MOLECULAR AND MICROBIAL STRATEGIES TO INCREASE AVAILABILITY OF IRON AND OTHER MINERAL NUTRIENTS TO PLANT ROOTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007260
• Project Start Date: Aug 3, 2015
• Project End Date: Jul 31, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Agronomy & Horticulture

Non Technical Summary
Iron deficiency is a problem for plant growth and often results from alkaline soil pH. In Nebraska, iron deficiency is particularly common in soybean, which in certain environments can experience Iron Deficiency Chlorosis (IDC). To find ways to ameliorate IDC, the Waters, Schachtman, and Graef labs will pursue a project that involves two different strategies. One strategy will be to look for genes in plants that respond to iron and/or copper deficiencies, and/or to alkaline stress. Further study will determine whether these genes could be used to enhance iron uptake or improve growth on alkaline soils. A second strategy will be to identify soil microbes (bacteria) that are found on plant roots in alkaline soils, but not in neutral-to-acidic soils. These microbes will have the potential to modify soil conditions or root growth in such a way that nutrient availability to roots is increased. If successful, both strategies will converge on the production of transgenic plant lines with new capabilities for enhanced nutrient uptake. An additional or alternative outcome will be a microbial culture that can be applied to soils to increase nutrient uptake. This will potentially reduce the yield losses in soybean and other plant species due to iron deficiency chlorosis.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	1820	1060	100%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1060 - Biology (whole systems);

Keywords

soybean

iron deficiency chlorosis

soil microbiome

alkaline stress

Goals / Objectives
The overall objective of this work is to find solutions to the problem of low mineral availability in certain soils. One broad approach is to use the plant's own genome to find transporter genes or genes that regulate uptake processes, and then modify expression or select favorable alleles of these genes to increase mineral uptake capacity, effectively increasing mineral availability. A second broad approach is to gain understanding into how plants interact with soil microorganisms to increase nutrient uptake or to influence nutrient availability. Part of this approach is to catalog the diversity of microbial life in soils with different chemical characteristics with differing nutrient availability, and then focus on characterizing specific parts of the microbial community. Another part of this approach is to address whether genotypes of plants that are more tolerant of adverse soils (e.g. IDC tolerant and IDC susceptible soybean lines) form different associations with the microbial population. The final approach is to characterize the species diversity and specific identities of microbial species in soils with high or low iron availability. We will try to culture members of the community and use these isolates to characterize the effects of these microbes on plant nutrient uptake or performance. If microbial species are found to be beneficial, selected genomes will be sequenced to discover novel biochemical pathways or compounds produced by these bacteria.Specific Aims:Aim 1. Determine genes that have increased or decreased expression in roots in response to Fe deficiency, Cu deficiency, and alkaline stress (Waters).Aim 2. Characterize phenotypes of mutants or overexpression lines for selected genes from Aim 1 (Waters, Schachtman, Graef).Aim 3. Identify the microbial communities from soils with differing characteristics that influence nutrient availability and from plant roots growing in these soils using 16s amplicon sequencing (Waters, Schachtman, Graef).Aim 4. Isolate microbes from Goal 3 and test them for ability to improve plant growth in adverse soils (Schachtman, Graef).Aim 5. Sequence genomes of selected microbial species (Schachtman).

