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Wanted: Micronutrients! Phytosiderophore-mediated acquisition strategies in grass crops

Periodic Reporting for period 1 - PhytoTrace (Wanted: Micronutrients! Phytosiderophore-mediated acquisition strategies in grass crops)

Reporting period: 2019-03-01 to 2020-08-31

Understanding how plants respond to micronutrient deficiency and which biogeochemical processes are induced at the root-soil interface, i.e. the rhizosphere, is crucial to improve crop yield and micronutrient grain content for high quality food and feed. Iron nutrition by grass species relies on the release and re-uptake of phytosiderophores, which are root exudates that form stable complexes with Fe but also other trace metals such as Zn and Cu. However, neither the importance of phytosiderophores under Zn and Cu deficient conditions nor the interplay of plant responses and rhizosphere processes are well understood as the majority of studies in the past was carried out under ‘soil-free’ hydroponic conditions. In this project, I aim to elucidate the mechanisms controlling phytosiderophore-mediated micronutrient acquisition of barley (Hordeum vulgare) under Zn, Cu, and as reference, Fe deficient conditions, with particular emphasis on soil environments. Barley is the fifth most produced crop worldwide and of great importance in regions that are characterized by harsh living conditions. In a holistic approach, my team and I will apply innovative soil-based and traditional hydroponic root exudation sampling approaches in combination with advanced plant molecular techniques to study the phytosiderophore release and uptake system under different experimental conditions. The chemical synthesis of otherwise commercially unavailable phytosiderophores in their natural and 13C-labelled form will allow us to trace their decomposition and metal solubilizing efficiency in the plant-microbe-soil system to uncover the interplay of plant genetic responses and rhizosphere processes affecting the time-window of PS-mediated MN acquisition. Moving beyond ‘soil-free’ experimental designs of the past, this project will generate key knowledge to improve selection of crops with highly efficient micronutrient acquisition traits to alleviate micronutrient malnutrition of people world-wide.
AIM 1 - Synthesize all known PS in their natural and 13C-labelled form (TU). All 8 PS were successfully synthesized within the planned period. Therefore, the first, most critical milestone of this project was achieved without any delay, with the developed synthetic strategy already being published https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202004004. Current activities focus on upscaling the developed synthesis and the implementation of the 13C label.
AIM 2 - Reveal PS release, re-uptake and molecular responses of contrasting barley cultivars under Zn, Cu and as a reference Fe deficiency in different experimental conditions (BOKU).
Selection of suitable barley lines & optimization of growth conditions: Fe, Zn & Cu seed tissue concentrations of barley grown on a micronutrient deficient soil at the James Hutton Institute (collaboration Tim George & Soren Husted) were determined to allow a pre-selection of several lines with highest and lowest micronutrient (MN) tissue concentrations. Micronutrient (MN) deficient soils from Turkey, Australia and Spain were imported and fertilization schemes and growth conditions were optimized for barley.
Maturity growth test & gene expression analysis: 3 MN efficient and 1 MN inefficient barley lines were grown to maturity on the Zn and Cu deficient soils including their respective fertilized controls. Plants were destructively sampled at 4 time points during plant development to link growth performance (biomass), MN uptake with PS exudation and gene expression. For gene expression analysis, gene specific primers were designed for genes participating in the PS synthesis, export, and import of PS-metal complexes. The gene selection was based on previous results from the literature and on in silico investigations in different crop species focusing on the most homologous genes of this pathway. Gene expression analysis is currently conducted.
AIM 3 - Uncover the efficiency and dynamics of PS-metal mobilization of all known PS in bulk and rhizosphere soil (BOKU).
PS analysis: An indirect Fe-binding assay that allows a quick screening of the sum of all Fe solubilizing metabolites in exudate samples was implemented. Using the synthesized PS, LC-MS/MS based method was successfully adapted and implemented.
Experimental soils: general soil parameters were determined as a basis for further geochemical experiments.
AIM 4 - Elucidate the partitioning dynamics of PS in the plant-soil-microbe system and identify the key microbial players involved (BOKU, UNIVIE). Experimental work is scheduled to start in March 2021.
- Successful development and implementation of a novel, unified, modular approach to chemically synthesize all eight identified phytosiderophores (PS). This modular approach now enables the installation of 13C2 isotopic labels into the middle fragments of all PS. Having achieved the most critical milestone of this project, allows us now to investigate and trace the fate and function of PS in the plant – microbe-soil system.
- Successful development and implementation of an advanced LC-MS/MS analysis of all eight PS including the use of a 13C labelled internal standard. This milestone now allows us tu accurately identify and quantify PS in root exudation and soil samples.
- Investigation of PS exudation patterns and related gene expression during plant development form barley lines differing in micronutrient use efficiency grown on Zn and Cu deficient soils