Progress 08/01/23 to 01/31/25

Outputs
Target Audience:Engaged undergraduate students, graduate students, postdoctoral associates, and faculty at the University of Wisconsin-Madison and Virginia Tech in the College of Agriculture and Life Sciences (CALS) in our applied technologies from objectives 1-2 and the creation of our illustrated videos from objective 3. Also engaged stakeholders/farmers, faculty researchers, Wisconsin legislatives/policy makers, industry scientists, graduate students, and the general public by presenting the applied technologies and illustrated videos at multiple presentations and seminars in Madison, Wisconsin and St. Louis, Missouri. This work was also presented within the reporting timeframe at the Plant Biology meeting (Honolulu, HI; June 22-26, 2024), USDA NIFA PD meeting (Honolulu, HI; June 26-27, 2024), the ICAR meeting (San Diego, CA; July 15-17,2024), and the Inositol Phosphates 5th annual meeting (Virtual; January 8-10, 2025) to university, industry, and government scientists. Additionally,engaged stakeholders/farmers, faculty researchers, USDA scientists, and graduate students (over 50 participants) at the Ohio State University Agricultural Research and Development Center, Wooster, OH at the all-day field day event proposed in objective 3. The efforts were to enhance public awareness of the phosphorus crisis through a series of talks and demonstrations (large and small-scale). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project involves one post-doctoral researcher (PD), one research scientist, and twoundergraduate students. Training: The research scientist and undergraduate student 1 have assisted in the transformation and maintenance of pennycress and Arabidopsis transgenics. Before this opportunity, the undergraduate student 1 had not been involved in plant biochemistry research. Through their work in our laboratory, this student has become proficiently skilled in growing and screening laboratory plants, collecting and analyzing phenotypic data (imaging, measuring, etc), performing assays to quantify the amount of P present in a sample, and other aspects of plant biochemistry and genetics in multiple plant species. Similarly, the undergraduate student 2 did not have a previous involvement in plant biochemistry research and aided in development of Phyte4Food illustrated videos.The PD has also been engaged in planning, developing, and implementing Extension activities through BMP Field Day. Additional extension activities include the creation of Phyte4Food, an illustrated video and podcast series. The post- doctoral researcher has written, illustrated, and narrated 10videos and created a website to share these videos with stakeholders and general public audiences. This was a highly enriching and educational experience, allowing this professional to gain experiences in but not limited to: hosting events across universities, disseminating information from the university to the general public and farmers across counties through a network of newspapers, radio stations, extension offices, and companies, leading demonstrations and demonstration sessions, a poster session, a seminar series, and coordinating this with a variety of researchers, farmers, caterers, and other professionals. Additionally, this opportunity has provided the means to travel and share research and extension work with stakeholders/farmers, faculty researchers, Wisconsin legislatives/policy makers, industry scientists, graduate students, and the general public. This includes 9oral presentations/seminars and 7 poster presentations (August 2023- January 2025). In addition to disseminating information through presentations and seminars, this has also provided the means to publish the research conducted for this grant. The PD has published one first author, peer-reviewed publication (DOI: 10.1093/plphys/kiae582) to date. There are currently two additional publications in preparation that the PD willsubmit to publishers for peer review by the end of February. How have the results been disseminated to communities of interest?This work has been presented to a variety of audiences that are unfamiliar with the Phosphorus crisis. At the beginning of this project, the original focus had been to reach the general public audience and farmers. Given lawmakers, scientists (industry and faculty), and students are also unfamiliar with the phosphorus crisis, the scope has been expanded to include these audiences as well. Listed are the major presentations (oral and poster) that disseminate information regarding the phosphorus crisis through the research goals 1 & 2 as well as extension goals (3; Phyte4food podcast and field day) within the August 2023- January 2025 reporting period. · Plant Cellular and Molecular Biology Supergroup Seminar Series, September 2023, Madison, Wisconsin Audience: Faculty scientists, postdoctoral researchers, graduate students, and undergraduate students. Information was disseminated through an oral presentation and follow up discussion with undergraduate student botany class. · Bayer University