Project Methods
Aim 1: Datasets of root transcriptomes from Arabidopsis thaliana, melon (Cucumis melo), and soybean (Glycine max) roots will be analyzed to identify genes that are highly upregulated or downregulated in response to low Fe and/or low Cu supply. Standard bioinformatics methods have been employed to determine genes that are statistically upregulated or downregulated in each condition relative to controls. From these lists of genes, we will use GO (gene ontology) analysis to identify genes that are overrepresented relative to the genome, for categories of biological process, molecular function, and cellular compartment localization. Key genes will be those that have the greatest differential expression and/or those that are differentially expressed and coincide with previously identified QTL, and will be chosen for further analysis in Aim 2. We will also use Venn diagrams to identify genes that have specific regulatory patterns, e.g. upregulated in both roots and leaves by Fe deficiency, upregulated by multiple stress treatments, upregulated by one metal deficiency and downregulated by another. Genes that have expression patterns that indicate importance in increasing mineral uptake will be subjected to further analysis. Co-expression databases will be queried to identify modules of genes that are similarly regulated to provide possible links between genes and processes to identify networks or metabolic/physiological pathways.Aim 2: Genes that are highly responsive to Fe and/or Cu deficiency, and/or bicarbonate, and that respond similarly in all species will be of high interest. For a subset of these key genes, we will order T-DNA knockout lines from the Arabidopsis Biological Resource Center, and generate overexpression lines in Arabidopsis. These lines will be tested for altered responses to Fe and Cu deficiencies and altered growth on bicarbonate. Genes that cause altered responses when knocked out or overexpressed will be characterized in greater detail, and we will generate soybean knockout or overexpression lines for the most promising genes. The additional characterization methods will depend on the type of gene. We will also test the effects of microbes discovered in later Aims on the knockout and overexpression lines. If there are genes that respond to low Fe and/or bicarbonate treatment only in soybean, and differently between the IDC susceptible and tolerant lines, these will be of special interest. In this case, we will seek funding for studies to follow up on a limited number (two to three) of the genes by making overexpression soybean lines. We will also search for knockouts in our genes of interest in soybean mutant databases as they become available. We will test mutant and overexpression lines for genes of interest for altered responses to Fe deficiency and bicarbonate treatmentAim 3: Two different types of sites will be identified in Nebraska for the collection of root, rhizosphere and bulk soil samples; from natural ecosystems in the sand hills of Nebraska and from agricultural soils that differ in incidence of soybean IDC. Both types of sites contain soils that have high alkalinity. Soybean roots will be collected from alkaline and neutral pH sites at two times during the season. Plant samples of selected species will be collected from land bordering sandhill lakes that have high pH. If time and funding permits, roots from the same plant species present will be collected from the shores of alkaline and non-alkaline lakes. The soil surrounding the roots will be collected. Roots will be placed into a phosphate buffer with a surfactant, and the rhizosphere soil will be washed off the roots. Finally the washed roots will be sterilized with bleach and ethanol to identify the microbial taxa that are located in the endosphere of the roots. DNA will be extracted from these samples. The V4 region of the 16S genes will be amplified to classify the microbial taxa that comprise these different samples. We expect to identify taxa of bacterial species that are specific to soils and rhizosphere samples that differ across the pH and bicarbonate concentrations at different sites. This research will address whether plants grown on different soil types have different root microbiomes, and if so, may provide clues to which specific bacterial species aid plant growth in high pH soils by improving the acquisition of nutrients.Aim 4: Soil and plant samples collected in Aim 3 will be used to culture the bacteria present in those samples. Bulk soil, rhizosphere soil and roots will be used to culture bacteria. For soybean we will culture bacteria from IDC resistant and susceptible varieties/lines from alkaline sites and non-alkaline sites. Metadata will be collected on the chemical analyses for soils and visual symptoms of the plants. Bacteria will be extracted from soils and roots and then plated onto 5 - 7 different types of media to promote the growth of different microbes including low nitrogen media to enrich for nitrogen fixing bacteria. Note that this is a new area of research, and culture protocols are still being developed. We acknowledge that likely only a small fraction of the total microbiome will be culturable. The bacterial colonies that grow on plates will be characterized to determine the genus and species by sequencing the 16s gene using standard Sanger sequencing methods. A sterile potting mix or soil will be developed where sodium bicarbonate or calcium oxide will added to increase pH. Individual microbes or consortia of microbes will then be added to the pots in which various accessions or varieties, mutants, or overexpression lines of Arabidopsis or soybean are growing. We will test polymers mixed with freshly grown bacterial cultures to adhere the microbes to the seed to optimize this seed treatment process. All coatings will be done immediately before planting with freshly grown bacteria. Depending on the success of seed treatments, we may also apply bacterial mixtures using a sprayer to the base of each plant just following germination. These plants will be assessed for symptoms of IDC to determine which strains or which consortia improve growth on alkaline soils or reduce the incidence of IDC. If we are unable to culture important microbes from roots or soils we will take a backup approach of soaking bacteria out of soils to create different bacterial teas from different soils. This bacterial tea will then be reapplied to plants growing in a sterile environment to determine whether the bacterial consortia that come from different soils have any effect on ameliorating iron deficiency symptoms for plants grown under alkaline conditions.Aim 5: If we find microbes with unique features in Aim 3 and are able to culture these microbes in Aim 4 these bacteria will be genome sequenced using both Illumina HiSeq and Pacific Biosystems methods. The PacBio system provides longer sequencing reads to more easily assemble the shorter HiSeq reads. The goal of the sequencing will be to identify unique iron acquisition mechanisms or metabolic pathways for the production of unique siderophores or reduction mechanisms that could be used to aid crop plants in the acquisition of iron. This may be done through studies on gene expression or specific genes may be identified through comparative genomics or through identification of homologues of already known genes encoding siderophores, iron reductases and/or ion transporters. In the long term, if we aim to identify unique microbes and test them directly on plants (in pot experiments as above) to aid in increasing iron acquisition. As a follow-up to this project, we will work toward a potentially more reliable approach to identify microbial genes that enhance plant growth and nutrient availability and engineer those into crop plant roots. This process would potentially create new plant based mechanisms for increasing the ability of roots to take up iron or other nutrients from alkaline soils.

