BIOLOGICAL NITROGEN FIXATION IN THE MUCILAGE OF MAIZE AERIAL ROOTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1024073
• Grant No.: 2020-67013-32675
• Cumulative Award Amt.: $1,068,999.00
• Proposal No.: 2020-08318
• Project Start Date: Sep 1, 2020
• Project End Date: Aug 31, 2025
• Grant Year: 2020
• Program Code: [A1171]- Plant Biotic Interactions

Project Director
ANE, J. M.

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Bacteriology

Non Technical Summary
The current yearly global nitrogen fertilizer application rate is estimated to be $115 Tg, of which, globally, 50-70% is lost from agricultural systems to the environment. Nitrogen, in the form of nitrate, can be lost via leaching and runoff at global rates estimated to account for 19% of total nitrogen fertilizer application. To avoid that, many synthetic fertilizers have in their composition nitrification inhibitors. Still, surprisingly, to our knowledge, efficient nitrification inhibition has not yet been demonstrated in maize, the third most important crop species after rice and wheat in terms of global fertilizer consumption and crop output. A maize variety that benefits from nitrogen fixation, together with the identification of microbes that play a significant role in the aerial roots, would improve the efficiency of biological nitrogen fixation to new levels, similar to what is reported for soybean, where 40% of nitrogen is derived from nitrogen fixation. In countries, where inoculants are widely used for cereals, 25 % of nitrogen supply comes from inoculation with Azospirillum. Therefore, optimization of inoculants and maize varieties would decrease the amount of nitrogen applied by at least 20% representing a significant economic gain for the farms, particularly in subsistence farming systems, but more than that would reduce the global reliance on synthetic nitrogen fertilizers and their environmental impact. Taken together, this may cause a change in condition because the food production can increase, by decreasing by at least 20% the effect on the environment, without augmenting the land use.The goal of our project is to better understand and improve the process of nitrogen fixation in the aerial root mucilage of SuperMuc maize varieties to enhance the sustainability of US agriculture but also to provide nitrogen to farmers in developing countries where nitrogen availability is still limiting their yields.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	1510	1080	50%
206	1510	1020	50%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1510 - Corn;

Field Of Science
1080 - Genetics; 1020 - Physiology;

Keywords

corn

nitrogen-fixation

symbiosis

Goals / Objectives
The primary goal of this project is to gain a better understanding of the environmental factors, the genes and the molecular mechanisms controlling aerial root development, mucilage production and nitrogen fixation in the mucilage produced by aerial roots of specific corn landraces. To accomplish this, we will determine the genetic basis of this trait, determine how aerial roots produce this mucilage, how nitrogen fixed by the bacteria is transferred to the plant, and if we can improve this system using inoculation with natural or engineered bacteria.The specific objectives of this proposal are to:Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments to determine the environmental factors affecting aerial root development and mucilage production.Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. We will use double haploids and rAmpSeq to map the loci controlling the determinants of aerial root development, mucilage production, and associative nitrogen fixation.Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. This objective will be achieved using RNA-Seq and enzymatic assays to analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water.Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. We will address this objective using a combination of 16S and ITS amplicon sequencing, microbe isolation, and inoculation experiments to understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots.