Conference, October 2023, St Louis, Missouri Audience: Bayer scientists, graduate students, and postdoctoral researchers. Information was disseminated through an oral presentation and poster presentation. · Catalysts for Science Policy, April 2024, Madison, Wisconsin Audience: General public and college students (STEM and non-STEM majors). Information disseminated through an oral presentation and an illustration. · UW Day at the Capitol, April 2024, Madison, Wisconsin Audience: Wisconsin legislatives/policy makers, farmers, general public, faculty researchers, graduate students, and undergraduate students. Information was disseminated through a poster presentation. · Midwest Cellular Dynamics Meeting, June 2024, Madison, Wisconsin Audience: Faculty researchers, industry scientists, postdoctoral researchers, graduate students, and undergraduate students. Information was disseminated through a poster presentation. · ASPB Plant Biology Meeting & USDA NIFA PD Meeting, June 2024, Honolulu, Hawaii Audience: Faculty researchers, industry scientists, USDA scientists, postdoctoral researchers, graduate students, and undergraduate students. Information was disseminated through two poster presentations. · International Conference on Arabidopsis Research (ICAR) Meeting, July 2024, San Diego, California Audience: Faculty researchers, industry scientists, USDA scientists, postdoctoral researchers, graduate students, and undergraduate students. Information was disseminated through an oral presentation and a poster presentation. · Plant Cellular and Molecular Biology Supergroup Seminar Series, November 2024, Madison, Wisconsin Audience: Faculty scientists, postdoctoral researchers, graduate students, and undergraduate students. Information was disseminated through an oral presentation and follow up discussion with undergraduate student biochemistry class. · Inositol Phosphates 5th Annual Meeting, January 2025, Virtual Conference Audience: Faculty researchers, postdoctoral researchers, graduate students, and undergraduate students from a variety of countries (including Germany, UK, Switzerland, India, Taiwan). Information was disseminated through an oral presentation. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The Phosphorus crisis presents a major challenge to food security. While Phosphorus (P) is critical for crop growth, P is a nonrenewable and increasingly limited resource. Our global population is fed at the expense of the remaining mineable P reserve, which may be depleted in 30 years. Further, fertilizer runoff from farmland and urban areas poses a dangerous problem as increased nutrients in watersheds toxifies our water supply and aquatic ecosystems. Time is running out to preserve our P supply. New and innovative strategies that reduce fertilizer inputs and watershed pollution are key to securing the global food supply and protecting the environment. Emerging evidence shows plants use inositol pyrophosphates (PP- InsPs) as signaling molecules to detect P. My preliminary work demonstrates altering PP-InsPs can decrease plant P accumulation with a limited growth trade-off or cause plants to hyperaccumulate P. We propose to translate knowledge on this pathway into a strategy to reduce fertilizer inputs and reclaim P polluted soils. We will achieve this with three distinct aims. (1) First, we will artificially increase PP-InsPs to determine how this impacts crop requirements for fertilizer. (2) Additionally, we will engineer P-accumulating crops and convert them into a novel biochar product to assess crop P reclamation potential. (3) Further, we have developed an Extension strategy to spread awareness of the P crisis and agricultural sustainability on the local, regional, national, and global level. Ultimately, this integrated research and Extension project will enhance P usage to protect the global food supply and environment. This integrative project combines research and extension to remedy the impending P crisis. First, this research translates fundamental discoveries we have made with inositol pyrophosphate signaling in Arabidopsis thaliana in two crop species to enhance plant Phosphorus-use efficiency and reclaim Phosphorus from polluted environments. The extension portion of this project will allow me to engage a wide variety of stakeholders regionally and globally in topics surrounding P usage, climate change, and how fertilizer impacts the environment. To achieve these goals, we have the following specific objectives: Objective 1. Research: Enhance phosphate-use efficiency (PUE) in two crop species and assess whether we can develop crops with a reduced need for fertilizer. This objective is 93% complete. We have selected three plant species for this project: a laboratory model species, Arabidopsis, as well as two crop species, pennycress and soybean. The two crop species are the major focus of this project. We have constructed the appropriate gain-of-function genetic constructs and transformed these into Arabidopsis and Pennycress plants. We hypothesize will reduce the plant's need to acquire P from the soil. The Wisconsin