Progress 10/01/19 to 07/31/20

Outputs
Target Audience: Nothing Reported Changes/Problems:A Final Report was submitted and approved in November of 2022 What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? N/A

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Dr. Waters has separated from UNL

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience is agricultural and biological scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student presented research findings at a professional conference. Four undergraduates and one graduate student have been trained during 2017 in this project. How have the results been disseminated to communities of interest?Two journal publications; one in Frontiers in Plant Science and one in Metallomics. Six conference presentations or seminars. What do you plan to do during the next reporting period to accomplish the goals?Soybean and arabidopsis results from Aim 1 will be published in peer-reviewed journals. Materials for further study will be generated for Aim 2. For Aim 3 the next steps are to analyze the sequencing data to make adjustments and appropriate experimental plans for the 2019 field season. For Aim 4, we plan to test the microbial isolates for their ability to improve plant growth in adverse soil conditions. For Aim 5, we plan to sequence genomes of selected microbes using information from Aim 4.

Impacts
What was accomplished under these goals? One of the impacts of this work will be a deeper understanding of how alkalinity stress interacts with or causes iron deficiency chlorosis (IDC) in plants. A second impact will be further understanding of how interactions between Fe deficiency and copper (Cu) deficiency could influence nutrient uptake. This knowledge could lead to improved crop varieties with greater IDC tolerance. Another impact will be an understanding of how the soil microbiome differs between plant species or varieties within a species as related to IDC tolerance. Another impact will be an understanding of how different microbial populations influence Fe and other mineral uptake. This knowledge could lead to microbial soil treatments that improve mineral nutrient uptake in crops. Aim 1. Determine genes that have increased or decreased expression in roots in response to Fe deficiency, Cu deficiency, and alkaline stress (Waters, Hyten). We have been performing this Aim using three plant species; melon (Cucumis melo), Arabidopsis thaliana, and soybean (Glycine max). For melon, we have performed RNA sequencing on roots of wild-type plants and the fefe mutant, in which Fe uptake responses are blocked upstream of Fe uptake genes. We are writing a manuscript for publication of these results. For Arabidopsis, we analyzed gene expression data from a wild-type variety and a copper uptake mutant, in both roots and leaves, under Fe deficiency, Cu deficiency, or simultaneous Fe and Cu deficiencies. This work has led to further understanding of the interactions between these elements and their effects on regulation of nutrient uptake. A key finding was a family of bHLH transcription factors that control Fe uptake when plants were Fe deficient or Cu deficient were oppositely regulated by Fe and Cu deficiencies. This family of bHLH transcription factors are homologous to the melon fefe gene. This work was published in Metallomics in 2018. For soybean, we have analyzed gene expression data from one IDC tolerant and one IDC sensitive variety. Roots were treated with Fe deficiency, alkalinity stress, or a combination of these treatments. A key finding was the upregulation of the phenylpropanoid synthesis pathway by both Fe deficiency and alkalinity stress. The activity of this pathway likely produces fluorescent root exudates that may be involved in Fe uptake or may interact with the soil microbiome. The IDC tolerant variety produces greater quantities of these compounds. This work was published in Frontiers in Plant Science in 2018. We developed a genotyping-by-sequencing method for soybean that can be used to genotype IDC mapping populations. This method uses a panel of 1000 single nucleotide polymorphisms (SNPs) to genotype recombinant inbred line populations. These 1000 SNPs will produce enough polymorphic markers in any mapping population for QTL discovery. We have also developed a method to produce high-density SNP data on a panel of diverse accessions. Soybean has over ten million markers, but currently only 50,000 markers are available for genome-wide association study (GWAS) and they only capture about 41% of the genetic diversity. We developed methods to sequence accessions at a 1x coverage followed by whole genome imputation to obtain high quality marker genotyping on over ten million markers. This method will enable all the variation to be captured as we perform GWAS for IDC tolerance. Aim 2. Characterize phenotypes of mutants or overexpression lines for selected genes from Aim 1 (Waters, Schachtman, Graef). We have selected specific soybean genes for overexpression, some of which are predicted to be transporters that may efflux fluorescent compounds from roots, and some of which are predicted to be transcription factors that may upregulate the phenylpropanoid synthesis pathway. These genes are more highly expressed under Fe deficiency in the IDC tolerant variety. In Arabidopsis, we have a triple mutant for the transcription factor family of interest, but need to knock out one additional gene to have material for study. We have identified putative quadruple mutants. Aim 3. Identify the microbial communities from soils with differing