Project Methods
Our preliminary data indicate that the mucilage produced by aerial roots is the primary environment where nitrogen fixation happens in SuperMuc maize. The more aerial roots are developed, the more mucilage is produced. Therefore, the first experiment will determine the environmental factors affecting aerial root development and mucilage production. To do that we will test different environmental factors, such as the effect of soil nitrogen levels, ambient humidity, and water limitation on aerial root development by the SuperMuc accessions Oaxaca184, Oaxaca187, and the B73 inbred maize. To test the effect of soil nitrogen, seeds will be planted in pots containing 50% sterilized sand, and 50% sterilized Turface® (by volume) in a greenhouse. Plants will be watered nutrient solution containing specified concentrations of ammonium nitrate. Gel production will be induced by exposing the plants to a misting environment, and the same system will be used to test the effect of ambient humidity. We will maintain relative humidity at 90%, 70%, and 50%. To test the effect of water limitation, the plants will be watered with 500, 350, and 200 mL with nutrient solution. When the plants show about 3-4 full developed leaves, we will evaluate the number of nodes with aerial roots and the total number of roots.The SuperMuc lines show several phenotypes that result in high mucilage production and associated nitrogen fixation. These traits persist in F1 hybrids made with elite maize lines. We will create doubled-haploid mapping populations to map the genes controlling mucilage production and nitrogen fixation. To do that we need to identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. Thus, the first step will be to develop four BC1 (first backcross) mapping populations for SuperMuc traits. Co-PI Wallace has already started making F1 seed between four different SuperMuc varieties and the elite varieties B73 and PH207. BC1 seed (backcrossed to the elite parent to simplify the genetics) will be selected from four crosses with distinct genetics, good seed yield, and strong SuperMuc phenotypes. We will identify the trait in the four double haploid families from previous experiments for aerial root phenotypes under field conditions and for both aerial root and nitrogen fixation phenotypes under greenhouse conditions. The field conditions we will record (1) flowering time, (2) overall height, (3) number of aerial root whorls, (4) the total number of aerial roots, and (5) average diameter, length, and volume of individual aerial roots. For the greenhouse experiment, each DH family will be grown in two reps in the greenhouse to measure nitrogen update via 15N dilution. Since 15N is at a lower abundance in the atmosphere than in the pot, plants that take up atmospherically fixed nitrogen have a lower 15N ratio in their tissue than ones that do not.Our preliminary data also indicate that the mucilage viscosity as well as its degradation into simple sugars allow oxygen protection and provide a source of energy for diazotrophs respectively. We will grow SuperMuc, and the B73 inbred maize under optimal conditions to produce aerial roots and gel, with this material we will analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. We will add 1 mL of water to the tip of aerial roots at the second aboveground node. Aerial roots will be collected at different time points and RNA will be extracted and to analyse its expression pattern. From our experience, most of the gel is produced within 60-90 minutes after addition of water, but we expect the degradation of the gel to continue and maybe the production of enzymes to degrade it to continue over a longer period. Among the genes that we will particularly track are those encoding glycosyltransferases and glycosyl hydrolases potentially involved in mucilage production and degradation. We will send all these samples for the quantification of free sugars, and other components of the root exudate. Altogether, this objective should allow us to determine what are some of the mechanisms involved in mucilage production and degradation on the surface of aerial roots. A large number of microbes were identified from the mucilage of Sierra Mixe maize using metagenomic and barcoding approaches, in a previous publication by the PI's lab. Significant enrichment in diazotrophs was detected in mucilage samples compared to the soil or other parts of the maize plant. However, these surveys did not allow us to determine which microbes play the most critical roles in the process of nitrogen fixation. Thus, it is crucial to understand and improve the transfer of fixed nitrogen between diazotrophs and maize in aerial roots. To achieve that we want to determine which microbes correlate the most with high levels of nitrogen fixation. We will take advantage of the natural variability in nitrogenase activity observed between individual aerial roots. We will grow Oaxaca184 and Oaxaca187 maize at West Madison Agricultural Station and at the University of Georgia. At V7, 90 minutes after top irrigation, we will collect mucilage from aerial roots of each accession. We will evaluate nitrogenase activity by indirect measurement of acetylene reduction, in an experiment called acetylene reduction assay - ARA. In parallel, we will use some mucilage for DNA extraction and 0.2 mL to evaluate the viscosity of the sample. DNA extracted from the mucilage will be used to amplify specific genes of bacterial and fungal sequences that will categorize them in operational taxonomic units (OTUs). Then, we will perform a correlation analysis between ARA, operational taxonomic units, and viscosity to determine if the nitrogenase activity correlates with the abundance of specific OTUs and viscosity. From the same field experiment, we will isolate diazotrophs from the mucilage collected from SuperMuc maize at West Madison Agricultural Station and the University of Georgia. We will also obtain mucilage from other SuperMuc accessions from CIMMYT. Pure cultures of diazotrophic bacteria will be isolated by serial dilution and plated onto a nitrogen-free medium containing carbon sources to reflect the free sugars and their concentrations available in the mucilage. The presence of nifH genes will be confirmed by PCR. Isolated bacteria will be identified through DNA extraction and 16S rRNA sequence analysis. For each strain, we will evaluate its nitrogenase activity in the nitrogen-free semi-solid medium described previously at its optimal growth temperature. We will also access ammonium release using Nessler's reagent. To reduce the variability between aerial roots described earlier, and possibly improve the benefits of nitrogen fixation to SuperMuc maize accessions, we will perform inoculation experiments of SuperMuc maize accessions and try to improve the transfer of nitrogen to the plant by inoculating with diazotrophs selected to fix more nitrogen and to release it to the plant. Ten strains will be tested per year. Six of these will be chosen from the experiments described earlier. Two strains will be wild-type Azospirillum brasilense as well as the HM053 mutant that has been engineered to release more ammonium. Two others will be Herbaspirillum seropedicae and Paraburkholderia unamae that have been shown to fix nitrogen efficiently in the maize mucilage. During winter, we will perform 15N dilution experiments in the greenhouse to test the efficiency of selected strains in the SuperMuc maize.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:We have provided comprehensive training to undergraduate students from diverse backgrounds, equipping them to become future leaders in the field of plant science. The PD's laboratories host weekly lab meetings to delve into scientific topics relevant to ongoing projects. Additionally, small group meetings are held weekly to delve into specific project aspects, allowing students to engage in idea generation and discuss scientific findings and challenges. This training has contributed to their career development and solid scientific groundwork and nurtured their passion for practical scientific applications in agriculture. Furthermore, we have furnished undergraduates with background knowledge, methods, and project-specific data collection techniques. The outreach activities aimed to engage elementary school students and the general public in interactive science experiences, including a display at science open-house events. To coordinate these efforts, the postdoctoral researchers and the PDs designed teaching modules focused on nitrogen fixation in agriculture. The PD introduced the project to Wisconsin corn growers at the annual meeting of the Wisconsin Crop Improvement Association. The postdoctoral researchers, the PDs, and other colleagues from the University of Wisconsin convened with Mexican scientists and concerned citizens to review the project's advancement, the implications of the research, and the necessity for ongoing open dialogue. The scientific community focused on nitrogen fixation is the target audience for this project. This year, the PD and postdocs presented this project at the European Nitrogen Fixation Conference and the North American Symbiotic Nitrogen Fixation Conference. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project supported three postdoctoral researchers, two at the University of Wisconsin and one at the University of Georgia: Dr. Daniel Laspisa is a bioinformatician specializing in maize comparative genomics and epigenomics in Dr. Jason Wallace's lab. Dr. Laspisa presented a poster on the Brace Root QTL in the doubled haploid populations at the Maize Genetics Conference 2024 and a talk at the Plant Animal Genome Conference 2024. He also had the opportunity to mentor an undergraduate student, Kenzington Deal, in identifying endophytic bacteria from maize tissue. Dr. Rubens Diogo is a newly hired postdoctoral researcher in Dr. Ane's lab who will assume Dr. Kimberly Gibson's responsibilities following her departure from Dr. Ane's lab. Dr. Rafael Venado is a postdoctoral researcher specializing in molecular biology in Dr. Ane's lab. Dr. Venado presented at the 13th International Congress on Plant Molecular Biology. June 28th, 2024. This project provided professional development opportunities for several undergraduate students: Simone Pete, Dan Triplett, Chloe Mootz, and Dannyell Hendrix assisted Courtney Phillips with management and data collection in the greenhouse and UGA nursery for the backcross genetic population and the doubled haploids. Caelin Smith, Madigan Feng, Damien Zingaro, McKenna Riley, Andrea Young, and Kaitlyn Madden are assisting Dr. Rubens Diogo during the field season 2024. They are currently recording data for this project. On Oct 3rd-5th, 2023, Dr. Jason Wallace, Dr. Daniel Laspisa, and Courtney Phillips attended an in-person meeting at the University of Wisconsin Madison with Dr. Jean-Michel Ane, Dr. Kimberly Gibson, and Dr. Rafael Venado. We reviewed the project's current results and planned for future experimentation. There were opportunities for Georgia team members to meet with UWM Faculty to develop collaborative relationships. The field at Iron Horse in Georgia was used as part of the "Plant Breeding Practicum" required for all UGA Plant Breeding, Genetics, and Genomics students. During the practicum, the students were given the project's background and tasked to measure root phenotypes in the field, which were then analyzed as a group with Dr. Wallace. How have the results been disseminated to communities of interest?Dr. Ane and Dr. Laspisa presented this work this year at the Maize Genetics Conference (MGC) and the Plant and Animal Genome (PAG) conference in 2024. Dr. Venado shared his research at several conferences. He presented his work at the 13th International Congress on Plant Molecular Biology in Cairns, Australia, in June 2024. Additionally, he presented at the 15th European Nitrogen Fixation Conference in Naples, Italy, in September 2023. He also delivered a seminar at the Department of Plant Pathology at the University of Wisconsin-Madison. Dr. Ane was invited to present this work at the National Academies webinar, the Western Illinois University, the Bill and Melinda Gates Foundation, and several conferences (26th North American Symbiotic Nitrogen Fixation Conference, Phytobiomes Alliance Webinar Series). Growers are also interested in this project, and Dr. Ane was invited to present it at the Wisconsin Crop Improvement Association Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments. We have successfully investigated the factors influencing aerial root development through greenhouse experiments and the manuscript containing the results is under review. Goal 2: Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. -Doubled Haploids and mapping populations We will complete the field season and collect brace root phenotypes for the doubled haploids and backup genetic population. We will conduct a final analysis of brace root QTL for the populations over the fall/winter of 2024. We will publish our results after completing the final analysis this fall. -Single-Nuclei RNA-seq We are currently working on validating the gene regulatory networks, and we hope to finish the validation and write a manuscript by the end of the year. Goal 3: Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. We plan to repeat one more set of experiments to evaluate the degradation of mucilage. Still, we have analyzed the molecular and biochemical basis of mucilage production and degradation and are currently analyzing data and preparing to publish our results. Goal 4: Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. We have analyzed the transfer of fixed nitrogen between diazotrophs and corn in aerial roots, and we are analyzing data and preparing to publish our results.

