO, COL4, and MYC64, each associated with more than 350 intcractors. Additional TFs such as RAP2.1, SHN3, and NF-YA8 also ranked high, suggesting a potential role in coordinating drought-responsive networks (FIGS. 15G-J). In contrast, when plants were exposed to high temperature stress, the transcriptional network shifted, with RVE1, RAP2.4, and RAP2.12 emerging as the most influential TFs. Notably, PHR1 appeared as a common regulator in both conditions, underscoring its central role in integrating responses across water deficit and heat stress environments (FIGS. 15G-J).

[0258] GO terms enrichment of the predicted targets of the top 10 ranked TFs for each stress condition, further distinguished the transcriptional strategies between drought and heat responses in soybean. Under low irrigation, enriched categories primarily involved photosynthesis, galactolipid biosynthesis, and chloroplast organization (FIGS. 15G-J). These processes reflect the prioritization of maintaining photosynthetic efficiency and membrane integrity during water deficit. Conversely, under high heat stress, interactors of key TFs were enriched in circadian rhythm regulation, response to heat, protein folding, and chloroplast organization (FIGS. 15G-J). This indicates a stronger emphasis on protein protection, and temporal regulation under elevated temperatures. Together, these results suggest that plants adopt distinct transcriptional strategies for drought and heat stress, both of which can be activated by Phi treatments.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0259] Example 2.13. The expression of stress-tolerance response genes is potentiated in ptxD plants fertilized only with Phi

[0260] The overall evaluations of plant performance suggest that when Phi was used in low concentration or in combination with Pi, the beneficial effects elicited by Phi are diminished in ptxD plants. However, benefits to plant growth, canopy development, and biochemical responses are fully recovered by ptxD plants when Phi is used as the sole source of P. Applicant decided to investigate and compare the transcriptional responses of ptxD plants, when using exclusively Phi as the P source (LPi + Phi), and Wt plants, when grown under optimal Pi and treated with Phi (HPi -t-Phi), subjected to high temperature and water limitation. This analysis will provide information about the transcriptional responses specifically elicited by Phi in both plant genotypes in the context of the stress condition.

[0261] In the water limitation experiments, Applicant’s Venn analysis compares DEGs from Wt plants treated with Phi under optimal Pi fertilization and watering level (HPi + Phi_WC80 vs. HPi_WC80) and ptxD plants under Phi and Pi fertilization and optimal watering (LPi + Phi_WC80 v.y. HPi_WC80). The analysis revealed that Wt and ptxD share a substantial proportion of DEGs (1246 genes, 21.8%). However, the majority of DEGs are unique to each genotype, with Wt showing 59.8% (3,411 genes) and ptxD 18.4% (1,048 genes) (FIG. 16A). This indicates that although the genotypes share a core drought-responsive transcriptional program modulated by Phi, there are genotype- specific responses likely due to the ability of ptxD plants to metabolize Phi effectively (FIG. 16A).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0262] A similar trend was found in the case of the high-temperature experiments. Fewer genes are shared between the two genotypes (1,202 genes, 10.6%), while ptxD plants exhibit a more pronounced transcriptional response (9,159 genes, 80.5%) compared to Wt (1,016 genes, 8.9%) (FIG. 16B). To further investigate the potential role of gene sets commonly regulated, Applicant performed GO enrichment analysis (FIGS. 16C-D). In the case of the shared genes in the water limitation experiments between Wt and ptxD plants treated with Phi, enriched GO terms are associated mainly with photosynthesis, chlorophyll biosynthesis, response to heat and cold stress, ROS-related responses, and tetrapyrrole metabolism. GO terms related to defense responses, like terpenes and innate immune responses, are also activated in both genotypes (FIG. 16C). In the case of the high temperature experiments, enriched GO terms arc primarily related to abiotic stress responses, such as cellular responses to hypoxia, oxidative stress, salt stress, osmotic stress, and heat response, hormones like ABA, and nutrients like response to phosphate starvation. Additionally, GO terms such as response to wounding, defense response to fungus, and defense response to bacterium indicate common stress-mitigating pathways (FIG. 16D), reinforcing the notion that Wt and ptxD plants share core signaling and metabolic adjustments under high temperature stress conditions.