characteristics that influence nutrient availability and from plant roots growing in these soils using 16s amplicon sequencing (Waters, Schachtman, Graef). To study how the microbial communities on soybean plants differ based on their ability to grow on alkaline soils a field experiment was conducted. Valley, Nebraska was selected as the field site because it has alkaline sections in different fields. Eight genotypes were grown in neutral pH and high pH areas of the Valley field site. The alkaline area of the field had an average pH of 8.42 while the neutral area had an average pH of 7.76. The eight genotypes were selected from a previously scored IDC population based on their sensitivity/tolerance to high pH. Four replicates were grown in neutral and high pH soil. Roots, rhizosphere, and soil samples were collected at three soybean growth stages V4 (vegetative), R3 (pod initiation), and R5 (seed initiation). Roots and rhizosphere were collected from every plot while five samples of soil were collected from the neutral and high pH areas of the field. Extracted DNA was sent for Illumina MiSeq sequencing of the 16s rDNA gene. In 2017 we completed a power analysis which indicated that we would need 8 - 10 replicates to successfully see the genotypic difference more clearly. Therefore 10 reps were harvested and we are now working on processing 8 reps to send for sequencing. In North Bend in 2018, the plots were either lost due to flooding in the early season and showed generally poor visible symptoms indicating that some environmental factor reduced the severity of the stress. Aim 4. Isolate microbes from Goal 3 and test them for ability to improve plant growth in adverse soils (Schachtman, Graef). A culture collection of key microbes in the alkaline soils has been developed. These isolates will be directly applied to future experiments to test plant-microbial interactions and possible amelioration of the symptoms due to soil alkalinity. Soil, roots and rhizosphere from the Sandhills were cultured. Isolates were stored in glycerol and the 16s gene was amplified using the 27F primer for each isolate to determine the species of each isolate. In total, over 1000 isolates have been isolated and 490 sequenced from roots, rhizosphere and soil from the Sandhills and from the North Bend alkaline site collected in 2016 and 2017. The sequencing of these isolates continues along with a more concentrated effort on the microbes that we can culture on high pH plates. We see interesting shifts in phylum representation of culturable microbes at more alkaline pH. There are a higher percentage of Firmicutes at high pH in the culturable fraction. Aim 5. Sequence genomes of selected microbial species (Schachtman). About 30 isolates were sent to JGI for sequencing. Many of these are sequenced and we are currently analyzing the data and doing comparative analyses.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ramamurthy RK, Xiang Q, Hsieh E-J, Liu K, Zhang C, Waters BM (2018) New aspects of Fe-Cu crosstalk uncovered by transcriptomic characterization of Col-0 and the copper uptake mutant spl7 in Arabidopsis thaliana. Metallomics DOI: 10.1039/C8MT00287H
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Waters BM, Amundsen K, Graef G (2018) Gene expression profiling of iron deficiency chlorosis sensitive and tolerant soybean indicates key roles for phenylpropanoids under alkalinity stress. Frontiers in Plant Science 9:10
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hyten Jr, D. (Presenter & Author), Wang, H. (Author Only), Happ, M. (Author Only), McConaughy, S. (Author Only), Curtolo, M. (Author Only), Amundsen, K. (Author Only), Posadas, L. (Author Only), Lorenz, A. (Author Only), Song, Q. (Author Only), Graef, G. (Author Only), The Plant & Animal Genome XXVI conference, San Diego, CA, "Development of Sequencing for Genotyping within Soybean Breeding Programs"
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hyten Jr, D., 2018 Soybean Breeder's Workshop, St. Louis, MO, "Development of sequencing for genotyping within soybean breeding programs", Research/Creative Activity, Workshop, National, Invited. (February 12, 2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hyten Jr, D., University of Guelph Department of Plant Agriculture seminar, University of Guelph, Guelph, Canada, "Development of High-throughput SNP Genotyping Technologies for Soybean"
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hyten Jr, D., VIII Brazilian Soybean Conference, Brazilian Soybean Congress, Goi�nia, Goi�s State, Brazil, "Soybean Breeding  New tools, challenges and future"
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Happ, M. (Author Only), Wang, H. (Author Only), Graef, G. (Author Only), Hyten Jr, D. (Presenter & Author), 17th Biennial Conference on the Molecular and Cellular Biology of the Soybean, Athens, GA, "Generating high density, low cost genotype data in soybean"
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: McHale, L. (Presenter & Author), Lorenz, A. (Author Only), Graef, G. (Author Only), Rainey, K. M. (Author Only), Beavis, W. (Author Only), Hyten Jr, D. (Author Only), Hudson, M. (Author Only), Clough, S. (Author Only), Ma, J. (Author Only), Campbell, B. (Author Only), Chen, P. (Author Only), Diers, B. (Author Only), Scaboo, A. (Author Only), Schapaugh, W. (Author Only), Singh, A. (Author Only), Wang, D. (Author Only), 17th Biennial Conference on the Molecular and Cellular Biology of the Soybean, Athens, GA, "Increasing the rate of genetic gain for yield in soybean breeding programs"