Impacts
What was accomplished under these goals? Goal 1: Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments. We evaluated various landraces in the greenhouse by measuring the number of nodes with aerial roots, root diameter, and the transition from juvenile to adult stages. These experiments were replicated at West Madison Agricultural Research Station in Wisconsin and the University of Georgia Iron Horse Plant Sciences Farm. The studies provided valuable insights into how environmental factors influence aerial root development. Building on this work, we conducted two experiments to investigate the effects of humidity and nitrogen on aerial root development. We grew the promising landrace Oaxa524 and the exPVP PHP02 in greenhouse conditions with two relative humidity levels: 30% and 70%. Higher humidity significantly increased the number of nodes with aerial roots, especially in the landrace. In a separate experiment, we assessed nitrogen's impact on three landraces (GRIN19770, Oaxa139, and Oaxa229) with low (70.36 ppm), medium (123.14 ppm), and high (175.91 ppm) nitrogen treatments. Nitrogen had minimal effect on aerial root development in these landraces. Overall, our findings confirmed that higher humidity is key to increasing the number of nodes with aerial roots, while nitrogen has a lesser impact. These results are included in a manuscript currently under revision at Crop Science. Goal 2: Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. -Doubled Haploids: Significant progress was made this year. Multidimensional scaling of our doubled haploid populations showed no genetic contamination, simplifying downstream analyses. We grew populations 1 and 7 in Wisconsin (Dr. Kimberly Gibson) and Georgia (Dr. Laspisa and Courtney Phillips), quantifying aerial root diameter, nodes with aerial roots, aerial root number, and stalk diameter. These traits exhibited moderate heritability (0.25-0.45), consistent with published estimates. We identified 17 quantitative trait loci (QTL) across the two populations--nine from population 1 and eight from population 7. These QTL were validated with RMIP analysis, and 52 of the 409 published loci associated with root architecture overlapped with our QTL. We will focus on these overlapping loci to identify genes of interest. Currently, we are in the second year of phenotyping in Georgia (Dr. Laspisa and Courtney Phillips) and Wisconsin (Dr. Diogo). Last year's challenges with poor germination in Georgia due to high rainfall and cool temperatures were addressed by treating seeds with fungicide, resulting in much better germination rates this year. Data from the 2024 field season will be analyzed this fall. In winter 2023, we generated F1 hybrids from doubled haploid genotypes with differing brace root phenotypes to explore dominance or additive effects. These hybrids are now in the field and will be phenotyped soon. -Genetic population development and evaluation: Last year's challenging conditions did not halt the development of the "backup" genetic population, which is currently in three stages: BC1S1, BC1S2, and BC1S3. The most advanced lines (BC1S3) are being grown in Georgia, where they will be phenotyped for brace root traits this season. We are also genotyping the population using Freedom Markers' GBS service to cross-check the QTL identified in the doubled haploids. -Single-Nuclei RNA-seq: We also performed single-nuclei RNA sequencing (snRNA-seq) to investigate the genetic determinants of mucilage production. We compared roots that did not produce mucilage with those that did. After stringent cluster annotation, we identified all cell clusters generated from our snRNA-seq experiment. We then generated gene regulatory networks associated with mucilage production and water perception. Currently, we are conducting experimental validation to confirm the function of these regulators using RNA interference (RNAi) in the model system Medicago truncatula. Goal 3: Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. We quantified the degradation of mucilage by measuring the reduction of simple sugars using Benedict's reaction. We collected mucilage from the Oaxa524 landrace grown in the greenhouse, triggering its release and then sampling every 20 minutes over two hours. Using Benedict's reaction, we quantified the free sugars at each time point and observed that free sugars increased over time. This suggests that plants release enzymes responsible for mucilage breakdown. Pankievicz et al. (2022) identified several genes encoding carbohydrate degradation enzymes, and our assays confirmed that mucilage is degraded into simple sugars, which could be utilized by the specific microbiome established in the mucilage. Goal 4: Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. We conducted an acetylene reduction assay (ARA) and a 15N isotope dilution experiment to investigate nitrogen transfer between diazotrophs and plants. In the ARA experiment, we collected mucilage from Oaxa524 landraces grown in the greenhouse and individually inoculated it with different diazotrophs to study nitrogen fixation in cereals. We tested ten different strains, including six traditional model diazotrophs isolated from other crops: Azospirillum brasilense FP2, Azorhizobium caulinodans, Herbaspirillum seropedicae, Paraburkholderia silvatlantica, Pseudomonas stutzeri, and Azotobacter vinelandii. Additionally, we included two mucilage strains, Klebsiella variicola and Klebsiella michiganensis, isolated from sorghum and maize mucilage, respectively, and two mutant strains unable to fix nitrogen: Azospirillum brasilense FP2 and Klebsiella variicola Δnif. Our results indicated that all strains could fix nitrogen in the maize mucilage. Notably, K. michiganensis, a maize mucilage strain, exhibited higher nitrogenase activity than other strains, including K. variicola, a mucilage strain isolated from sorghum. Negative controls did not show a significant signal compared to the different strains. These findings suggest that maize mucilage provides an excellent environment for diazotrophs, with some strains performing better than others. For the 15N isotope dilution experiment, we grew Oaxa524 and PHP02 with Hoagland solution and 15N ammonium sulfate as a unique nitrogen source. In two separate rooms, we inoculated the plants with a cocktail of nitrogen-fixing diazotrophs at the V8 stage, where adventitious roots were observed. Room one received K. michiganensis and Azotobacter vinelandii, while room two received a mix of mutant strains unable to fix nitrogen. The 15N isotope was determined using isotope ratio mass spectrometry and reported as Atom %15N excess. After tasseling (flowering), the Oaxa524 landraces inoculated with diazotrophs had the lowest Atom%15N excess compared to the other three conditions (Oaxa524 with mutant strains and PHP02 with diazotrophs and mutant strains). These results suggest that upon inoculation with diazotrophs, the maize landrace Oaxa524 provides an environment conducive to nitrogen fixation through its aerial roots and mucilage. Additionally, we calculated the Nitrogen derived from the atmosphere (Ndfa), which indicates the percentage of total plant nitrogen. The Ndfa for Oaxa524 was 49.99%, whereas the value for PHP02 was 14.97%. These results are consistent with the range reported by Van Deynze et al. (2018), which ranged between 29% and 82%. Based on these findings, we have selected Oaxa524 as the parent in our breeding program.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Rafael Venado - Single-cell insights on the aerial roots of nitrogen-fixing maize and sorghum. 13th International Congress on Plant Molecular Biology (June 28, 2024)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Daniel Laspisa - Genetic Determinants of Aerial Root Formation in Maize: Prospects for Harnessing Biological Nitrogen Fixation. Plant and Animal Genome Conference hosted in San Diego, California (January 16, 2024)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Jean-Michel Ane - Leveraging bacterial and plant genetics to optimize associative nitrogen fixation for cereal crops. 26th North American Symbiotic Nitrogen Fixation Conference. Burlington, VT, USA (June 3, 2024).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Jean-Michel Ane -Engineering nitrogen-fixing microbial communities associated with maize and sorghum roots. Phytobiomes Alliance Webinar Series (May 23, 2024).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Jean-Michel Ane -Biological Nitrogen Fixation on the Aerial Roots of Maize for Sustainable Agriculture. Western Illinois University, online (April 10, 2024).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Jean-Michel Ane - Biological Nitrogen Fixation on the Aerial Roots of Maize and Sorghum for Sustainable Agriculture. Plant and Animal Genome 31 (PAG31) conference. San Diego, CA, USA (January 15, 2024).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Jean-Michel Ane -Discoveries to Improve Biological Nitrogen Fixation in Cereal Crops. Wisconsin Crop Improvement Association Annual Meeting. Wisconsin Dells, WI, USA (November 28, 2023).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Jean-Michel Ane - Engineering Biological Nitrogen Fixation for Cereals. Bill and Melinda Gates Foundation. Seattle, WA, USA (October 9, 2023).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Jean-Michel Ane - Engineering Plant-Microbe Symbioses for Enhanced Plant, Soil, and Human Health. National Academies webinar. Online (September 18, 2023).
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Van Gelder K, Oliveira-Filho ER, Messina CD, Venado RE, Wilker J, Rajasekar S, An� JM, Amthor JS, Hanson AD. Running the numbers on plant synthetic biology solutions to global problems (2023). Plant Science 335:111815 DOI: 10.1016/j.plantsci.2023.111815
• Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Kloppenburg J, Calder�n CI, An� JM. The Nagoya Protocol and nitrogen fixing maize: Close encounters between Indigenous Oaxacans and the men from Mars (Inc.) (2024). Elementa: Science of the Anthropocene 12 (1): 00115. DOI: 10.1525/elementa.2023.00115
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Wilker J, Venado RE, Infante V, McLimans C, Robbins F, Phillips C, Calder�n CI, Wallace J, An� JM. Aerial root formation in Oaxacan maize (Zea mays) landraces persists into the adult phase and is minimally affected by soil nitrogen and ambient humidity. Crop Science (submitted).

