UNDERSTANDING CRITICAL SOIL-SIDEROPHORE ANTAGONISTIC INTERACTIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022163
• Grant No.: 2020-67019-31162
• Cumulative Award Amt.: $497,668.00
• Proposal No.: 2019-06522
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2025
• Grant Year: 2020
• Program Code: [A1401]- Foundational Program: Soil Health

Project Director
Duckworth, O.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Crop & Soil Sciences

Non Technical Summary
Siderophores, chelating agents produced by fungi, bacteria, and grassy plants, play critical roles in the acquisition of iron and other trace metals and are essential to proper plant nutrition as well as the maintenance of soil quality and the suppression of plant diseases. Many studies have focused on the ability of siderophores to solubilize metals from minerals and the details of cellular uptake systems. However, for a siderophore to function, it must travel from the cell or root to an iron source and then back to the organism. Because soils are complicated, dynamic systems, antagonistic reactive agents, including soil enzymes, mineral surfaces, and soil organic matter, may disrupt this nutrient uptake process. Plants, fungi, and bacteria produce structurally distinct siderophores, and thus we anticipate specific differences in their fates and ability to deliver iron in soils. Understanding these differences will provide a new angle for managing agricultural systems so that nutrient deficiencies and crop disease susceptibility are minimized. In the proposed work, we utilize recent breakthroughs in analytical approaches to develop a cohesive analytical plan designed to gain a quantitative understanding of the reaction rates and mechanisms of important reactive soil components and whole soils with plant, fungal, and bacterial siderophores, as well as their corresponding iron complexes. The outcomes of this research will help us to better understand and control iron cycling in the root zone of crops, leading to improved yields and disease supression.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	0110	2000	75%
102	0110	1103	25%

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

Subject Of Investigation
0110 - Soil;

Field Of Science
1103 - Other microbiology; 2000 - Chemistry;

Keywords

biogeochemistry

iron

micronutrients

siderophores

Goals / Objectives
Acquisition of nutrient metals by plants, beneficial microbes, and pathogenic fungi are fundamental soil biogeochemical processes that have impacts on food security and critical ecosystem services at the global scale. Iron nutrition in plants is critical to food security and human heath, and is becoming an acute issue as the global population increases and climate change progresses. Additionally, competition for iron uptake also plays a direct role in microbial antagonism and disease suppression. Plants and rhizosphere microbes exude specific agents, including organic acids, extracellular enzymes, and siderophores (a structurally diverse group of Fe(III) chelating agents produced by fungi, bacteria, and graminaceous plants), to facilitate the solubilization and uptake of essential nutrients, especially Fe, from soil. However, because soils are complicated, dynamic systems, a myriad of antagonistic reactive agents, including soil enzymes, mineral surfaces, and soil organic matter (SOM), may disrupt nutrient uptake processes. To date, there has been little effort made to track siderophore fate (which, in principle, span a range of reactivities) in soil, a knowledge gap which limits our ability to understand how to better manage agroecosystems to inprove nutritative values of crops and limit plant disease.Our overall goal is to develop a mechanistic understanding of the antagonistic effects of reactive soil components on exudate-mediated micronutrient uptake by plants, fungi, and bacteria. This information is essential to understanding not only crop nutrition, but also the balance between beneficial and pathogenic organisms in the rhizosphere. Our specific objectives are to: (1) identify which soil components (viz. mineral surfaces, soil organic matter, and extracellular soil enzymes) have the most significant antagonistic effects on siderophore-mediated micronutrient uptake; (2) elucidate major mechanisms for reactions of siderophores, and their Fe(III)-complexes, with antagonistic soil components; and (3) verify these findings in soil. We will achieve our objectives through three independent tasks that inform each other and will be synthesized into a realistic conceptual model of siderophore activity in soils.