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience is agricultural and biological scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four undergraduates and one graduate student have been trained during 2017 in this project. How have the results been disseminated to communities of interest?Results for melon fefe mapping were published in Frontiers in Plant Science in 2017. What do you plan to do during the next reporting period to accomplish the goals?Soybean and arabidopsis results from Aim 1 will be published in peer-reviewed journals. Materials for further study will be generated for Aim 2. For Aim 3 the next steps are to analyze the sequencing data to make adjustments and appropriate experimental plans for the 2018 field season. For Aim 4, we plan to test the microbial isolates for their ability to improve plant growth in adverse soil conditions. For Aim 5, we plan to sequence genomes of selected microbes when we have appropriate information from Aim 4.

Impacts
What was accomplished under these goals? One of the impacts of this work will be a deeper understanding of how alkalinity stress interacts with or causes iron deficiency chlorosis (IDC) in plants. A second impact will be further understanding of how interactions between Fe deficiency and copper (Cu) deficiency could influence nutrient uptake. This knowledge could lead to improved crop varieties with greater IDC tolerance. Another impact will be an understanding of how the soil microbiome differs between plant species or varieties within a species as related to IDC tolerance. Another impact will be an understanding of how different microbial populations influence Fe and other mineral uptake. This knowledge could lead to microbial soil treatments that improve mineral nutrient uptake in crops. Aim 1. Determine genes that have increased or decreased expression in roots in response to Fe deficiency, Cu deficiency, and alkaline stress (Waters, Hyten). For melon, in roots of the fefe mutant, in which Fe uptake responses are blocked upstream of Fe uptake genes, alkaline stress or Fe deficiency up-regulation of certain Fe uptake and riboflavin synthesis genes was inhibited, indicating a central role for the FeFe protein. These implicate shoot-to-root signaling of Fe status to induce Fe uptake gene expression in roots. In 2017, we published an article in Frontiers in Plant Science that identified the fefe gene as a bHLH transcription factor called bHLH38. For Arabidopsis, we have analyzed gene expression data from a wild-type variety and a copper uptake mutant, in both roots and leaves, under Fe deficiency, Cu deficiency, or simultaneous Fe and Cu deficiencies. This work has led to further understanding of the interactions between these elements and their effects on regulation of nutrient uptake. A key finding was a family of bHLH transcription factors that control Fe uptake when plants were Fe deficient or Cu deficient were oppositely regulated by Fe and Cu deficiencies. This family of bHLH transcription factors are homologous to the melon fefe gene. For soybean, we have analyzed gene expression data from two varieties, one tolerant to IDC and one sensitive to IDC. A manuscript for this work was under review at the end of 2017. The results are from roots treated with Fe deficiency, alkalinity stress, or a combination of these treatments. A key finding was upregulation of the phenylpropanoid synthesis pathway by both Fe deficiency and alkalinity stress. The activity of this pathway likely produces fluorescent root exudates that may be involved in Fe uptake or may interact with the soil microbiome. The IDC tolerant variety produces greater quantities of these compounds. To test a selected set of genes we are developing and testing molecular inversion probes. This sequencing method can potentially be adapted to target a specific set of 4000 genes on a larger number of replicates to increase statistical power. Aim 2. Characterize phenotypes of mutants or overexpression lines for selected genes from Aim 1 (Waters, Schachtman, Graef). We have selected specific soybean genes for overexpression, some of which are predicted to be transporters that may efflux fluorescent compounds from roots, and some of which are predicted to be transcription factors that may upregulate the phenylpropanoid