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The maize and the nitrogen fixation communities are a specific target audience for this research. This year, Dr. Laspisa presented his work at the Maize Genetics Conference (MGC), and Dr. Wilker presented her work at the Plant and Animal Genome (PAG) conference. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported three postdoctoral researchers, two at the University of Wisconsin and one at the University of Georgia. Dr. Daniel Laspisa is a bioinformatician specializing in maize comparative genomics and epigenomics in Dr. Jason Wallace's lab. Dr. Laspisa presented a poster on the backcross population at the Maize Genetics Conference 2023. He also had the opportunity to mentor an undergraduate student Kenzington Deal to isolate endophytic bacteria from maize tissue. Dr. Jennifer Wilker is a plant breeder and geneticist in Dr. Ane's lab. Dr. Wilker presented a poster at PAG 2023 on aerial root formation in Sierra Mixe maize landraces. She has a permanent position at CIAT (Centro Internacional de Agricultura Tropical, Spanish acronym) Dr. Kimberly Gibson is a newly hired postdoctoral researcher in Dr. Ane's lab who will assume Dr. Jennifer Wilker's responsibilities following her departure from Dr. Ane's lab. This project provided professional development opportunities for several undergraduate students: Fran Idaewor has assisted Dr. Daniel Laspisa in mapping brace root traits in the "backup" BC1S2 population. Zoey Stephens, Kofi Reeves-Miller, Stephen Hedden, and Dan Triplett assisted Courtney Phillips with management and data collection in the greenhouse and UGA nursery for the backcross genetic population and the doubled haploids. Caelin Smith, Madigan Freng, Andrea Young, and Kaitlyn Madden assisted Dr. Jennifer Wilker during the planting season of 2023. They are currently recording data for this project. On November 6-8, 2022, Dr. Jean-Michel Ane, Dr. Jennifer Wilker, Rafael Venado, and Fletcher Robbins from Wisconsin attended an in-person meeting at UGA with Dr. Jason Wallace, Dr. Daniel Laspisa, Courtney Phillips, and Hanxia Li. We reviewed the state of the project, current results, and planned experiments for the coming year. There were opportunities for Wisconsin team members to meet with UGA Faculty to develop collaborative relationships and for team-building exercises. The field at Iron Horse in Georgia was used as part of the "Plant Breeding Practicum" required for all UGA Plant Breeding, Genetics, and Genomics students. During the practicum, the students were given the project's background and tasked to measure root phenotypes in the field, which were then analyzed as a group with Dr. Wallace. How have the results been disseminated to communities of interest?The results of our research activities were made available to the scientific community through presentations at the Maize genetics conference, Plant and Animal Genome Conference, and the Wisconsin Land and Water Conference. Results were also made available in the Pankievicz et al. 2022 publication submitted to Frontiers in Plant Science. Two additional manuscripts are currently in preparation. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments. We have successfully investigated the factors influencing aerial root development through greenhouse experiments. Currently, Dr. Jennifer Wilker is working on a manuscript that will encompass various environmental factors impacting the growth of aerial roots, such as nitrogen levels, humidity, and plant developmental stages. We aim to submit this comprehensive manuscript by the end of the year. Goal 2: Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. -Doubled haploids The climate-related setback in Georgia has reduced the amount of data and data quality that can be collected from the doubled haploids at Iron Horse. We plan to grow this population again in spring 2024 to gather more, higher-quality data. Seeds will also be pre-treated with a fungicide to prevent the problems with germination that we experienced this season. Regardless, the data collected from Georgia and Wisconsin will be used to conduct preliminary genetic mapping on the doubled haploid populations to identify genetic determinants of aerial root development. In Wisconsin, severe drought is affecting plant growth. We are concerned that some data will be lost, but we will generate some preliminary data, and we are planning to increase seed from each plot by self-pollination. -Genetic population development and evaluation We will continue the advancement of the "backup" backcross population and advance the remaining BC1S1 and BC1S2 plants in the spring of 2024. We will also phenotype and genotype the BC1S3 plants with Freedom Markers GBS at this time. These data will be used to conduct genetic mapping experiments. If successful, the remaining backcross groups will be genotyped and phenotyped when advanced to the BC1S3 stage to refine the mapping results and compare them to the doubled haploid mapping experiments. - Mapping population F2:3 We will have one year of data from our mapping population this year. We plan to perform QTL mapping and identify the genetic determinants controlling aerial root development. We will generate the genotype using DArTseq, and for the analysis, we plan to use the package R/qtl within R. Goal 3: Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. Our study of Pankievicz et al. 2022 identified multiple genes encoding enzymes essential for mucilage synthesis. In addition, we have conducted single-nuclei RNA sequencing (snRNA-seq) to generate data from aerial roots both with and without mucilage. Currently, this data is undergoing analysis. We plan to conduct RT-qPCR on mucilage samples to validate the genes identified in Pankievicz's study and the snRNAseq data. As for the factors influencing degradation, we will perform two biochemical assays to quantify free sugars and total carbohydrates. Building on the findings from the Pankievicz et al. publication, which indicated the presence of various plant enzymes responsible for carbohydrate breakdown, we hypothesize that plants may secrete these enzymes to support nitrogen-fixing bacteria. In our experiment, we aim to collect mucilage at different time points and examine how these plant enzymes influence the content of free sugars over time. We will obtain mucilage samples from a more controlled environment, specifically the greenhouse, to minimize the impact of enzymes from the mucilage microbiome. Goal 4: Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. We have identified Klebsiella michiganensis as a promising candidate for genetic engineering due to its significant nitrogenase activity in maize mucilage. This strain will be utilized in two separate experiments: i) We plan to assess nitrogen uptake in the landrace Sierra Mixe using a 15N dilution assay. To conduct this study, we will grow Sierra Mixe and PHP01 in two high-humid greenhouse rooms. We will inoculate the plants in one room with the wild-type K. michiganensis strain. In contrast, in the other room, we will inoculate with a mutant K. michiganensis strain lacking the nifH gene, which is essential for nitrogenase activity. To create the mutant strain, we will perform targeted genetic modifications. The plants will be fertilized with 15N, and subsequently, we will measure and compare the ratio of 15N to 14N in the plants using isotope ratio mass spectrometry. This experiment aims to evaluate the effectiveness of K. michiganensis in providing nitrogen to the landrace Sierra Mixe. ii) The second experiment focuses on engineering K. michiganensis to act as ammonium-excreting diazotrophs. This modification aims to enhance the bacterium's capacity to supply nitrogen to the plant through the mucilage on its aerial roots. However, developing this engineered strain involves establishing several protocols, making it a long-term research project. These experiments aim to gain valuable insights into K. michiganensis' potential as a plant nitrogen supplier. We will hold our next in-person meeting regarding the project Tuesday, October 3rd - 5th, 2023, in Madison, Wisconsin.