Project Methods
Overview. We conduct a targeted set of laboratory-based biochemical and chemical experiments that will track the fate of several different classes of microbial and plant siderophores (2'-deoxymugineic acid (DMA), desferrioxamine B (DFOB), and pyoverdin from Pseudomonas putida GB-1 (PVDGB1)). These studies will be designed to isolate and quantify the effects of specific reactive components, which will then be verified by studies in soils with a range of compositions. The quantitative kinetic and thermodynamic parameters from these experiments will be synthesized into a new conceptual model of siderophore dynamics in soils.Task 1. Quantify the effects of SOM and minerals on siderophores and Fe(III)-siderophore complex availability. We will perform sorption experiments to better understand the interactions of siderophores with important soil components, including clay minerals, and SOM. Additionally, we will also use spectroscopic and diffraction methods to probe the nanoscale mechanisms of sorption to clays and SOM. To better understand the mechanism of sorption to clays, we will ATR-FTIR spectroscopy to probe structure of complexes at the surface. In addition X-ray diffraction will be used to examine in d-spacing of swelling clays upon adsorption of siderophores or siderophore metal complexes.We will also use X-ray spectroscopic methods to probe the fate of metal complexes in SOM and clays.Task 2. Quantification of reaction rates and identification of reaction products of extracellular soil enzymes and manganese oxides with siderophores and Fe(III)-siderophore complexes.Siderophores and Fe(III)-siderophore complexes will be incubated with purified enzymes in solutions in activities equivalent to those in soils. Siderophore and Fe-siderophore complex degradation will also be tested in water or buffer extracted soil solutions that contain an array of native hydrolytic and oxidative enzymes. In addition, reactions with oxidants present in soil environments may represent a major sink for siderophores that limits their ability to promote nutrient uptake. Thus, reaction kinetics will be quantified using continuous-flow stirred tank reactors (CFSTR) to measure steady-state dissolution of the Mn oxide phases using procedures previously described. Degradation products will be characterized UPLC-MS as described in task 3.Task 3. Quantification of siderophores and degradation products in soil. In this task, we will determine the concentration of added siderophore in soils as function of time, as well as look for degradation products. By using a range of soils with different edaphic properties, we anticipate a range of reactivities with differing siderophores.Native and sterilized soil samples incubated, extracted, and products measured by UPLC-MS with electrospray ionization. Results will provide kinetic information about both the loss of siderophores (as quantified in tasks 1 and 2) and the formation of degradation products.

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:The target audience this period was the scientific community that was reached via conference and seminar presentations. Changes/Problems:Dr. Baars will be leaving academia in May 2024. However, he will remain availble to complete projects,and the project will continue to progress to completion under the guidance of Drs. Duckworth and Shi. What opportunities for training and professional development has the project provided?The project is the primary focus of two Ph.D. students, providing the research vehicle for their education. One NSF funded REU studentwas mentored by one of the Ph.D. students for 10 weeks over the summer. In addition, one undergraduate from NC State worked on the project during the spring 2024 semester as part of her curriculum. How have the results been disseminated to communities of interest?Presentations were given at the ASA, CSSA and SSSA Annual Meetings, the Pan American Light Sources for Agriculture, the ACS national meeting, andthe Soil Science Society of North Carolina annual meeting. Duckworth also attended and presented at the A1401 Soil Health Project Director Meeting. What do you plan to do during the next reporting period to accomplish the goals?We plan to analyze possible degradation products for DFOB, PDMA, and protochelin when they come in contact with ferrihydrite, goethite, birnessite, kaolinite, montmorillonite, and organic matter through LC-MS. This will help us understand which minerals have the potential to degrade siderophores, as well asdetermine the structure of siderophore degradation products in soils. We also plan to combine previously collected XRF data of protochelin, PDMA, and DFOB sorbed to soils with high resolution EXAFS to get a clearer picture of the structure of siderophore sorption in soils. In addition, we will continue our study of fungal siderophore degradation, which has revealed a new mechanism for the degradation of hydroxamate siderophores by a wheat root colonizing fungus, presumably during reductive iron uptake (see French et al., 2024). We believe that this new mechanism of siderophore degradation is widespread among fungi. To establish our hypothesis and mechanisms involved in the degradation we will utilize the model S. cerevisiae which has been extensively studied for its iron acquisition mechanisms. S. cerevisiae wild-type and mutants that lack components of the iron uptake machinery are currently cultured and degradation of siderophores will be analyzed by LC-MS.