[0263] To determine whether these genes have similar expression in Wt and ptxD plants under water limitation and high temperature stress, Applicant analyzed the normalized expression of upregulated genes shared by both genotypes (Wt plants in HPi + Phi_WC80, and ptxD plants in LPi + Phi_WC80). Interestingly, Applicant found that under both high temperature stress and water limitation, the expression of these upregulated genes is significantly higher in ptxD plants than in Wt plants (FIGS. 16E-F). These findings indicate that the ptxD soybean, when treated with Phi as its sole P source, shows increased transcriptional activation of Phi-responsive genes. This suggests that the Phi-ptxD system, which combines Phi fertilization with / x -cxprcssing plants, offers even greater benefits than using well-Pi fertilized Wt plants treated with Phi. The ability of ptxD plants to tolerate higher Phi concentrations without negative effects further enhances these beneficial responses, particularly in low Pi environments.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0264] Example 2.15, Discussion[002651 In this Example, Applicant demonstrated that the Phi- / x system provides plant protection against two of the most aggressive abiotic stresses, high temperature and water limitation (FIGS. 9A-D and 13A-G). Applicant showed that when fertilized with Phi only, ptxD plants exhibit a chemical-primed state, activating more strongly plant responses related to multiple stresses at the transcriptional level. This, in addition to providing P for the plant, enhances the tolerance and performance of ptxD plants under conditions of low watering and high temperature. The Phi-ptxD system is a promising tool to improve crop production under challenging climatic conditions.

[0266] The Phi-ptxD system provides unprecedented benefits to soybean cultivation. Low water availability and high temperature stresses severely affect plant growth, physiology, and gene expression in soybean. Applicant observed growth inhibition under water deficit and elevated temperature, characterized by decreased plant height, canopy area, and chlorophyll content, along with increased activities of antioxidant enzymes such as GPX. These findings reflect well-known plant responses to drought and heat stresses, which result in reduced photosynthesis and biomass, protein denaturation, and oxidative damage. Both stresses cause oxidative stress, which soybean plants combat by increasing ROS-scavenging enzymes.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0267] When plants were treated with Phi, plants showed higher growth under stressed conditions, which can be interpreted as an enhanced stress tolerance mechanism. In this Example, Phi supplementation under low irrigation and high temperature notably improved Wt plant performance, resulting in greater height and larger canopies in some cases, as well as higher chlorophyll content compared to controls under stressed conditions without Phi (FIGS. 9A-9D), suggesting improved carbon and nitrogen metabolism. These results suggest that Phi helps alleviate drought and high-temperature stresses by maintaining photosynthetic capacity and enhancing the stability of the photosynthetic apparatus. This might be influenced by a higher content of antioxidant enzymes such as SOD and GPX, which mitigate oxidative damage. Furthermore, this improved plant performance may also be attributed to enhanced nutrient use efficiency and root growth, which can help plants tolerate stress by securing water and nutrients, thereby sustaining growth.

[0268] In Applicant’s experiments, Phi-treated plants indeed retained higher chlorophyll under stress, indicating less photosynthetic decline, and showed higher SOD and GPX activity, consistent with those mechanisms. Additionally, Phi triggers phosphate starvation signaling, which primes stress responses, as previously reported. These PSR genes have been reported to have the ability to activate plant responses to drought through ABA and TFs like PHR1 (Phosphate Starvation Responses 1), which is a master regulator of the PSR. Overall, Applicant’s results suggest that the Phi stress-mitigating effect in soybean likely involves improved resource uptake, maintenance of photosynthesis and the photosynthetic apparatus, and activation of antioxidant defenses.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0269] In both types of stress experiments, the Phi-ptxD system provides unprecedented benefits to soybean cultivation, which might contribute to making the soybean industry more sustainable. ptxD plants fertilized exclusively with Phi showed outstanding performance mainly under stress conditions, which is reflected in plant height, canopy development, and other physicochemical parameters (FIGS. 9A-D and 10A-E). The potential and benefits of Phi application were enhanced, particularly under stress conditions, where ptxD plants coped with the stresses better than non-treated plants. On the contrary, Phi application to Wt plants grown under Pi starvation and subjected to stress conditions (high temperature or water limitation) led to the death of most plants. Additionally, Applicant found that the Phi supplementation to plants grown with optimal Pi fertilization and no stress provides additional benefits, as it acts as a biostimulant molecule.