Impacts
What was accomplished under these goals? Goal 1: Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments. An experiment was conducted to assess how humidity influences the development of nodes with aerial roots. Two greenhouses were utilized, each maintaining a distinct relative humidity level: high (75%) and low (30%). The landraces Sierra Mixe: CIMMYTMA-BANK-017456 and exPVP: PHP02 were planted in both greenhouses. The number of nodes, the number of roots at the top node, and the diameter of aerial roots were measured. The results revealed that high humidity significantly impacted the number of nodes in the maize landrace Sierra Mixe, while it did not affect PHP02. There were no significant differences observed in the other traits. This information will be included in an upcoming manuscript, which discusses the formation of aerial roots in the Sierra Mixe landrace and incorporates previous findings concerning the influence of nitrogen. Goal 2: Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. -Doubled haploids Eight populations of doubled haploids were successfully generated and genotyped by Limagrain and distributed between Wisconsin and Georgia locations. The two best doubled haploid populations were planted at Iron Horse Farm in Georgia and West Madison Agricultural Research Station in Wisconsin. Higher rainfall and a cooler-than-average spring in Georgia stimulated fungal growth on newly planted seeds, significantly reducing germination rates (10.2% and 33.3% for DH populations 1 and 2, respectively). This issue will be addressed in future field experiments by applying antifungal seed treatments before planting. DH populations successfully germinated in Wisconsin, but intense drought is delaying the development of these populations. We will obtain as much data as possible. -Genetic population development and evaluation The abnormal spring conditions also affected the advancement of the "backup" genetic population, which is currently in three stages of development BC1S1, BC1S2, and BC1S3 (Germination rates of 15.4%, 9.0%, and 13.2%, respectively). The limited number of plants made it impractical to advance the populations, which will be resumed in the field next year. The most advanced population is at the final BC1S3 stage. In the last year, the BC1S2 population was genotyped with Repeat Amplification Sequencing (rAmpSeq) and phenotyped to generate preliminary genetic mapping data for the population to compare to the doubled haploid analysis and to evaluate the efficacy of rAmpSeq as a genotyping method. The rAmpSeq genotyping method relies on amplifying and sequencing repetitive transposable elements, specifically the Huck retrotransposon. Read mapping experiments and investigation of SNPs indicated that aligning repetitive sequences to repeats in a physical map resulted in inaccurate genotyping. As such, rAmpSeq will not be used in future genotyping, and we will use the third-party GBS service Freedom Markers for genotyping the backcross population. Regardless, we identified a few putative QTLs of low significance associated with aerial root development phenotypes we can cross-check with future experiments. -Mapping population F2:3 We will perform QTL mapping with an F2:3 population derived from the cross Sierra Mixe maize landrace CIMMYTMA-BANK-017456 (Van Deynze et al. 2018) and the exPVP PHZ51. For this experiment, we have two locations in Wisconsin: West Madison Agricultural Research Station and Hancock agricultural research station. We have two replicates in a randomized design in each location augmented with PHZ51 checks. Goal 3: Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. Last year, we published a paper detailing mucilage production's cellular and genetic aspects in the maize landrace Sierra Mixe (Pankievicz et al. 2022). Our findings revealed that border cells, a specific cell type, release mucilage upon water exposure. Furthermore, we successfully identified multiple genes encoding enzymes essential for mucilage synthesis. Goal 4: Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. We conducted a comprehensive microbiome study using mucilage samples from maize, and subsequently, we analyzed the bacterial composition, organizing it into Operational Taxonomic Units. Our analysis revealed the predominant phyla as Pseudomonadota, followed by Cyanobacteria and Bacillota. These findings will be published in a paper focusing on mucilage in sorghum, a close relative of maize. We aim to submit this manuscript before the end of August. In a combined study involving maize and sorghum mucilages, we successfully isolated 500 different bacteria. Only nine bacteria exhibited diazotrophic characteristics, and we evaluated their nitrogenase activity through an acetylene reduction assay (ARA). Notably, the strains Klebsiella variicola and Klebsiella michiganensis demonstrated promising nitrogenase activity. To confirm our findings, we conducted a second ARA experiment using maize mucilage as a growth medium. As a control, we used a mutant variation of Klebsiella variicola (ΔnifH), a non-fixing bacterium. Our results demonstrated that K. michiganensis exhibited higher nitrogenase activity than K. variicola in maize mucilage. We plan to conduct further experiments with the K. michiganensis strain based on these promising results. Additionally, we conducted a metabolomic study to gain insights into the components within the mucilage that facilitate the establishment of diazotrophs and other bacteria. These valuable findings will also be included in the same manuscript that elucidates the role of mucilage in sorghum.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Pankievicz, V. C. S., Infante, V., Delaux, P.M., Hirsch, H. H., Rajasekar, S., Zamora, P., Jayaraman, D., Calderon, C. I., Bennett, A., An�, J.M. (2022). Nitrogen fixation and mucilage production on maize aerial roots is controlled by aerial root development and border cell functions. Frontiers in Plant Science, 3871.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: The ultimate fix to the nitrogen problem in agriculture: improving the ability of non-leguminous plants to host diazotrophs. EUGLOH Plant Science Meeting.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Jennifer Wilker, Valentina Infante, Caitlin McLimans, Ally Murray, Fletcher Robbins, Claudia Calderon, Jason Wallace, Jean-Michel An� (2023). Aerial root formation persists into the adult phase and is nitrogen-level dependent in Sierra Mixe corn landraces. Plant and Animal Genome 30 conference.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Jennifer Wilker. Nitrogen Fixation in Corn. 70th Annual Wisconsin Land and Water Conference.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Daniel Laspisa, Courtney Phillips, Holly Griffis, Jason Wallace. Mapping Maize Brace Root Traits Associated with Nitrogen-fixing Bacterial Symbiosis. Annual Maize Genetics Conference.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Rafael Venado. Nitrogen Fixation on the Aerial Roots of Maize and Sorghum for Sustainable Agriculture. Plant Cellular and Molecular Biology (PlantCMB) supergroup.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:-We trained undergraduate students from diverse backgrounds to form the next generation of plant scientists. The PD's laboratories organize weekly lab meetings to discuss scientific topics related to the projects. Also, small group meetings occur weekly to discuss specific aspects of the project, where students brainstorm ideas and discuss their scientific results and problems. This training has contributed to their career development and strong scientific foundation while fueling their desire to learn about practical scientific applications in agriculture. We also trained undergraduates in the background, methods, and data collection specific to this project. -Elementary school students and the general public were exposed to science knowledge and experience through outreach activities, including an interactive display at science open-house events. To manage the outreach activities, a group of postdocs in the lab and the PD developed teaching activities about nitrogen fixation in agriculture. -The PD introduced the project to Wisconsin Corn Growers Association members. -The PDs, postdocs and other researchers from the University of Wisconsin met with Mexican scientists and concerned citizens to discuss the project's progress, the research's implications, and the need for continued open dialogue. -The nitrogen fixation scientific community is a specific target audience for this project. This year, the PD and postdocs presented on this project at the European Nitrogen Fixation Conference and the North American Symbiotic Nitrogen Fixation Conference. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project supported two postdoctoral researchers at the University of Wisconsin - Madison: Dr. April MacIntyre is a microbiologist who worked in Dr. Ané's lab until January 15, 2022. Dr. MacIntyre trained an undergraduate student to phenotype the plant-growth-promoting traits of mucilage bacterial isolates. Dr. Jennifer Wilker is a plant breeder and geneticist in Dr. Ané's lab. Dr. Wilker presented a poster summarizing the nitrogen fixation and mucilage production function of border cells at the 2021 European Nitrogen Fixation Conference and a poster describing the genetic studies on aerial root traits at the North American Symbiotic Nitrogen Fixation Conference. The Georgia field location was used for practical demonstration as part of the "Plant Breeding Practicum" that all graduate students in UGA's Institute of Plant Breeding, Genetics, and Genomics must take. The demonstration included the background of the project and brief training on how to measure the relevant phenotypes, followed by students collecting basic phenotype data on aerial roots, which were then collected and analyzed as a class. This project has also provided professional development opportunities for several undergraduate students: Kai Sanders has assisted Courtney Phillips, who managed this project's field and greenhouse aspects for the UGA team. Fran Idaewor has assisted Hanxia Li in labeling images of brace roots for machine learning applications. Quinlan Kiefer assisted Dr. April MacIntyre in screening mucilage isolates and DNA extractions for 16S studies; Bella Rupnick, Caitlin McLimens, Lucas Crawford, Ally Murray, Martha Barta, Esha Mahalingam, and Jack Terlap have assisted Dr. Jennifer Wilker with laboratory, greenhouse, and field studies. On September 16-18, 2021, the CoPD Wallace came to the University of Wisconsin - Madison for our annual in-person meeting. We reviewed progress, went to the field and greenhouses,and discussed future directions. Dr. Wallace also gave a seminar on his work at the Plant Breeding Plant Genetics (PBPG) seminar. How have the results been disseminated to communities of interest?Results are made available to the nitrogen fixation and scientific community through the manuscript submitted to the Frontiers in Plant Sciences journal. Two additional manuscripts are in preparation. As indicated earlier, Dr. Ane and Dr. Wallace also gave several presentations on this project. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments. Preliminary genetic mapping studies using an F2:3 mapping population identified a gene of interest associated with aerial root development. A construct was generated using GoldenGate cloning, and CRISPR-Cas9 was used to transform Hi-II (High type II callus) following standard methods at the Wisconsin Crop Innovation Center. Four transformed lines were generated, and we obtained seeds. Molecular characterization of the transformed lines will be carried out, including confirmation of the expected deletion. The successfully transformed lines will be grown-out under greenhouse or field conditions and evaluated for aerial root production. Goal 2: Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. Doubled haploids: - Phenotypic evaluation of doubled haploid (DH) lines in the field will be carried out if the seed is provided in time for the 2023 summer field season. - Genetic comparison of landraces, teosinte, and modern corn genotypes: Maize has undergone selection during domestication. To test if the aerial root mucilage production results from several selection cycles, we will look for regions with a high allelic variation using the index of fixation (Fst) at the genome-wide level. Currently, SNP data is available for different landraces that produce mucilage. Exploring this data should reveal genomic regions that have been selected and generate the hypothesis that explains this unique trait in these landraces. - Genetic population development and evaluation:Mapping populations will be advanced in the winter nursery (Tuniche, Chile), where the F2 plants will be grown and selfed. we will plant the F2:3 seed in Madison, Wisconsin, for field evaluation in the summer of 2023. A parallel set of "backup" backcross populations will be advanced to another generation in Georgia in the summer of 2023. The most advanced of these (BC1S2 in 2022) was genotyped this year, and we will do the genetic mapping over the coming months. Assuming positive results, the other populations will be genotyped and phenotyped in 2023. - Identify the genetic components required to produce mucilage on aerial roots:Identification of genetic players controlling different agronomic traits has been possible using comparative transcriptomics (Du et al., 2019; Kost et al., 2020; Xu et al., 2020). Therefore, we will use this approach to identify the genetic components required to produce mucilage on aerial roots. We will compare the transcriptome of three different landraces against exPVP corn varieties that also develop aerial roots but lack the production of carbohydrate-rich mucilage. These landraces have consistently produced mucilage across different environments. In addition, we will compare the transcriptome of underground roots with that obtained from the aerial roots to look at possible differences between these tissues. Ideally, single-cell RNAseq will be conducted in isolated border cells as it has been described that in underground roots, this type of cell is responsible for producing mucilage on the root cap (Mravec et al., 2017). A preliminary study performed bulk RNAseq in both root types and accessions. Bioinformatic analysis is ongoing to identify preliminary candidate genes. Single-cell and bulk RNAseq are complementary approaches that could be useful for dissecting the genetic players responsible for mucilage production. Therefore, both sequencing methods will provide valuable information to understand this trait. Once candidate genes are identified, genetic characterization will be carried out using different molecular, biochemical, and cell biology methods and mutant analysis. Goal 3: Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. We are planning to evaluate the enzymatic activities of enzymes involved in mucilage degradation as described in the proposal. Root border cells release their mucilage using large vesicles from the trans-Golgi network (Wang et al., 2017a). There is no evidence of how mucilage is exported on aerial root border cells. Thus, we plan to test the hypothesis that mucilage is secreted from vesicles similarly to root border cells. To confirm this, we will purify vesicles using a sugar ultracentrifuge gradient described in ginger (Zingiber officinale) roots (Cao et al., 2019; Zhuang et al., 2015). Different fractions could be recovered and tested for the presence of carbohydrates identified in the aerial roots of the Sierra Mixe maize landrace. After successfully purification of these fractions, gas chromatography coupled with mass spectrometry will be performed as previously reported (van Deynze et al., 2018). Alternatively, immuno-TEM may be performed on tissue from the aerial roots (Wang et al., 2017b), and specific monoclonal antibodies can be used in the purified vesicles to detect the presence of the carbohydrates in the vesicles. Goal 4: Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. In addition to wet-lab experiments for characterizing mucilage and bacterial characteristics, we are planning a set of microbiome studies. These studies will 1) correlate the microbiome, mucilage viscosity, and acetylene reduction to determine which bacterial community members fix the most nitrogen and the optimal viscosity for nitrogen-fixing behavior, and 2) compare the differences in microbial community composition between corn grown in Georgia vs. Wisconsin and 3) determine the origin of bacterial community members (air vs. soil vs. rain) with implications for future methods of introducing synthetic communities to the field.For the diazotroph inoculation experiment, leaf samples will continue to be prepared and sent for isotope analysis. We will analyze phenotypic data and determine nitrogen fixation levels for each genotype and inoculation treatment. This experiment will be replicated next year, depending on the results obtained this season. Using the transcriptome information generated from Goal 2, we will conduct gene ontology and gene enrichment analyses to look for biological processes related to nitrogen metabolism and transport. Within these clusters of genes, we will primarily search for nitrate and ammonium transporters as these proteins are responsible for nitrogen uptake in the plant (Masclaux-Daubresse et al., 2010). In addition, we will expand the search by exploring available transcriptomic data or predicted transporters. Validation of the possible candidate genes will include qRT-PCR in different tissues and conditions. We will use the Xenopus laevis oocytes system to characterize the nitrate or ammonium transporters. This will reveal the activity of transferring nitrogenated compounds in vitro. Once the activity of the candidate transporters is confirmed, we could use overexpression and CRISPR lines to explore the function in planta. Finally, we will evaluate the effect of different synthetic communities in nitrogen nutrition using a 15N abundance assay to understand if certain diazotrophs enhance nitrogen uptake. We will conduct our next in-person annual meeting onNovember 7 - 9, 2022.