synthesis pathway. These genes are more highly expressed under Fe deficiency in the IDC tolerant variety. In Arabidopsis, we have a triple mutant for the transcription factor family of interest, but need to knock out one additional gene to have material for study. We are in the process of using a CRISPR-Cas9 system to make this mutation, since it cannot be done by crossing due to tandem positioning of two of the gene loci. Aim 3. Identify the microbial communities from soils with differing characteristics that influence nutrient availability and from plant roots growing in these soils using 16s amplicon sequencing (Waters, Schachtman, Graef). To better understand interactions between microorganisms and plant root systems in alkaline soil ecosystems, a field experiment was conducted in the Sandhills of Nebraska. The Sandhills provide a unique environment to study microbial and plant root system interactions within alkaline soils. The samples collected from 2016 were analyzed and the general conclusions were: significant differences were present in microbial communities of all sample types associated with pH difference and plant species; significant differences in microbial communities were found at each pH across the 3 locations; there were non-uniform significant differences in microbial communities between the 3 locations. In 2017 sites were sampled twice - once in mid May and in late June, three sites were sampled in Grant County, Nebraska. Site pH ranged 6.7 - 10.1, 7.1 - 9.6, and 6.95 - 10. In total, 336 samples were collected . The roots, rhizosphere, and soil from each sample were collected, processed, and stored on ice for processing. Four species of plants were sampled. The roots, rhizosphere, and soil were processed using standard lab protocols. Extracted DNA was cleaned up, quantified with a quantiflour protocol, and sent for Illumina MiSeq sequencing of the 16s rDNA gene. To study how the microbial communities on soybean plants differ, Glycine max alkalinity tolerant and sensitive soybean lines and 6 progeny lines were chosen. The field site is known to have alkaline sections. Eight genotypes were grown in neutral pH and high pH areas. The alkaline area had an average pH of 8.42 while the neutral area had an average pH of 7.76. Roots, rhizosphere, and soil samples were collected at three soybean growth stages V4 (vegetative), R3 (pod initiation), and R5 (seed initiation). Roots and rhizosphere were collected from every plot while 5 soil samples were collected from the neutral and high pH areas. The roots, rhizosphere, and soil were processed using standard lab protocols. DNA was extracted and sent for Illumina MiSeq sequencing of the 16s rDNA gene. We also planted the soybean lines at North Bend, NE in a field that in the past showed stronger alkaline soil effects on soybean. This field was severely damaged by hail early in the season in 2017. Only a few replicates in the alkaline treatment survived and those samples were collected, DNA was extracted and sent for amplicon sequencing. Aim 4. Isolate microbes from Goal 3 and test them for ability to improve plant growth in adverse soils (Schachtman, Graef). A culture collection has been developed that seeks to culture the key microbes in the alkaline soils. These isolates will be directly applied to future experiments to test plant-microbial interactions and possible amelioration of the symptoms due to soil alkalinity. Soil, roots and rhizosphere from the Sandhills were cultured on R2A, PDA, and TSA media. Roots were washed in phosphate buffer before plating and two replicates were combined, and then cut into 1 - 2 cm pieces and plated. Isolates were stored in glycerol and the 16s gene was amplified using the 27F primer for each isolate to determine the species of each isolate. In total, over 1000 isolates have been isolated and 490 sequenced from roots, rhizosphere and soil from the Sandhills and from the North Bend alkaline site collected in 2016 and 2017. Aim 5. Sequence genomes of selected microbial species (Schachtman). We have not started on this Aim yet.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kastoori Ramamurthy, R and Waters, BM (2017) Mapping and characterization of the fefe gene that controls iron uptake in melon (Cucumis melo L.). Frontiers in Plant Science 8:1003

