Development of a mugineic acid family phytosiderophore analog as an iron fertilizer

Alkaline/calcareous soils, which cover a large fraction of global arable land, limit plant iron (Fe) availability because Fe precipitates as insoluble ferric hydroxides/oxides. Poaceae species use a Strategy II Fe-uptake system, secreting mugineic acid family phytosiderophores (MAs) that chelate Fe(III) and are taken up via YS1/YSL transporters. Rice is particularly susceptible to Fe deficiency due to low secretion of the MA 2'-deoxymugineic acid (DMA). Although transgenic approaches enhancing MA biosynthesis improve tolerance to low Fe, these are limited by species transformability and environmental adaptation. Conventional synthetic chelates (EDTA, DTPA, EDDHA) can supply Fe but pose environmental persistence concerns and are less effective in Poaceae that preferentially take up Fe(III)-MAs. Prior studies with purified MAs largely involved hydroponics, and soil application has been hindered by instability and high cost. This study aims to: (1) test soil application of synthetic DMA versus other chelates in calcareous soil using rice as a model, and (2) design and validate a more stable, lower-cost DMA analog suitable for agricultural use.

Background work established Strategy II Fe uptake via MAs and YS1/YSL transporters in grasses, rice sensitivity due to low DMA secretion, and benefits of MA-biosynthesis transgenes in rice. Synthetic chelates (EDTA, DTPA, EDDHA) are widely used in alkaline soils, but persistence in the environment and limited efficacy in Poaceae are concerns. Non-Poaceae may also utilize MAs released by Poaceae in intercropping systems, suggesting broader applicability of MAs. Past applications of purified MAs focused on hydroponics, with soil use considered impractical due to degradation and cost. Chemical syntheses of DMA are known but expensive, often relying on L-azetidine-2-carboxylic acid. Stability constants for MA-Fe complexes and transporter specificities have been reported, with hydroxylated derivatives showing higher Fe(III) affinity. These insights motivated a redesigned analog to reduce ring strain, improve stability, and lower cost.

- Plant growth experiments in calcareous soil (pH ~9) using rice (Oryza sativa cv. Nipponbare or Koshihikari): multiple pot experiments compared Fe sources and chelating agents. Treatments included Fe-DMA, Fe-PDMA (proline-2'-deoxymugineic acid), Fe-EDTA, Fe-EDDHA, Fe-citrate, desferrioxamine (DFO), FeSO4 alone, and unchelated PDMA at varying concentrations (1–30 µM). Applications were either repeated (six times between days 3–14 post-transplant) or single (at 4–6 days post-transplant). SPAD values (proxy for chlorophyll/Fe status) on newest leaves were measured over time. Virgin calcareous soil previously characterized was used, with controlled-release NPK-micronutrient fertilizer included. - Transport assays: Fe(III)-complex transport assessed using HvYS1-expressing Sf9 insect cells with 55Fe-labeled complexes (DMA, PDMA, GDMA, MGDMA). After 1 h incubation and washing, intracellular radioactivity quantified by scintillation counting. Electrophysiology in Xenopus laevis oocytes expressing HvYS1, ZmYS1, or OsYSL15 measured steady-state currents upon perfusion with 200 µM Fe(III)-DMA or Fe(III)-PDMA at pH 6.0. - In planta uptake: LC/ESI-TOF-MS of xylem sap after root supply of Fe-DMA or Fe-PDMA, derivatized with FMOC-Cl, to detect PDMA in xylem. - Gene expression: qPCR of Fe-deficiency marker genes (OsNAS2, OsIRO2) in roots of hydroponically grown rice supplied with Fe-DMA, Fe-PDMA, or Fe-EDTA (30 µM Fe) after Fe depletion. - Stability/complexation: Potentiometric titrations determined PDMA proton dissociation constants; Bjerrum method estimated the 1:1 Fe(III)-PDMA stability constant (log K). - Biodegradation: OECD 301A test compared biodegradability of PDMA, citrate, and EDTA via Japan Food Research Laboratories. - Soil metal solubilization: Incubation of calcareous soil with chelators (PDMA, EDTA, Fe-EDTA, Fe-EDDHA) measured Fe, Zn, Mn, Cu in supernatant by ICP-OES. - Leaf metal analysis: After chelator applications (Fe- or Zn- complexes with PDMA or EDTA; unchelated PDMA; controls), newest leaves were harvested (n=8–10 pots per treatment), dried, digested (HNO3/H2O2), and Fe, Zn, Mn, Cu quantified by ICP-OES. - Analog synthesis and characterization: PDMA synthesized from N-Boc-L-allylglycine tert-butyl ester via ozone oxidation, reductive amination with L-proline, HCl-mediated ester formation, subsequent reductive amination with an aldehyde from L-malic acid, deprotection/hydrolysis to PDMA·HCl. Scale-up yielded 42.5 g PDMA·HCl. Additional analogs (GDMA, MGDMA, six-membered PiDMA, acyclic AvDMA and ADMA) were synthesized; Fe(III) complexation confirmed by HRMS. - Field pilot: 1 m × 1 m × 0.5 m blocks of calcareous soil with irrigated conditions; 35-day-old rice transplanted. Treatments applied 14 days later: unchelated PDMA (3 or 30 µM), Fe-EDDHA (30 µM), Fe-DTPA (30 µM), or control. Weekly SPAD measurements over 4 weeks; soil redox potential monitored. - Statistics: One-way ANOVA with Tukey’s HSD for SPAD and metal concentration comparisons; replicates as specified per experiment. - Safety: Cytotoxicity of PDMA vs DMA assessed in HEK-293EBNA cells via ATP content and LDH release across 1 µM–1 mM; Dunnett’s test used.