Impacts
What was accomplished under these goals? Goal 1: Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments. Greenhouse experiments this year investigated the juvenile-adult growth phase change in corn and whether aerial roots continue to be produced beyond the end of the juvenile vegetative stage. The maize scientific community widely assumes that brace and aerial root production ceases once maize plants transition from the juvenile vegetative to the adult vegetative stage. However, we hypothesize that landrace maize genotypes with high numbers of nodes with aerial roots continue to produce these roots after the juvenile vegetative stage. An experiment was conducted to determine whether aerial roots are made by Oaxacan landraces beyond the juvenile vegetative stage. Six genotypes were chosen, including 3 Oaxacan-derived landrace maize accessions, two expired Plant Varietal Protection certificate (exPVP) inbreds, and one giant heirloom corn variety. The plant growth stage was monitored by observing morphological indicators of the transition between juvenile and adult stages, including 1) leaf epicuticular wax production and 2) toluidine blue (TBO) staining of leaf epidermis. Additionally, we recorded the number of nodes with aerial roots weekly and calculated the days to tasseling. The number of nodes with aerial roots, the number of roots on the top-most complete node, the diameter of roots, stalk diameter, and plant height were recorded after the plants flowered.Data collection of leaf epidermis staining and further statistical analyses are ongoing. Goal 2: Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. - Doubled haploids Doubled haploid lines were initiated to be used in studies to characterize the genetic determinants of aerial root traits. We chose three BC1 crosses between elite inbred lines and Oaxacan landraces. The generation of these lines, which takes several months, is still ongoing, and it is expected that double haploid lines will be available in 2022 for the summer 2023 field season. - Genetic population development and evaluation Mapping populations continue to be advanced in both summer and winter nurseries. The most advanced of these are now at the BC1S2 stage (1 generation of backcrossing, two generations of selfing) and are evaluated for mapping potential in Summer 2022. We gathered both genotype and phenotype information on 200 lineages, and this data will be used for genetic mapping in the next reporting period. Additional populations (at the BC1 and BC1S1 stage) were selfed to advance another generation and be phenotyped to assess their genetic diversity. - Genetic comparison of landraces, teosinte, and modern corn genotypes Single nucleotide polymorphism (SNP) data from 527 landraces, teosinte, and exPVP corn inbred DNA samples were obtained and prepared for further analyses. We compared the landraces to the corn inbreds and carried out a phylogenetic analysis, revealing the genetic distance among these genotypes. The landrace genotypes we use to develop genetic mapping populations fall among different branches of the phylogenetic tree, supporting the observation that the aerial root traits of interest are found in genetically diverse accessions. Whole-genome sequencing of a GRIN landrace accession was completed, and genome resequencing using a bioinformatics pipeline was initiated. The landrace reference sequence will facilitate genetic comparison of landrace genotypes with the corn (Zea mays) reference genome and related species teosinte (Zea mays ssp mexicana and Zea mays ssp parviglumis). Additionally, RNAseq reads previously generated in this study will be mapped to our landrace reference sequence. - Genetic population development and evaluation Four F2 populations are being developed to examine the genetic basis of aerial root diameter and the number of nodes with aerial roots. Two elite inbred parents and three Oaxacan landrace parents are being used, which differ in their aerial root characteristics. The genotypes differ in aerial root diameter and the number of nodes with aerial roots they produce. Developing mapping populations with these genotypes will allow us to explore the genetic basis for these traits. Two populations are currently at the F1 stage and will be self-pollinated this summer, and We will send the F2 seed to the winter nursery to obtain F2:3 families. Two other populations are at the F2 stage and will be self-pollinated this summer to generate F2:3 families. All F2:3 families will be genotyped and phenotype for traits of interest in multiple locations in the summer of 2023. Goal 3: Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. We obtained and identified via 16S amplicon sequencing greater than eighty-five unique environmental isolates from the mucilage, including diazotrophs. These isolates were screened for plant-growth-promoting traits like nitrogen-fixation, auxin, siderophore production, organic and inorganic phosphate solubilization, and ACC deaminase activity. We wanted to develop a more refined artificial mucilage media with more suitable carbon sources. We quantified the levels of succinate, sucrose, maltose, and lactose in the mucilage, which are common carbon sources added to diazotrophic media. While not present at high levels, these carbon sources were present in the mucilage as free sugars, expanding our knowledge of the carbon sources present in mucilage. We also quantified microelements iron and molybdenum levels, ions that serve as cofactors in the nitrogenase enzyme. The artificial mucilage media will be supplemented with these carbon and microelement sources. At the start of this project, we determined through untargeted metabolomics that there are many more metabolites present than just monosaccharides in the mucilage. We continued these metabolomic studies by looking at the effect of environmental factors such as soil nitrogen levels.In 2020-2021 we performed a 16S survey in mucilage samples to determine that we had isolated a wide diversity of microbes from mucilage. Later in 2021, we developed a better DNA extraction protocol from the mucilage and performed 16S, nifH, and ITS meta-amplicon sequencing studies between microbial communities in nitrogen treatment, genotype, and location. These data are currently being analyzed. Goal 4: Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. To investigate the effect of inoculation with diazotrophs on nitrogen fixation, a field study was carried out at Hancock Agricultural Research Station, WI, in 2021. Three elite inbreds and four Oaxacan-derived landraces were planted in a randomized split-plot design with 6 replications per treatment (non-inoculated and inoculated).Plots were side-dressed with labeled 15N ammonium sulfate fertilizer five times throughout the growing season to allow for quantification of nitrogen fixation using the 15N dilution method. The number of nodes with aerial roots was recorded on 3 randomly chosen plants per plot at 10, 14, and 17 weeks after planting. At 10, 14, and 17 weeks after planting, one landrace genotype had significantly more nodes with aerial roots than all other genotypes. Further, by 17 weeks after planting, that landrace genotype had 7 nodes with aerial roots on average, whereas other landrace genotypes averaged less than 4.5 and the exPVPs less than 3. Leaf samples were taken from 5 randomly chosen plants per plot, twice before aerial root formation (at 4 and 6 weeks after planting) and twice more (at 12 and 16 weeks after planting) approximately one week after each inoculation. Dried leaf samples were ground and processed for isotope ratio mass spectrometry (IRMS) analysis, providing d15N values for quantification of nitrogen fixation. The data are still being analyzed.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Va?nia Carla Silva Pankievicz; Pierre-Marc Delaux; Hayley H. Hirsch; Shanmugam Rajasekar; Valentina Infante; Pablo Zamora; Dhileepkumar Jayaraman; Claudia Irene Calderon; Alan Bennett; and Jean-Michel Ane?. Nitrogen fixation and mucilage production on maize aerial roots is controlled by aerial root development and border cell functions. Frontiers in Plant Sciences.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Jean-Michel Ane?. Associative nitrogen-fixation for cereal crops: old challenges and new opportunities. European Nitrogen Fixation Conference
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Jean-Michel Ane?. Associative nitrogen-fixation for cereal crops: old challenges and new opportunities. International Symposium for the 40th Anniversary of CCG/CIFN, UNAM.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Jean-Michel Ane?. Discoveries to Improve Biological Nitrogen Fixation in Cereals. Cell Presss webinar.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Jean-Michel Ane?. A New Model for Associative Nitrogen Fixation in Cereal Crops. 25th North American Nitrogen Fixation Conference. Madison, WI
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: V�nia Carla Silva Pankievicz; Pierre-Marc Delaux; Hayley H. Hirsch; Shanmugam Rajasekar; Valentina Infante; Pablo Zamora; Dhileepkumar Jayaraman; Claudia Irene Calderon; Jennifer Wilker; Alan Bennett; and Jean-Michel An�. Nitrogen fixation and mucilage production on maize aerial roots is controlled by aerial root development and border cell functions. Poster at European Nitrogen Fixation Conference.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Jennifer Wilker, Jason Wallace, Natalia de Leon, Claudia Irene Calder�n, and Jean-Michel An�. Genetic Control of Maize Aerial Node Root Number and Diameter. Poster at North American Symbiotic Nitrogen Fixation Conference. Madison, WI
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Jennifer Wilker, Jason Wallace, Natalia de Leon, Claudia Irene Calder�n, and Jean-Michel An�. Genetic Control of Node Number and Root Diameter in the Aerial Nodes of Maize. Poster at North American Plant Breeders association conference. Ames, IA