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience is agricultural and biological scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One undergraduate and one graduate student have been trained during this project. Waters had professional development by participating in the PAG meeting and visits to other universities to present seminars. How have the results been disseminated to communities of interest?Waters made a conference presentation at the Plant and Animal Genome meeting Legumes Workshop to an audience of scientists. Waters gave two seminars to an audience of scientists (faculty, postdocs, and graduate students); one at Lehman College in New York, and one at Korea University in Seoul, South Korea. What do you plan to do during the next reporting period to accomplish the goals?Results from Aim 1 will be pursued further and published in a peer-reviewed journal. Materials for further study will be generated for Aim 2. For Aim 3 the next steps are to analyze the sequencing data to make adjustments and appropriate experimental plans for the 2017 field season. One potential option is to add a soybean mutant (nodule-less) with little to no nitrogen-fixing bacteria to the genotypes planted in Valley, NE. This would provide a comparison of the microbial communities present in soybean root systems with and without nitrogen fixing bacteria. No cultures were made with the soybean roots. For Aim 4, we plan to test the microbial isolates for their ability to improve plant growth in adverse soil conditions. For Aim 5, we plan to sequence genomes of selected microbes when we have appropriate information from Aim 4.

Impacts
What was accomplished under these goals? One of the impacts of this work will be a deeper understanding of how alkalinity stress interacts with or causes iron deficiency chlorosis (IDC) in plants. A second impact will be further understanding of how interactions between Fe deficiency and copper (Cu) deficiency could influence nutrient uptake. This knowledge could lead to improved crop varieties with greater IDC tolerance. Another impact will be an understanding of how the soil microbiome differs between plant species or varieties within a species as related to IDC tolerance. Another impact will be an understanding of how different microbial populations influence Fe and other mineral uptake. This knowledge could lead to microbial soil treatments that improve mineral nutrient uptake in crops. Aim 1. Determine genes that have increased or decreased expression in roots in response to Fe deficiency, Cu deficiency, and alkaline stress (Waters, Hyten). We have been performing this Aim using four plant species; cucumber (Cucumis sativus), melon (Cucumis melo), Arabidopsis thaliana, and soybean (Glycine max). For cucumber and melon, we studied expression of specific Fe deficiency responsive genes to test whether they are also regulated by alkalinity stress in leaves and roots. These genes are involved in Fe uptake or riboflavin synthesis. An outcome of this work was a publication in the Journal of Experimental Botany. Alkaline stress abolished the normal root response to low Fe supply. Riboflavin synthesis genes responded to Fe deficiency, but not alkalinity. Iron deficiency increased expression of some common genes in roots and leaves, but up-regulation of these genes was blocked in Fe-deficient leaves by alkaline stress. In roots of the melon (Cucumis melo L.) fefe mutant, in which Fe uptake responses are blocked upstream of Fe uptake genes, alkaline stress or Fe deficiency up-regulation of certain Fe uptake and riboflavin synthesis genes was inhibited, indicating a central role for the FeFe protein. These implicate shoot-to-root signaling of Fe status to induce Fe uptake gene expression in roots. For Arabidopsis, we have analyzed gene expression data from a wild-type variety and a copper uptake mutant, in both roots and leaves, under Fe deficiency, Cu deficiency, or simultaneous Fe and Cu deficiencies. This work has led to further understanding of the interactions between these elements and their effects on regulation of nutrient uptake. A key finding was a family of transcription factors that control Fe uptake when plants were Fe deficient or Cu deficient were oppositely regulated by Fe and Cu deficiencies. For soybean, we have analyzed gene expression data from two varieties, one tolerant to IDC and one sensitive to IDC. This data is from roots treated with Fe deficiency, alkalinity stress, or a combination of these treatments. A key finding was the upregulation of the phenylpropanoid synthesis pathway by both Fe deficiency and alkalinity stress. The activity of this pathway likely produces fluorescent root exudates that may be involved in Fe uptake or may interact with the soil microbiome. The IDC tolerant variety produces greater quantities of these compounds. To test a selected set of genes with more biological replication we have begun developing and testing molecular inversion probes. This sequencing method can potentially be adapted to target a specific set of 4000 genes on a larger number of replicates to increase statistical power. Aim 2. Characterize phenotypes of mutants or overexpression lines for selected genes from Aim 1 (Waters, Schachtman, Graef). We have selected specific soybean genes for overexpression, some of which are predicted to be transporters that may efflux fluorescent compounds from roots, and some of which are predicted to be transcription factors that may upregulate the phenylpropanoid synthesis