- Soil application of Fe-DMA alleviated Fe deficiency in rice grown in calcareous soil: at the final application day (14 days after transplant; time 0), SPAD ~30 with Fe-DMA vs ~12 control; Fe-EDDHA and Fe-EDTA showed moderate improvement; Fe-citrate and Fe-DFO were ineffective. Over the next 5 days without further application, Fe-DMA maintained higher SPAD than Fe-EDDHA or Fe-EDTA (ANOVA P ≤ 0.0001 at multiple time points). - PDMA design and transport: A five-membered ring analog (PDMA) retained 1:1 Fe(III) complexation and was transported by HvYS1 with uptake comparable to DMA in Sf9 cells; GDMA and MGDMA showed much lower transport. Electrophysiology showed Fe-PDMA transported by HvYS1, ZmYS1, and OsYSL15 (significant current responses, e.g., p-values 0.0001–0.0182 vs water). PDMA was detected in xylem sap after Fe-PDMA supply, whereas not detected after Fe-DMA; DMA was detectable in both due to endogenous biosynthesis. qPCR indicated Fe-PDMA reduced Fe-deficiency gene expression (OsNAS2, OsIRO2) similarly to Fe-DMA and more than Fe-EDTA. - Stability constant: Fe(III)-PDMA 1:1 complex log K = 17.1 (slightly lower than DMA log K ≈ 18.4), indicating strong Fe(III) binding. - Single soil application efficacy: Fe-PDMA and Fe-DMA raised SPAD to ~30 within days; Fe-PDMA increased SPAD to ~40 by day 12 and maintained elevated values for ~2 weeks, outperforming single-application Fe-EDDHA which showed minimal enhancement (ANOVA P < 0.0002 at ≥7 days). Biodegradation tests showed PDMA degraded slower than citrate, while EDTA showed no biodegradation. - Unchelated PDMA effectiveness: Single applications of 1–30 µM PDMA (without added Fe) prevented Fe-deficiency chlorosis and raised SPAD similarly to Fe(II)- or Fe(III)-PDMA, whereas disodium EDTA did not. 1–3 µM PDMA achieved effects comparable to 30 µM Fe-EDDHA, indicating >10-fold greater efficacy than Fe-EDDHA. Soil solution analyses confirmed PDMA solubilized native Fe, forming Fe-PDMA taken up by plants. - Micronutrient impacts: PDMA solubilized Fe, Zn, and Cu in soil incubations. In plants, metal-free PDMA and Fe-PDMA treatments increased leaf Fe concentrations and SPAD; Fe-EDDHA slightly raised SPAD but not leaf Fe, suggesting preferential allocation to chlorophyll under deficiency. Zn-PDMA and Zn-EDTA increased leaf Zn, but only Zn-PDMA raised SPAD and also increased Fe, implying exchange to Fe-PDMA in Fe-rich soil. Mn concentrations decreased with PDMA- and Zn-chelate treatments; Cu tended to increase with metal-free PDMA. SPAD correlated with leaf Fe rather than other micronutrients. - Analog structure–activity: Six-membered ring analog (PiDMA) was effective in soil similar to PDMA; acyclic analogs were substantially less effective, underscoring the importance of a cyclic amino acid moiety. - Scalable synthesis: Practical large-scale PDMA·HCl production demonstrated (42.5 g product from 77.8 g starting material), enabling broader research and application compared to prohibitively costly DMA (~$1000/mg). - Pilot field validation: In outdoor blocks of calcareous soil, 30 µM unchelated PDMA significantly improved SPAD within 1 week and maintained superiority over 3 µM PDMA, 30 µM Fe-EDDHA, and 30 µM Fe-DTPA across 4 weeks (ANOVA P < 0.0001 at all weeks). Fe-DTPA was ineffective in the timeframe assessed. Subsequent prolonged flooding reduced soil redox potential and confounded further assessment of grain Fe.