Impacts
What was accomplished under these goals? In the fourth year of the project, we made substantial progress in our understanding of the interactions of siderophores with reactive soil components. Highlight of activities are as follow: We have continued to develop gallium as a spectroscopic tracer for siderophore complexes. Trivalent gallium is similar in size and makes complexes of comparable affinity to ferric iron with siderophores. We have thus utilized Ga-siderophore complexes in X-ray absorption spectroscopy (XAS) experiments so that we can follow the fate of the metal ion in siderophore complexes even in the presence of iron. We received competitive beamtine to the Stanford Synchrotron Radiation Lightsource (SSRL) to expand this work. We have built a spectral library of Ga-DFOB, Ga-PDMA, and Ga-Protochelin complexes interacting with mineral and DOM. We conducted spatially resolved micro-X-ray fluorescence measurements to determine the elemental associations in soils with differing edaphic properties and reactive component contents. One paper was published describing siderophore degradation by a pathogenic fungus and another was drafted probing the degradation of siderophore by extracellular enzymes.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: French, K.S., Chukwuma, E., Linshitz, I., Namba, K., Duckworth, O.W., Cubeta, M.A., and Baars, O. (2024) Inactivation of siderophore iron chelation by the fungal wheat root symbiont Pyrenophora biseptata, Environmental Microbiology Reports, e13234.
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Kohn, J., Evers, A., Baars, O., Harrington, J.M., Namba, K., and Duckworth, O.W. (2024) Stability of metal ion complexes with the synthetic phytosiderophore proline-2?-deoxymugineic acid, submitted.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Evers, A. and Duckworth, O.W., Elucidating the fate of metal-siderophore complexes by using gallium XAS and �-XRF, Pan American Light Sources for Agriculture (PALSA 2023), Ithica, NY, July 2023.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Chukwuma, E., French, K.S., Linshitz, I., Cubeta, M.A., Duckworth, O.W., and Baars, O. Assessing The Fate of Siderophores after secretion and potential impacts for plant growth, SM 3903395 Technical program for American Chemistry Society (ACS) Fall Conference, San Francisco, CA, August 2023.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Chukwuma, E., French, K.S., Linshitz, I., Cubeta, M.A., Duckworth, O.W., and Baars, O. Enzymatic degradation of siderophores: Assessing the fate of siderophores after secretion and their potency as iron chelating agents for Iron nutrition, increased crop yield, and plant disease resistance, SM 3903396 American Chemistry Society (ACS) Fall conference - AGRO division seminar series, San Francisco, CA, August 2023.
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Evers, A. and Duckworth, O.W., Probing the Fate of Siderophores in Soils through Gallium X-Ray Absorption Spectroscopy, Soil Science Society of America Annual Conference, St. Louis, MO, October 2023.
• Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Evers, A. and Duckworth, O.W., Probing the Fate of Siderophores in Soils through Gallium X-Ray Absorption Spectroscopy, Soil Science Society of North Carolina Annual Meeting, April 2024.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Evers, A., Chukwuma, E., French, K.S., Linshitz, I., Kohn, J.,Baars, O., Shi,W., and Duckworth, O.W. Understanding Critical Soil-Siderophore Antagonistic Interactions, A1401 Soil Health Project Director Meeting, Kansas City, Mo, April 2024.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The target audience this period was the scientific community that wasreached via conference and seminar presentations. Changes/Problems:COVID-19 related issues delayed recruitment of students and progress on the project. We anticipate that we will request ano-cost extension to continue the project until 6/1/2025. What opportunities for training and professional development has the project provided?The project is the primary focus of two Ph.D. students, providing the reserach vichile for this eduation. Two NSF funded REUs, each one mentored by one of the Ph.D. student, worked on the project during 10 weeks over the summer. In addition, a visiting student PhD from Brazil,who is supported by her home state and is reseraching the role of siderophores in iron accumulation by esturine plants, has benefit from working in andcontriubted to the project. How have the results been disseminated to communities of interest?Presentations were given at the ASA, CSSA and SSSA Annual Meetings, the GSA annual meeting, and the University of Sao Paulo. What do you plan to do during the next reporting period to accomplish the goals?Following the enzymatic and fungal siderophore degradation experiments, we will characterize fungal enzymes involved in the siderophore degradation this summer together with an NSF REU student. The study will then be expanded to test siderophore degradation by root exudates and soil enzyme extracts. In final experiments, we will investigate siderophore degradation in plant-microbe cocultures in defined media and in soils. We plan to conduction adsorption isotherms for the siderophores DFOB, PDMA, and protochelin in both their unbound and iron chelated states to ferrihydrite, goethite, birnessite, kaolinite, montmorillonite, and organic matter, in order to understand how structure effects sorption. We also plan to expand our use of Ga-complexes in XAS and micro-XRF/XAS experiments to study the reactivity of PDMA and protochelin complexes in soil. Further plans include focusing on the reactions that occur with organic matter to determine whether soil organic matter acts as an iron source, or a siderophore sink. In addition, we currently have three papers in advanced preparation that we intend to submit in calendar year 2023.