[0270] Low Pi, high temperature and water limitation stresses have shared transcriptional responses with Phi application. Soybean plants experience significant physiological changes and alteration of molecular responses when subjected to mixed Pi starvation and water limitation or high temperature stresses, which under field conditions are commonly present at the same time. Physiologically, mixed stresses significantly impede photosynthesis, resulting in leaf chlorosis, decreased stomatai conductance, expedited senescence, and considerable reductions in biomass and production, illustrating how integrated stresses amplify the impacts of individual stresses (FIGS. 9A-9D and 13A-13G). At the transcriptional level, these combined stresses activate some overlapping pathways, including PSRs, oxidative stress control, and hormone signaling. Applicant also found that key TFs like PHR1 and HD-Zip family members like HB13, ERF, WRKY, and NF-YA7 are upregulated, which coordinates stress-responsive genes for osmoprotection, ROS coping, and metabolic changes related to nutrient recycling and energy production. At the same time, production of antioxidant enzymes like SOD, CAT, and POD, osmoprotectant molecules such as proline, sugars, and polyols, and defense hormones like ABA, ethylene, and JA, is enhanced to cope with ROS stress, maintain cellular integrity, and osmotic balance (FIGS. 9A-D and 10A-E).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0271] The Phi-ptxD system greatly helps plants cope with the negative effects of mixed abiotic stresses. ptxD plants fertilized exclusively with Phi maintain their internal phosphorus levels high to sustain growth, but interestingly, activate some PSRs. These response genes help detoxify ROS, respond to heat shock, signal hormones, while maintaining active photosynthesis. Because of this, ptxD plants supplemented with Phi as their only P source are better prepared to handle stress than Wt plants under the same conditions.

[0272] Phi activates molecular programs that help plants cope with high temperature and low- water irrigation. Under low watering conditions, soybean plants displayed significant transcriptional changes reflecting a classic drought response, including modulation of cell wall organization, glutathione metabolic process, and response to auxin. These changes are indicative of a molecular strategy of oxidative stress and a reorganized cell wall that may be compromised during limited water availability (FIG. 10A). When Phi is applied, the plant responses to water limitation change (FIGS. 10C-D), enriching the expression of genes related to photosynthesis, photosystem stabilization, and chlorophyll biosynthesis. Phi-treated plants were also enriched in processes such as cell redox homeostasis, response to hydrogen peroxide, and removal of superoxide radicals, which could indicate that Phi application allows plants to better cope with low irrigation-induced oxidative stress and maintain active photosynthesis. These beneficial effects of Phi were also present under optimal irrigation, where Phi treatment enriched terms mainly related to photosynthetic processes (FIG. 10C). A higher photosynthetic rate could explain the enhanced growth under Phi treatments without stress.