The results demonstrate that a natural MA (DMA) applied to calcareous soil can alleviate Fe deficiency in rice, validating Strategy II chelation-based fertilization in soil. However, DMA’s instability and high synthesis cost limit practicality. The newly developed PDMA preserves functional characteristics crucial for Poaceae uptake: strong Fe(III) chelation and recognition by YS1/YSL transporters across species (barley, maize, rice). PDMA’s five-membered ring reduces ring strain compared with DMA’s azetidine, enhancing resistance to biodegradation while remaining biodegradable, balancing efficacy and environmental fate. Critically, unchelated PDMA chelates native soil Fe(III), obviating the need to co-apply Fe—a unique property not shared by conventional synthetic chelators—and is markedly more efficient than Fe-EDDHA on a molar basis. PDMA also aids Zn nutrition and modulates Mn and Cu uptake, suggesting broader micronutrient management potential. Field block data corroborate pot experiments, showing sustained improvement of chlorosis under variable irrigation. Collectively, PDMA offers an effective, potentially environmentally friendlier Fe fertilization strategy for alkaline/calcareous soils, with applicability to Poaceae and possibly non-Poaceae via intercropping or external MA uptake.

This work establishes PDMA, a proline-based analog of 2'-deoxymugineic acid, as a practical, effective iron fertilizer for alkaline/calcareous soils. PDMA is transported by YS1/YSL transporters, forms strong Fe(III) complexes (log K 17.1), alleviates Fe deficiency in rice more effectively and durably than Fe-EDDHA, and uniquely functions when applied without Fe by chelating native soil Fe. Scalable synthesis was demonstrated, addressing prior supply and cost bottlenecks associated with DMA. PDMA also enhances Zn uptake and could serve as a universal micronutrient chelator. Future work should optimize industrial synthesis and formulations, evaluate long-term field performance across crops and soil types, quantify impacts on yield and grain nutrient content, fully assess environmental fate and safety, and explore PDMA’s role in multi-micronutrient management and intercropping systems.

- Soil biodegradation of PDMA, while slower than citrate, still occurs and may limit persistence; formulation strategies may be needed to optimize release. - Field pilot conditions had variable water status and later reduced redox potential, preventing assessment of effects on grain Fe and complicating long-term conclusions. - Most efficacy data are from rice; although transporters in other Poaceae responded to PDMA, broader crop testing is needed, including non-Poaceae. - Stability constant for PDMA is slightly lower than DMA, potentially affecting performance under certain competitive chelation environments. - Safety assessment is preliminary (in vitro cytotoxicity only); comprehensive toxicology and environmental impact studies are required.