Crop Innovation Center has constructed the proposed vectors and has introduced them into soybean Williams82. We have obtained homozygous plant lines and have assessed the growth of these lines. Based on our analyses, we conclude that there are alterations in plant growth and physiology in these lines depending on the construct type. We are in the process of analyzing the nutrient data from these lines and will be able to determine the link between PUE and plant physiology as whether specific germplasms have increased PUE compared to WT once we conclude the nutrient analysis. We have also characterized a few of these lines in Arabidopsis and have a manuscript in preparation containing this data (to be submitted for peer review by the end of February 2025). Objective 2: Research. Develop two crop species that accumulate high amounts of inorganic phosphate (Pi) from the environment This objective is 95% complete. Appropriate gain-of-function genetic constructs that encode genes that impact the plants' ability to hyperaccumulate P have been assembled and have been transformed into all three plant species. Similar to objective 1, the two crop species, pennycress and soybean, are the major focus of this project. We have obtained homozygous soybean lines and are assessing alterations in PP-InsPs and genes involved in P sensing. We have obtained soybean ipk1 mutants seeds from the University of Missouri. Unfortunately, we were unable to recover this germplasm due to the age of the seeds and have pivoted to studying our gain of function plants that support this objective. Our group has assessed biochar made from pennycress lines and have optimized the conditions for biochar production to decrease the rate of phosphate release from these biochars. We are in the process of observing the chemical forms of phosphorus in the pennycress biochar as well as analyzing the biochars created from the soybean lines. We currently have two manuscripts in preparation (one to be submitted by the end of February 2025). Objective 3. Extension. Implement a two-part Extension strategy to increase public awareness of the Phosphorus crisis This work is 100% complete. We completed the Best Management Practice (BMP) field day on March 29th, 2023 at the OSU OARDC in Wooster, OH. This Field Day included a series of presentations that highlight difference aspects of P- use efficiency and -use inefficiency, interactive surveys, and field site demonstrations. The seminar components of BMP Field Day are accessible on the BMP Field Day website: https://bmpfieldday.carrd.co/ We have created 10 videos for the video series, Phyte4Food, as well as the website: https://phyte4food.com/ to store these videos and other resources. These videos address what P is and why it's important, the P crisis, how P cycles through the environment, how P and nutrients impact plant growth, phytoremediation strategies we can use to protect the environment, what biotechnology is and how it has impacted society, and how biotechnology can be applied to resolving the P crisis. We have kept to the original plan to include ten illustrated videos in this series. A future aim for Phyte4Food is to create podcasts that will include interviews with a variety of professionals with expertise in the specific areas discussed in the videos. Online distribution streams will include the Phyte4Food website made and maintained by our lab group for these videos, the American Society of Plant Biologists official website (Plantae), YouTube, and various social media/podcast platforms.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Freed C, Craige B, Donahue J, Cridland C, Williams SP, Pereira C, Kim J, Blice H, Owen J Jr, Gillaspy G (2024) Using Native and Synthetic Genes to Disrupt Inositol Pyrophosphates and Phosphate Accumulation in Plants. Plant Physiology kiae582 https://academic.oup.com/plphys/article/197/1/kiae582/7849690

Progress 08/01/23 to 07/31/24

Outputs
Target Audience:Engaged undergraduate students, graduate students, postdoctoral associates, and faculty at the University of Wisconsin-Madison and Virginia Tech in the College of Agriculture and Life Sciences (CALS) in our applied technologies from objectives 1-2 and the creation of our illustrated videos from objective 3. Also engaged stakeholders/farmers, faculty researchers, Wisconsin legislatives/policy makers, industry scientists, graduate students, and the general public by presenting the applied technologies and illustrated videos at multiple presentations and seminars in Madison, Wisconsin and St. Louis, Missouri. This work was alsopresented within the reporting timeframe at the Plant Biology meeting (Honolulu, HI; June 22-26, 2024), USDA NIFA PD meeting (Honolulu, HI;June 26-27, 2024), and the ICAR meeting (SanDiego, CA;July 15-17)2024 to university, industry, and government scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has so far engaged one post-doctoral researcher (PD), one research scientist, and one undergraduate student. Training: The research scientist and undergraduate student have assisted in the transformation