[0273] Under high-temperature stress, soybean plants induced strong molecular mechanisms related to protein folding and heat acclimatization responses. High temperature increased the expression of genes involved in chaperone activity, protein refolding, and endoplasmic reticulum- associated degradation (ERAD) for recycling, alongside strong induction of responses to ROS such as hydrogen peroxide (FIG. 13A). Phi treatment under temperature stress conditions also elicited these protective molecular responses, evidenced by enhanced expression of protein stabilization genes, and antioxidative responses (FIG. 13B).PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0274] In non-stressed plants grown at 25°C, Phi application induced the upregulation of genes related to chloroplast organization, water transport, and carbohydrate metabolism, as well as the synthesis of unsaturated fatty acids, which could help plants resist water loss, suggesting an underlying mechanism of metabolic priming and stress preactivation in anticipation of future stresses (FIG. 13C). These molecular responses show that Phi treatment confers a dual advantage to plants; it helps maintain photosynthetic processes under water limitation and high temperature, while enhancing protein stability and antioxidant protection mechanisms. These changes in key metabolic and stress-responsive pathways are part of the chemical priming elicited by Phi, which helps plants withstand abiotic stresses while enhancing growth.

[0275] Transcription factors that help plants cope with high temperature and low irrigation are activated. Many stress-responsive TFs are involved in orchestrating plant responses to drought and high temperature. Applicant’s transcriptomic data show that Phi, LPi, and low irrigation treatments activate a shared set of TFs that have been previously reported to enhance plant tolerance against drought and nutrient stresses (FIGS. 14A-F and 15A-F). These TFs might be orchestrating the multiple transcriptional responses observed in response to the stress conditions (FIGS. 10A-E and 14A-F). Plants under these stresses induced TFs linked to abiotic stress responses, notably AtHB13, NF-YA7, and ERF74. Overexpression of AtHB13 in plants has been shown to increase their tolerance to drought and salinity. This tolerance is linked to the induction of proteins that stabilize cell membranes. Moreover, AtHB13 is known to regulate the expression of JUNGBRUNNEN1 (JUB1), a NAC transcription factor, which also contributes to drought tolerance. Likewise, NF-YAs TFs has been recognized for positively regulating drought resistance by modulating stress-responsive genes involved in maintaining cellular homeostasis. Meanwhile, the overexpression of ERF74 enhances tolerance to drought and other abiotic stresses.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0276] Additionally, several stress-responsive TFs involved in nutrient stress signaling were notably induced, including PHR1, WRKY40, and WRKY42. PHR1, the central regulator of PSRs, was markedly upregulated in response to LPi, low watering, and Phi treatments. This TF coordinates phosphate uptake and the systemic PSR responses, and activates some drought responses. Studies have shown that overexpression of some WRKY TFs in plants can improve their drought tolerance by regulating stress-related genes and reducing reactive oxygen species (ROS) levels. Applicant’s results suggest that Phi, especially under low irrigation, primes soybean plants by activating TFs involved in ABA-mediated drought responses, enhanced cuticle formation, and nutrient homeostasis mechanisms, collectively contributing to improved adaptation to these combined stresses.

[0277] When Applicant analyzed transcriptomic data from high temperature and Phi treatments, Applicant found 43 shared TFs with significant upregulation known to modulate heat tolerance mechanisms in plants (FIGS. 15A-F). Prominent among these TFs were members of the Heat Shock Factor (HSF) family, particularly HSFA6B and HSFB1, known to regulate heat shock proteins (HSPs) and promote cellular protection against protein aggregation and damage under elevated temperatures. These TFs function as critical components of the heat stress transcriptional network, activating genes that stabilize proteins and membranes, thus maintaining cellular homeostasis. Other important TFs identified were DREB2C (CBF2), belonging to the AP2 / ERF family, and MYB94 and MYB70, belonging to the MYB-type family (FIGS. 15A-F). DREB2C is recognized for mediating stress-responsive pathways through binding to dehydration-responsive elements (DRE) and conferring improved thermotolerance by coordinating crosstalk between heat and drought stress signaling. The overexpression of MYB94 induced increased cuticular wax loads and reduced water loss through the cuticle in Arabidopsis. MYB70 works by interacting with both ABA and auxin signaling pathways, therefore, regulating water loss and root development under this stress. Collectively, these TFs orchestrate complex regulatory networks to ensure robust heat-stress responses, involving chaperone synthesis, redox balance, and modulation of hormonal pathways, ultimately enhancing plant survival under thermal stress conditions.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0278] The ptxD plants are able to activate the similar regulatory programs as in Wt when supplemented with Phi as the main P source. The shared transcriptional responses observed between Wt and transgenic ptxD soybean plants when treated with Phi suggest that the ptxD plants effectively activate stress-responsive pathways when Phi is their sole P source. Experiments with both Wt and ptxD lines, treated with HPi + Phi and LPi + Phi, respectively, showed upregulation of genes associated with multiple processes. For instance, processes such as photosynthesis, chlorophyll biosynthesis, electron transport, and abiotic stress tolerance, along with responses to oxidative, osmotic, and salt stress, as well as cellular homeostasis pathways like autophagy (FIGS. 16A-F), were activated. These data indicate that ptxD plants can activate stress-coping genes related to ROS detoxification, enabling effective management of oxidative stress, in addition to metabolizing Phi. Overall, these transcriptional responses demonstrate that transgenic ptxD soybeans can respond to Phi, benefiting from it, and showing the agronomic potential of Phi-based fertilization paired with ptxD plant strategies under challenging environmental conditions.