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:1. We trained undergraduate students from diverse backgrounds to form the next generation of plant scientists. The PI's laboratories organize weekly lab meetings to discuss scientific topics related to the projects. Also, small group meetings occur every week. Specific meetings are dedicated to this project where students brainstorm ideas and discuss their scientific results and problems. This has contributed to their career development and their strong foundation while fueling their desire to learn about practical scientific applications in agriculture. We also trained undergraduates in the background, methods, and data collection specific to this project. 2. During a " plant breeding practicum " mini-class, graduate students from diverse backgrounds were exposed to the project background and goals of our project during a "plant breeding practicum" mini-class. They were trained in basic sample collection and carried out a preliminary sampling and data analysis on these materials, including a discussion of its implications. 3. Middle-school students were exposed to science knowledge and experience through outreach activities, including a summer science camp segment. To manage the outreach activities, a group of postdocs in the lab and the PI developed a plan of teaching activities about nitrogen fixation in agriculture. 4. The nitrogen fixation scientific community is a specific target audience for this project. Over the last decades, different approaches have been proposed to solve the nitrogen problem in non-leguminous crops and cereals in particular. The information obtained in our project will answer long-term questions about nitrogen fixation in cereals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided professional development opportunities for several undergraduate students: Quinlan Kiefer has been assisting Dr. April MacIntyre in screening mucilage isolates and DNA extractions for 16S studies. Bella Rupnick, Caitlin McLimens, and Keeley Kuru have been assisting Dr. Jennifer Wilker and Valentina Infante with laboratory, greenhouse, and field studies. This project also supported two postdoctoral researchers: Dr. April MacIntyre is a microbiologist in Dr. Ané's lab. Dr. MacIntyre is presenting a poster introducing the microbial side of the project at the 2021 UW-Madison Raper Symposium, as well as at the 2021 European Nitrogen Fixation Conference. Additionally, Dr. MacIntyre is currently training an undergraduate student to phenotype the plant-growth-promoting traits of mucilage bacterial isolates. Dr. Jennifer Wilker is a plant breeder and geneticist in Dr. Ané's lab. Dr. Wilker is presenting a poster summarizing the nitrogen fixation and mucilage production function of border cells at the 2021 European Nitrogen Fixation Conference. How have the results been disseminated to communities of interest?Results are made available to the nitrogen fixation and scientific community through the manuscript submitted to the Molecular Plant-Microbe Interactions (MPMI) journal. Dr. Ane also gave several presentations on this topic as indicated earlier. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments. It is widely assumed by the maize scientific community that brace and aerial root production ceases once maize plants transition from the juvenile vegetative to the adult vegetative stage. However, we hypothesize that landrace maize genotypes with high numbers of nodes with aerial roots continue to produce these roots after the juvenile vegetative stage. An experiment will be carried out to determine whether aerial roots are produced by Oaxacan landraces beyond the juvenile vegetative stage. Three conventional maize varieties and three Oaxacan-derived maize accessions will be planted in the greenhouse in a randomized complete block design with 3 replicates. The plant growth stage will be monitored through the observation of morphological indicators of the transition between juvenile and adult stages. These morphological indicators will include 1) leaf cuticular wax production, 2) toluidine blue (TBO) staining of leaf epidermis, and 3) leaf macro hair production. Additionally, the number of nodes with aerial roots will be recorded weekly, and the days to tasseling and silking will be calculated. Experience with controlled-environment trials in recent years has indicated that mucilage production occurs in high-humidity conditions. Greenhouse growing conditions will continue to be modified in order to optimize mucilage production. Goal 2: Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. Double haploids: Phenotypic evaluation of double haploid (DH) lines in the field will be carried out if the seed is provided in time for the summer field season. Genetic comparison of landraces, teosinte, and modern corn genotypes: We will use the genomic data (SNPs) obtained this year to compare the 527 landrace and teosinte lines to 302 modern corn inbred genotypes ("Goodman panel") using the corn reference genome and our landrace sequence. We will use TASSEL and fastSTRUCTURE software to develop a phylogenetic tree and determine the genetic distance of landraces from modern genotypes. Genetic population development and evaluation: Mapping populations will be advanced in the winter nursery (Tuniche, Chile) where the F2 plants will be grown and selfed. The F2:3 seed will be planted in Madison, Wisconsin for field evaluation in the summer of 2022. EMS mutant population development: A population of EMS mutants will be generated using an elite inbred with thick aerial roots. The mutant population will be used to screen for mutations affecting this trait. Goal 3: Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. We are planning to evaluate the enzymatic activities of enzymes involved in mucilage degradation as described in the proposal. Goal 4: Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. In addition to wet-lab experiments for characterizing mucilage and bacterial characteristics, we are planning a set of microbiome studies. These studies will 1) correlate the microbiome, mucilage viscosity, and acetylene reduction to determine which bacterial community members fix the most nitrogen and the optimal viscosity for nitrogen-fixing behavior, 2) compare the differences in microbial community composition between corn grown in Georgia vs. Wisconsin and 3) determine the origin of bacterial community members (air vs. soil vs. rain) with implications for future methods of introducing synthetic communities to the field. For the diazotroph inoculation experiment, leaf samples will continue to be prepared and sent for isotope analysis. Phenotypic data will be analyzed and levels of nitrogen fixation will be determined for each genotype and inoculation treatment. This experiment will be replicated next year, depending on the results obtained this season.