and maintenance of pennycress and Arabidopsis transgenics. Before this opportunity, the undergraduate student had not been involved in plant biochemistry research. Through their work in our laboratory, this student has become proficiently skilled in growing and screening laboratory plants, collecting and analyzing phenotypic data (imaging, measuring, etc), performing assays to quantify the amount of P present in a sample, and other aspects of plant biochemistry and genetics in multiple plant species. The post-doctoral researcher has also been engaged in planning, developing, and implementing Extension activities through BMP Field Day. Additional extension activities include the creation of Phyte4Food, an illustrated video and podcast series. The post-doctoral researcher has written, illustrated and narrated 5 videos and created a website to share these videos with stakeholders and general public audiences. This was a highly enriching and educational experience, allowing this professional to gain experiences in but not limited to: hosting events across universities, disseminating information from the university to the general public and farmers across counties through a network of newspapers, radio stations, extension offices, and companies, leading demonstrations and demonstration sessions, a poster session, a seminar series, and coordinating this with a variety of researchers, farmers, caterers, and other professionals. Additionally, this opportunity has provided the means to travel and share research and extension work with stakeholders/farmers, faculty researchers, Wisconsin legislatives/policy makers, industry scientists, graduate students, and the general public. This includes 7 oral presentations/seminars and 7 poster presentations (August 2023-August 2024). How have the results been disseminated to communities of interest?This work has been presented to a variety of audiences that are unfamiliar with the Phosphorus crisis. At the beginning of this project, the original focus had been to reach the general public audience and farmers. Given lawmakers, scientists (industry and faculty), and students are also unfamiliar with the phosphorus crisis, the scope has been expanded to include these audiences as well. Listed are the major presentations (oral and poster) that disseminate information regarding the phosphorus crisis through the research goals 1 & 2 as well as extension goals (3; Phyte4food podcast and field day) within the August 2023- August 2024 reporting period. Plant Cellular and Molecular Biology Supergroup Seminar Series, September2023, Madison, Wisconsin Audience: Faculty scientists, postdoctoral researchers,graduate students, and undergraduate students. Informationwas disseminated through an oral presentation and follow up discussion with undergraudate student botany class. Bayer University Conference, October 2023, St Louis, Missouri Audience: Bayer scientists, graduate students, and postdoctoral researchers. Informationwas disseminated through an oral presentation and poster presentation. Catalysts for Science Policy, April 2024, Madison, Wisconsin Audience: General public and college students (STEM and non-STEM majors). Information disseminated through an oral presentation and an illustration. UW Day at the Capitol, April 2024, Madison, Wisconsin Audience: Wisconsin legislatives/policy makers, farmers, general public, faculty researchers, graduate students, and undergraduate students. Information was disseminated through a poster presentation. Midwest Cellular Dynamics Meeting, June 2024, Madison, Wisconsin Audience: Faculty researchers, industry scientists, postdoctoral researchers, graduate students, and undergraduate students. Information was disseminated through a poster presentation. ASPB Plant Biology Meeting & USDA NIFA PD Meeting, June 2024, Honolulu, Hawaii Audience: Faculty researchers, industry scientists, USDA scientists, postdoctoral researchers, graduate students, and undergraduate students. Information was disseminated through two poster presentations. International Conference on Arabidopsis Research (ICAR) Meeting, July 2024, San Diego, California Audience: Faculty researchers, industry scientists, USDA scientists, postdoctoral researchers, graduate students, and undergraduate students. Information was disseminated through an oral presentation and a poster presentation. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We are currently assessing the P use efficiency by measuring the total P content in the plant biomass when grown under different conditions. Additionally, we are assessing the seed properties of these plants. We hypothesize that by altering the InsPs in specific areas of the plant, we can decrease the amount of P allocated to seeds. Objective 2:We are assessing the P reclamation potential of transgenic soybean and pennycress plants. We are also assessing this by growing these plants under a variety of P conditions that imitate field conditions and assess their P reclamation potential. Objective 3:We will complete the final illustrated video andpodcasts for Phyte4Food.