[0279] Previous studies have shown transgenic ptxD crops can maintain yield with less Pi input, yet achieve similar productivity to Pi-fertilized controls. Moreover, when grown in competition with weeds (which cannot utilize Phi), ptxD crops gain a significant advantage, accumulating 2- 10 times more biomass than when grown with regular Pi. These findings align with Applicant’s observations of ptxD soybeans outperforming Wt under Phi fertilization. Phi itself is considered environmentally benign; it is already used as a fungicide on crops and oxidizes into Pi in a couple of months. Using Phi in agriculture could lower the risk of eutrophication since less Pi runoff occurs. ptxD plants do not accumulate any novel toxic compounds; they simply convert Phi to Pi internally. Thus, the harvested crop should be as safe as conventional crops. Overall, ptxD transgenic crops perform well with Phi as a nutrient, are agronomically competitive, and present a promising strategy to reduce fertilizer use and herbicide reliance while helping plants cope with multiple stresses, with minimal safety downsides.PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128

[0280] While catalyzing the conversion of Phi into Pi, the enzyme PTXD uses NAD+as a cofactor and produces NADH. The PTXD mutant (E175A / A176R) utilizes both NAD+and NADP+as cofactors, thereby producing both NADH and NADPH. Both NADH and NADPH, are ubiquitous molecules involved in energy metabolism and redox signaling. NADH directly participates in the mitochondrial electron transport chain, facilitating ATP production essential for stress responses, while also influencing redox homeostasis by serving as a cofactor for antioxidant enzymes and metabolic pathways that eliminate reactive oxygen species (ROS). A high NADH / NAD+ratio might provide plants protection against stresses like drought and heat, by improving the plant's ability to synthesize reduced GSH and ascorbate, which helps the ascorbate-glutathione cycle and protects from oxidative damage. NADH has also been linked to stress adaptation by functioning as a signaling molecule that induces changes in gene expression. NADPH serves as the source of reducing equivalents for the two major antioxidant systems in plant cell s, the ascorbate-glutathione cycle and the thioredoxin-dependent networks. This could initiate transcriptional programs that activate ROS coping mechanisms, the production of osmoprotectants, and also heat-shock proteins. Without being bound by theory, Applicant hypothesizes that the Phi metabolism in ptxD plants could provide a benefit by providing phosphorus and increasing NADH / NADPH-mediated redox signaling pathways essential for tolerance to concurrent heat and drought stress.

[0281] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-128WHAT IS CLAIMED IS:

1. A method of treating a plant or seed, said method comprising: applying a composition to the plant or seed, wherein the composition comprises a fertilizer composition comprising a phosphite (Phi), and wherein the plant or seed comprises a transgenic crop expressing a phosphite oxidorcductasc-cncoding gene.

2. The method of claim 1, wherein the applying comprises applying the composition onto a field, soil or substrate that comprises the plant or seed.