Impacts
What was accomplished under these goals? Goal 1: Determine the environmental factors affecting aerial root development and mucilage production using greenhouse experiments. Greenhouse experiments this year investigated the effect of nitrogen fertilizer rate on aerial root traits. Our objective was to quantify the effect of different nitrogen fertilization levels on the aerial root development of maize plants in a controlled greenhouse setting. Three Oaxacan-derived landrace maize accessions were selected based on the high number of nodes with aerial roots observed in the field in 2019. Three nitrogen levels (low, medium, and high) were tested using Hoagland's solution as the nitrogen source. There were three replicates per fertilizer treatment, and the number of nodes with aerial roots was recorded on three different dates. The number of roots per node, the diameter of roots per node, stalk diameter, and plant height was recorded after the plants flowered. We found that the number of nodes with aerial roots ranged from 2 to 6. The high nitrogen treatment had significantly more nodes with aerial roots than the medium (p=0.0227) and low (p=0.0074) treatments, while there was no significant difference in the number of nodes with aerial roots between low and medium nitrogen treatments. There was no significant effect of the nitrogen treatments over the number of roots per node and the aerial root diameter, but this will be interesting to test with more replicates per treatment. Further greenhouse trials are ongoing to test and optimize greenhouse growth conditions to increase humidity and encourage mucilage production. Goal 2: Identify the genetic determinants of aerial root development, mucilage production, and associative nitrogen fixation. Double haploids Double haploid lines were initiated to be used in studies to characterize the genetic determinants of aerial root traits. Three BC1 crosses between elite inbred lines and Oaxacan landraces were chosen. Generation of these lines will take a number of months and it is expected that double haploid lines will be available in 2022 in time for either the summer or winter field season. Genetic comparison of landraces, teosinte, and modern corn genotypes Single nucleotide polymorphism (SNP) data on 527 landrace and teosinte lines was obtained in the spring. The data is being prepared for genomic analyses. Whole-genome sequencing (Novogene) of a GRIN landrace accession was also initiated. The landrace reference sequence will facilitate genetic comparison of landrace genotypes with the corn (Zea mays) reference genome, as well as related species teosinte (Zea mays ssp mexicana and Zea mays ssp parviglumis). Genetic population development and evaluation Four F2 populations are being developed to examine the genetic basis of aerial root diameter and the number of nodes with aerial roots. Two elite inbred parents and two Oaxacan landrace parents are being used, which differ in their aerial root characteristics. The genotypes differ in aerial root diameter and the number of nodes with aerial roots they produce. Developing mapping populations with these genotypes will allow us to explore the genetic basis for these traits. Two populations are currently at the F2 stage and have been self-pollinated this summer; the other two populations have just been initiated by making crosses. Backup populations previously created in Georgia by crossing Oaxacan-derived landraces with an elite inbred were advanced to the BC1S1 and BC1S2 generations. Additionally, backup populations previously created in Wisconsin by crossing Oaxacan-derived landraces with three elite inbreds were similarly advanced. These populations are not a central focus of the grant but will be used to complement the primary analyses. Goal 3: Analyze the molecular and biochemical basis of mucilage production and degradation on aerial roots in response to water. We demonstrated that mucilage production depends on root cap and border cells sensing water, as observed in underground roots. The diameter of aerial roots correlates with the volume of mucilage produced and the nitrogenase activity supported by each root. Young aerial roots produce more mucilage than older ones, probably due to their root cap's integrity and their ability to produce border cells. Transcriptome analysis on aerial roots at two different growth stages before and after mucilage production confirmed the expression of genes involved in polysaccharide synthesis and degradation. Genes related to nitrogen uptake and assimilation were upregulated upon water exposure. Altogether, our findings suggest that in addition to the number of nodes with aerial roots reported previously, the diameter of aerial roots and abundance of border cells, polysaccharide synthesis and degradation, and nitrogen uptake are critical factors to ensure efficient nitrogen fixation in maize aerial roots. Goal 4: Understand and improve the transfer of fixed nitrogen between diazotrophs and corn in aerial roots. We obtained and identified via 16S amplicon sequencing greater than eighty-five unique environmental isolates from the mucilage, including diazotrophs. All predicted diazotrophs have been screened for nitrogen-fixation using acetylene reduction assays in vitro. In addition, we confirmed that we isolated a wide diversity of microbes from the mucilage with a preliminary 16S meta-amplicon sequencing study of the communities in the mucilage. These isolates have also been screened for auxin and siderophore production, phosphate solubilization, and ACC deaminase activity which are common plant-growth-promoting traits. We have determined the average pH and viscosity of the mucilage. These values will assist us when determining what factors correlate with the highest nitrogen fixation in the mucilage, and under what conditions N is best transferred to the plant. To determine the 'nitrogen currency' of the mucilage (i.e. what form of nitrogen is available for the plant in mucilage) we quantified the nitrate and ammonium levels in the mucilage. Nitrate concentration in the mucilage is 100X greater than ammonium, suggesting 1) a strong presence of nitrifying bacteria (which were not present in the 16S meta-amplicon screen except in one mucilage sample), 2) environmental nitrate contamination, or 3) that ammonium more rapidly absorbed by the aerial roots than nitrate. The 'nitrogen currency' question deserves further study. To investigate the effect of inoculation with diazotrophs on nitrogen fixation, a field study is in progress at Hancock Agricultural Research Station, WI. Three elite inbreds and four Oaxacan-derived landraces were planted in a randomized split-plot design with 6 replications per treatment; non-inoculated and inoculated. Twice during the growing season after aerial roots had formed Azospirillum brasilense commercial inoculant (MicroAZ-IF Liquid™, TerraMax) was applied on the lower part of the stalk where brace and aerial roots had formed. Plots were side-dressed with labeled 15N ammonium sulfate fertilizer five times throughout the growing season to allow for quantification of nitrogen fixation using the 15N dilution method. Leaf samples were taken from all plots, twice before aerial root formation, and twice more approximately one week after each inoculation. Dried leaf samples will be ground and processed for isotope analysis.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Vania Carla Silva Pankievicz; Pierre-Marc Delaux; Hayley H. Hirsch; Shanmugam Rajasekar; Valentina Infante; Pablo Zamora; Dhileepkumar Jayaraman; Claudia Irene Calderon; Alan Bennett; and Jean-Michel Ane. Nitrogen fixation and mucilage production on maize aerial roots is controlled by aerial root development and border cell functions. Molecular Plant-Microbe Interactions (under review).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Jean-Michel Ane. Discoveries to Improve Nitrogen Fixation in Cereals. Bayer Crop Science. Online (September 24, 2020).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Jean-Michel Ane. Discoveries to Improve Nitrogen Fixation in Cereals. University of Manitoba. Online (October 27, 2020).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Jean-Michel Ane. Discoveries to Improve Nitrogen Fixation in Cereals. University of Nebraska Lincoln. Online (October 28, 2020).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Jean-Michel Ane. Discoveries to Improve Nitrogen Fixation in Cereals. EPA-USDA working group. Online (November 2, 2020).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Jean-Michel Ane?. Nitrogen fixation in maize landraces from Oaxaca, Mexico: from Indigenous knowledge to the Wisconsin Idea. Latin American, Caribbean, and Iberian Studies Program (LACIS). Online (February 9, 2021).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Jean-Michel Ane. Discoveries to Improve Nitrogen Fixation in Cereals. 5th Microbiome Movement  AgBioTech Summit. Online (February 22, 2021).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Jean-Michel Ane. Nitrogen Fixation on the Aerial Roots of Maize and Sorghum. Center for Genomic Sciences UNAM (Centro de Ciencias Gen�micas UNAM), Mexico. Online (August 26, 2021)