pathway. These genes are more highly expressed under Fe deficiency in the IDC tolerant variety. We have not yet begun to make the overexpression lines. In Arabidopsis, we have a triple mutant for the transcription factor family of interest, but need to knock out one additional gene to have material for study. We are in the process of using a CRISPR-Cas9 system to make this mutation, since it cannot be done by crossing due to tandem positioning of two of the gene loci. Aim 3. Identify the microbial communities from soils with differing characteristics that influence nutrient availability and from plant roots growing in these soils using 16s amplicon sequencing (Waters, Schachtman, Graef). To better understand interactions between microorganisms and plant roots in alkaline soil, a field experiment was conducted in the Sandhills of Nebraska, which provide a unique environment with alkaline soils. From June 15 - 16th, 2016, three sites (1 - 3) were sampled in Grant County, Nebraska. Sampling sites were chosen based on potential high pH values and accessibility. Location 1 had a pH range of 6.7 - 10.05, location 2 ranged 7.1 - 9.6, and location 3 ranged 6.95 - 10. Samples of roots, rhizosphere, and soil from each sample of nine species of plants, both monocots and dicots, were harvested. These species include Bromus inermis (Smooth Brome grass), Convalvulus arvensis (Bindweed), Carex praegracilis (Clustered field sedge), Elymus smithii (Western Wheatgrass), Hordeum jubatum (Foxtail Barley), Plantago patagonica (Woolly Plantain), Silene tatifolia (White Campion), Stipa comata (Needle and Thread grass), and Trifolium pretense (Red Clover). The roots, rhizosphere, and soil were processed using standard lab protocols. DNA was extracted, quantified, and sent for Illumina MiSeq sequencing of the 16s rDNA gene. A field experiment was conducted on Glycine max (soybean) to study microbial communities on eight soybean lines that differ in ability to grow on alkaline soils. The lines were derived from tolerant and sensitive soybean parents. The eight genotypes of soybean were selected from a previously scored IDC population based on their sensitivity/tolerance to high pH. These included two parent lines, one sensitive to IDC (U06-625083) and one tolerant to IDC (U06-105454), along with six recombinant inbred lines that displayed varying IDC scores (3 genotypes more sensitive and 3 genotypes more tolerant). The field site was in Valley, Nebraska, in a randomized block design in neutral pH and high pH areas of the field. The alkaline area had an average pH of 8.42 while the neutral area had an average pH of 7.76. Four replicates were grown in each pH. Roots, rhizosphere, and soil samples were collected at three soybean growth stages: V4 (vegetative), R3 (pod initiation), and R5 (seed initiation). Roots and rhizosphere were collected from every plot while 5 samples of soil were collected from the neutral and high pH areas of the field (64 roots + 64 rhizosphere + 10 soil samples = 138 samples total at each time point). The roots, rhizosphere, and soil were processed, and DNA was sent for Illumina MiSeq sequencing of the 16s rDNA gene (414 total). Aim 4. Isolate microbes from Goal 3 and test them for ability to improve plant growth in adverse soils (Schachtman, Graef). A culture method was developed to culture the key microbes in the alkaline soils. These isolates can be directly applied to future experiments to test plant-microbial interactions and possibly ameliorate IDC or improve nutrient uptake. Soil, roots and rhizosphere from the Sandhills were cultured on R2A, PDA, and TSA media. Roots were washed in phosphate buffer before plating and two replicates were combined, and then cut into 1 - 2 cm pieces and plated. Isolates were stored in glycerol until they were used for sequencing. The 16s gene was amplified using the 27F primer to determine the species of each isolate. In total, 357 isolates have been sequenced from the Sandhills roots collected in June 2016. Aim 5. Sequence genomes of selected microbial species (Schachtman). We have not started on this Aim yet.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Hsieh EJ and Waters BM (2016) Alkaline stress and iron deficiency regulate iron uptake and riboflavin synthesis gene expression differently in root and leaf tissue: implications for iron deficiency chlorosis. Journal of Experimental Botany 67 (19): 5671-5685
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Waters, BM. Expression Profiling of Iron Deficiency Chlorosis (IDC) in Soybean (Glycine max): Similarities and Differences Between Low Iron Supply and Alkaline Stress, Plant and Animal Genome, Legumes Workshop (2016)

Progress 08/03/15 to 09/30/15

Outputs
Target Audience:The target audience is agricultural and biological scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue working on the stated aims. This reporting period covers the first two months only, so we are basically getting strated on the proposed plan at this time.

Impacts
What was accomplished under these goals? Aim 1. We have generated RNAseq data from soybean roots treated with normal Fe or low Fe, with or without alkaline stress. We are in the process of anayzing this data. Aim 2. We have not started on this aim. Aim 3. We have sampled roots, rhizomes, and soil from an alkaline soybean field. A graduate student is characterizing the microbial populations from these samples. Aim 4. We have not started on this aim. Aim 5. We have not started on this aim.

Publications