3. The method of claim 1, wherein the applying comprises applying the composition onto a plant as a foliar application, or onto a seed as a seed coat or soaking treatment.

4. The method of claim 1, wherein the transgenic crop is selected from the group consisting of maize, a legume, soybean, tobacco, cotton, tomato, sorghum, potato, pepper, rice, wheat, lettuce, common beans, grapes, onion, strawberry, banana, spinach, mustard, varieties thereof, or combinations thereof.

5. The method of claim 1, wherein the transgenic crop comprises a legume.

6. The method of claim 1, wherein the transgenic crop comprises soybean.1144823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-1287. The method of claim 1, wherein the phosphite is selected from the group consisting of H2PO3 , [HPO3]"2, salts thereof, or combinations thereof.

8. The method of claim 1, wherein the phosphite is selected from the group consisting of KH2PO3, NH4H2PO3, Na2HPC>3, CaHPO ,, or combinations thereof.

9. The method of claim 1, wherein the composition comprises a phosphite concentration of at least 100 pM.

10. The method of claim 1, wherein the composition comprises a phosphite concentration ranging between 50 pM and 1 mM.

11. The method of claim 1, wherein the composition comprises a phosphite concentration of 10 to 150 parts per million when applied directly to soil.

12. The method of claim 1, wherein the composition comprises a mixture of phosphite and orthophosphate (Pi).

13. The method of claim 12, wherein the orthophosphate is selected from the group consisting of [PO4]'3, [HPO4]'2, [H2PO4]', H3PO4, salts thereof, or combinations thereof.1154823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-12814. The method of claim 12, wherein the orthophosphate is selected from the group consisting of KH2PO4, NH4H2PO4, Na2HPO4, CaHPO4, or combinations thereof.

15. The method of claim 1, wherein the composition comprises an orthophosphate concentration between 10 pM and 1 mM.

16. The method of claim 1, wherein the composition enhances the growth rate of the plant or seed relative to untreated plants or seeds.

17. The method of claim 1, wherein the composition enhances the plant’s or seed’s efficiency in utilizing nitrogen and / or phosphorus relative to untreated plants or seeds.

18. The method of claim 1, wherein the composition enhances the plant’s or seed’s resistance to one or more sources of environmental stress relative to untreated plants or seeds.

19. The method of claim 18, wherein one or more sources of environmental stress comprise biotic stress, wherein the biotic stress is selected from the group consisting of microbial infections, viral infections, fungal infections, bacterial infections, or combinations thereof.

20. The method of claim 18, wherein one or more sources of environmental stress comprise abiotic stress, wherein the abiotic stress is selected from the group consisting of drought, heat, or combinations thereof.1164823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-12821. The method of claim 18, wherein the one or more sources of environmental stress comprise drought.

22. The method of claim 18, wherein the one or more sources of environmental stress comprise heat.

23. The method of claim 1, wherein the composition reduces the growth of weeds along with the plant or seed relative to untreated plants or seeds.

24. The method of claim 1, wherein the phosphite oxidoreductase-encoding gene comprises a ptxD gene.

25. The method of claim 24, wherein the ptxD gene comprises SEQ ID NO: 1 or a sequence with at least 85% sequence identity to SEQ ID NO: 1.

26. The method of claim 24, wherein the ptxD gene comprises SEQ ID NO: 2 or a sequence with at least 85% sequence identity to SEQ ID NO: 2.

27. The method of claim 24, wherein the ptxD gene comprises SEQ ID NO: 3 or a sequence with at least 85% sequence identity to SEQ ID NO: 3.1174823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-12828. The method of claim 1, wherein the expressed phosphite oxidoreductase comprises one or more mutations that enable more efficient use of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as cofactors.

29. The method of claim 1, wherein the expressed phosphite oxidoreductase comprises SEQ ID NO: 4 or a sequence with at least 85% sequence identity to SEQ ID NO: 4.

30. The method of claim 1, wherein the expressed phosphite oxidoreductase comprises SEQ ID NO: 5 or a sequence with at least 85% sequence identity to SEQ ID NO: 5.