Impacts
What was accomplished under these goals? In the third year of the project we made substantial progress in our understanding of the interactions of siderophores with reactive soil components. Highlight of activities are as follow: We determined the stability constants of the model phytosiderophore proline 2′deoxymugineic acid (PDMA) with Mn, Co, Cu, Ni, and Zn to determine how these metals may impact iron acquisition. Furthermore, we determined that these stability constants correlate (log-log basis) with those of other phytosiderophores, the siderophore DFOB, and hydroxide. These relationships will allow for prediction of other constants and improve our understanding how these soil components may impact iron and other trace metal uptake. We have developed gallium as a spectroscopic tracer for siderophore complexes. Trivalent gallium is similar in size and makes complexes of comparable affinity to ferric iron with siderophores. We have thus utilized Ga-siderophore complexes in X-ray absorption spectroscopy (XAS) experiments so that we can follow the fate of the metal ion in siderophore complexes even in the presence of iron. We have built a spectral library of Ga-DFOB complexes interacting with mineral and DOM standards, as well as utilized spatially resolved micro X-ray absorption and micro-X-ray fluorescence spectroscopy to establish that we can track the fate of the complexes in soil. We have submitted a beamtime proposal to the Stanford Synchrotron Radiation Lightsource (SSRL) to expand this work. We have determined how a suite of extracellar enzymes break down siderophores and iron-siderophore metal complexes by UV-visible and mass spectrometry. To represent the wide variety of siderophore structures and chemistries, we tested three different structures, each characteristic for one of the three major chelating moieties found in siderophores (Fig. 1). Protochelin is a tris-catecholate siderophore, desferrioxamine B (DFOB) represents a tris-hydroxamate siderophore, and proline-2′- deoxymugineic acid (PDMA) is a carboxylate siderophore. Each of the siderophores was tested in its unbound form and as iron-siderophore complexes with each of the enzymes. We found that phenol oxidases are highly effective in the degradation of the catechol siderophore protochelin. Peroxidases were effective in both the degradation of protochelin and DFOB. Complexation of protochelin and DFOB with iron protected the siderophore and reduced the amount of degradation. PDMA was inert in all degradation reactions. Following these experiments we started testing degradation with cultures of fungi known to produce extracellular enzymes. We selected four fungi known to be prolific producers of extracellular enzymes for the acquisition of carbon, which can potentially degrade siderophores: (1, 2) Two strains of Phanerodontia chrysosporium, a white-rot fungus, (3) Linnemania elongata, a plant-growth-promoting fungus, and a new isolate from wheat and phenotypically in agreement with Drechslera biseptata. Of the four fungal cultures, only D. biseptata was able to efficiently break doewn protochelin and DFOB, while PDMA was only slightly degraded. Structures of breakdown products were determined in both the enzymatic and fungal degradation experiments using high-resolution LC-MS/MS. In summary, our work revealed siderophore breakdown by enzymes and selected fungi is possible, and particularly effective for protochelin, a representative of catechol siderophores. Hydroxamate siderophores, such as DFOB, were also degraded but their structure was protected when present as iron chelates. Siderophore breakdown products could be structurally characterized and were compromised or lost their ability to bind iron. The synthetic phytosiderophore analog PDMA remained relatively inert in all experiments.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kohn, J., Evers, A., and Duckworth, O.W., Determining Stability Constants of Metal-Proline 2?Deoxymugineic Acid (PDMA) Chelates via Potentiometric and Spectrophotometric Titration, Geological Society of America Meeting, Denver, Colorado, October 9-12, 2022
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Evers, A. and Duckworth, O.W Understanding Critical Soil-Siderophore Antagonistic Interactions. ASA, CSSA, SSSA International Annual Meeting, Baltimore, Maryland. November 6-9 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: French, K., Chukwuma, E., and Baars, O. (2022) Enzymatic Degradation of Siderophores in Soil by Fungi. ASA, CSSA, SSSA International Annual Meeting, Baltimore, Maryland. November 6-9 2022.