Impacts
What was accomplished under these goals? The Phosphorus crisis presents a major challenge to food security. While Phosphorus (P) is critical for crop growth, P is a nonrenewable and increasingly limited resource. Our global population is fed at the expense of the remaining mineable P reserve, which may be depleted in 30 years. Further, fertilizer runoff from farmland and urban areas poses a dangerous problem as increased nutrients in watersheds toxifies our water supply and aquatic ecosystems. Time is running out to preserve our P supply. New and innovative strategies that reduce fertilizer inputs and watershed pollution are key to securing the global food supply and protecting the environment. Emerging evidence shows plants use inositol pyrophosphates (PP-InsPs) as signaling molecules to detect P. My preliminary work demonstrates altering PP-InsPs can decrease plant P accumulation with a limited growth trade-off or cause plants to hyperaccumulate P. We propose to translate knowledge on this pathway into a strategy to reduce fertilizer inputs and reclaim P polluted soils. Wewill achieve this in three distinct aims. (1) First, we will artificially increase PP-InsPs to determine how this impacts crop requirements for fertilizer. (2) Additionally, wewill engineer P-accumulating crops and convert them into a novel biochar product to assess crop P reclamation potential. (3) Further, wehave developed an Extension strategy to spread awareness of the P crisis and agricultural sustainability on the local, regional, national, and global level. Ultimately, this integrated research and Extension project will enhance P usage to protect the global food supply and environment. This integrative project combines research and extension to remedy the impending P crisis. First, this research translates fundamental discoveries wehave made with inositol pyrophosphate signaling in Arabidopsis thaliana in two crop species to enhance plant Phosphorus-use efficiency and reclaim Phosphorus from polluted environments. The extension portion of this project will allow me to engage a wide variety of stakeholders regionally and globally in topics surrounding P usage, climate change, and how fertilizer impacts the environment. To achieve these goals, we have the following specific objectives: Objective 1. Research: Enhance phosphate-use efficiency (PUE) in two crop species and assess whether we can develop crops with a reduced need for fertilizer This objective is 65% complete. Wehave selected three plant species for this project: a laboratory model species,Arabidopsis, as well as two crop species, pennycress and soybean. The two crop species are the major focus of this project. We have constructed the appropriate gain-of-function genetic constructs and transformed these intoArabidopsisand Pennycress plants. We hypothesize will reduce the plant's need to acquire P from the soil. The Wisconsin Crop Innovation Center has constructed the proposed vectors and has introduced them into soybean Williams82. We have obtained homozygous plant lines and are currently assessing Phosphorus Use Efficiency (PUE) of these plants. Objective 2: Research. Develop two crop species that accumulate high amounts of inorganic phosphate (Pi) from the environment Appropriate gain-of-function genetic constructs that encode genes that impact that plants ability to hyperaccumulate P have been assembled and have been transformed into all three plant species. Similar to objective1, the two crop species, pennycress and soybean, are the major focus of this project. We have obtained homozygous soybean lines and are assessing alterations in PP-InsPs and genes involved in P sensing. Wehave obtained soybeanipk1mutants seeds from the University of Missouri. These are older seed stocks that unfortunately have a very poor germination rate, thus efforts are on-going to recover this germplasm. If we cannot recover this germplasm we will still have our gain of function plants that support this objective. Weestimate that this work is 55% completed. Objective 3. Extension. Implement a two-part Extension strategy to increase public awareness of the Phosphorus crisis We have completed the Best Management Practice (BMP) field day organized and scheduled to take place March 29th, 2023 at the OSU OARDC in Wooster, OH. This Field Day included a series of presentations that highlight difference aspects of P-use efficiency and -use inefficiency, interactive surveys, and field site demonstrations. The seminar components of BMP Field Day are accessible on the BMP Field Day website: https://bmpfieldday.carrd.co/ We have created six fully illustrated and narrated videos for the video series, Phyte4Food, as well as a website: https://phyte4food.com/. These videos address what P is and why it's important, the P crisis, how P cycles through the environment, how P and nutrients impact plant growth, phytoremediation strategies we can use to protect the environment. Our original plan was to have ten illustrated videos, but we have decided to combine some of the original videos into 7 videos and add some podcasts to the series. We have subsequently decided to create 5 podcasts in lieu of the videos, because this will allow us to expand on more information presented in the illustrated videos. These podcasts will include interviews with a variety of professionals with expertise in these specific areas. The script for the final video and illustrations have been created and the podcasts are in progress. Online distribution streams will include the Phyte4Food website made and maintained by our lab group for these videos, the American Society of Plant Biologists official website, YouTube, and various social media/podcast platforms. Weestimate that this work is about 75% completed.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Freed, C., Craige, B., Donahue, J., Cridland, C., Williams, P., Pereira, C., Kim, J., Blice, H., Owen, J., Gillaspy, G. USING NATIVE PLANT AND SYNTHETIC GENES TO DISRUPT INOSITOL PYROPHOSPHATES AND PHOSPHATE ACCUMULATION IN PLANTS. Submitted and under review.