31. The method of claim 1, further comprising a step of introducing the phosphite oxidoreductase- encoding gene into the plant or seed.

32. A plant or seed, wherein the plant or seed comprises a transgenic crop expressing a phosphite oxidoreductase-encoding gene, and wherein the plant or seed has been treated with a composition, wherein the composition comprises a fertilizer composition comprising a phosphite (Phi).

33. The plant or seed of claim 32, wherein the transgenic crop is selected from the group consisting of maize, soybean, tobacco, cotton, tomato, sorghum, potato, pepper, rice, wheat, lettuce, common beans, grapes, onion, strawberry, banana, spinach, mustard, varieties thereof, or combinations thereof.1184823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-12834. The plant or seed of claim 32, wherein the transgenic crop comprises soybean.

35. The plant or seed of claim 32, wherein the phosphite is selected from the group consisting of H2PO3 , | HPO'.|’2- salts thereof, or combinations thereof.

36. The plant or seed of claim 32, wherein the phosphite is selected from the group consisting of KH2PO3, NH4H2PO3, Na2HPO3, CaHPCh, or combinations thereof.

37. The plant or seed of claim 32, wherein the composition comprises a mixture of Phi and orthophosphate (Pi).

38. The plant or seed of claim 37, wherein the orthophosphate is selected from the group consisting of [PO4]'3, [HPO4]'2, [H2PO4]’, H3PO4, salts thereof, or combinations thereof.

39. The plant or seed of claim 37, wherein the orthophosphate is selected from the group consisting of KH2PO4, NH4H2PO4, Na2HPO4, CaHPO4, or combinations thereof.

40. The plant or seed of claim 32, wherein the plant or seed demonstrates enhanced growth rate relative to untreated plants or seeds.

41. The plant or seed of claim 32, wherein the plant or seed demonstrates enhanced resistance to one or more sources of environmental stress relative to untreated plants or seeds.1194823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-12842. The plant or seed of claim 41, wherein one or more sources of environmental stress comprise biotic stress, wherein the biotic stress is selected from the group consisting of microbial infections, viral infections, fungal infections, bacterial infections, or combinations thereof.

43. The plant or seed of claim 41, wherein one or more sources of environmental stress comprise abiotic stress, wherein the abiotic stress is selected from the group consisting of drought, heat, or combinations thereof.

44. The plant or seed of claim 32, wherein the one or more sources of environmental stress comprise drought.

45. The plant or seed of claim 32, wherein the one or more sources of environmental stress comprise heat.

46. The plant or seed of claim 32, wherein the plant or seed demonstrates efficiency in utilizing nitrogen and / or phosphorous relative to untreated plants or seeds.

47. The plant or seed of claim 32, wherein the phosphite oxidoreductase-encoding gene comprises a ptxD gene.

48. The plant or seed of claim 47, wherein the ptxD gene comprises SEQ ID NO: 1 or a sequence with at least 85% sequence identity to SEQ ID NO: 1.1204823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P078WOTexas Tech No. 2024-12849. The plant or seed of claim 47, wherein the ptxD gene comprises SEQ ID NO: 2 or a sequence with at least 85% sequence identity to SEQ ID NO: 2.

50. The plant or seed of claim 47, wherein the ptxD gene comprises SEQ ID NO: 3 or a sequence with at least 85% sequence identity to SEQ ID NO: 3.

51. The plant or seed of claim 32, wherein the expressed phosphite oxidoreductase comprises one or more mutations that enable more efficient use of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as cofactors.

52. The plant or seed of claim 32, wherein the expressed phosphite oxidoreductase comprises SEQ ID NO: 4 or a sequence with at least 85% sequence identity to SEQ ID NO: 4.

53. The plant or seed of claim 32, wherein the expressed phosphite oxidoreductase comprises SEQ ID NO: 5 or a sequence with at least 85% sequence identity to SEQ ID NO: 5.1214823-4406-5785V.3 13368-42

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