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:The target audience this period was the scientific community who was reached via a publication and a conference presentation. Changes/Problems:COVID-19 related issues delayed recruitment of students and progress on the project. We anticipate that the project would benefit from one or possibly two no-cost extensions. What opportunities for training and professional development has the project provided?Two PhD students (a female student in the Duckworth labanda minority student in the Baars lab) have recruited to the project and will use this project for thedissertationwork. Regular project meeting have occurred, and all three PI will serve on the mentoringcommittees for these students. How have the results been disseminated to communities of interest?A paper was published in Biometals and presenation given at theASA, CSSA and SSSA Annual Meetings. What do you plan to do during the next reporting period to accomplish the goals?We plan to accelerate the reach pace of the project as students advance in the project and COVID restriction loosen. We plan to systematically study the interactions of siderophore and siderophore-metal complexes with minerals and organic matter. We anticipate that this will also include submission of a beamtime proposal to a synchrotron facility lead by a student. Similarly, we plan to study the impact of extracellular enzymes on the degradation of siderophore, as well as the possible in activation of enzymes. These project will be complemented by the development of mass spectrometry pipelines to determine degradation products from these reactions. In addition, we have started two complementarycollaborations. The first involvesfaculty in material science interested in the reactivity of hydroxamate siderophores with organophosphates. We specifically will work with these faculty to better understand if siderophores can promote the degradation of xenobiotic organophosphates and phosphorus containing soil organic carbon. In the second collaboration, a visiting student from Brazil will seek to understand the impact of siderophores on the accumulation of iron by plants used for phytoremediation. Both of these projects leverage the knowledge associated with this project but are independently funded and will utilize resources from this project.

Impacts
What was accomplished under these goals? Despite issue from COVID delays, we have made significant process toward the project. As noted, the there were delays in recruiting PhD students to the project. Additionally, both postdoctoral scholars working on the project moved on to other opportunities. Thus, a research scholar contributed to the project by studying the interactions of siderophores with other competing metals, resulting in a publication. Additionally, two PhD students have joined the project and started to conduct research on the interactions of siderophores and siderophore-metal complexes with mineral surfaces and extracellular enzymes.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Doydora, S.A., Baars, O., Harrington, J.M., and Duckworth, O.W. (2022) Salicylate coordination in metal-protochelin complexes, Biometals, 35, 8798.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Doydora, S.A., Baars, O., Harrington, J.M., and Duckworth, O.W., Structure and Stability of Mo(VI)-Siderophore Complexes, Soil Science Society of America meeting, Salt Lake City, Utah, Nov 7-11 2021.

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:The target audience reached in this period was (1) the scietific community through a publication and (2) NC State University undergraduate students who received a "current topics" lecture based on this research project. Changes/Problems:The project started during a university shutdown due tocovid-19 policies. This delayed the start of our laboratory work and the recruitment of doctoral students to work on the project. What opportunities for training and professional development has the project provided?Recruiment of personel was delayed covid-19. Consequently, the intial phase of the project was carried out by existing postdoctoral scholars in the Duckworth and Baars laboratories, proving training for two female postdocs. For the fall, two PhD students have been recruited (1 female, 1 froman underrepresnted group). How have the results been disseminated to communities of interest?A paper was published inFrontiers in Microbiological Chemistry and Geomicrobiology. What do you plan to do during the next reporting period to accomplish the goals?Two PhD students supported by this projects will join us in the fall. A student in chemistry will continue to develop detection methods for siderophores, siderophore metal complexes, and breakdown products by liquid chromatography-mass spectrometry and liquid chromatography- inductively coupled plasma mass spectrometry. A student in soil science will continue experiments that probe the kinetics and breakdown products of siderophores with reactive soil components.

Impacts
What was accomplished under these goals? NC State University experienced an extended shutdown due in 2020 to COVID-19. To continue progress on the project during a shutdown, we expanded analysis of a preliminary dataset used as the bast of the foundation of the proposal. Publication of a paper on methods of recovery and detection of siderophores in soils resulted from this effort. After the opening of facilities, we initiated initial experiments initiated to study the interactions of desferrioxamine B (DFOB) and iron-DFOB complexes with soil components

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Rai, V., Fisher, N., Duckworth, O.W., and Baars, O. (2020) Extraction and analysis of structurally diverse siderophores in soil, Frontiers in Microbiological Chemistry and Geomicrobiology, 11, 581508.