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

Abstract

Iron (Fe) is an essential nutrient, but is poorly bioavailable because of its low solubility in alkaline soils; this leads to reduced agricultural productivity. To overcome this problem, we first showed that the soil application of synthetic 2'-deoxymugineic acid, a natural phytosiderophore from the Poaceae, can recover Fe deficiency in rice grown in calcareous soil. However, the high cost and poor stability of synthetic 2'-deoxymugineic acid preclude its agricultural use. In this work, we develop a more stable and less expensive analog, proline-2'-deoxymugineic acid, and demonstrate its practical synthesis and transport of its Fe-chelated form across the plasma membrane by Fe(III)•2'-deoxymugineic acid transporters. Possibility of its use as an iron fertilizer on alkaline soils is supported by promotion of rice growth in a calcareous soil by soil application of metal free proline-2'-deoxymugineic acid.

Fig. 1. Iron (Fe) uptake strategy in Poaceae plants.

Poaceae plants secrete mugineic acid family phytosiderophores (MAs) into the rhizosphere and absorb Fe-MAs complex. Fe deficiency occurs when secretion of MAs is inadequate. This problem may be overcome by adding synthetic 2′-deoxymugineic acid (DMA) or an analog thereof. DMA 2′-deoxymugineic acid, MAs mugineic acids, TOM1 transporter of mugineic acid 1, YS1/YSL yellow stripe 1/yellow stripe 1 like transporter.

Fig. 2. Effects of DMA application on plant growth in calcareous soil.

a Soil and plant analyzer development (SPAD) values of the newest rice leaves after the last of six applications of Fe-chelates at 14 days after transplantation (0 days after the last application). The chelates (DMA, citrate, and desferrioxamine [DFO]) were mixed with equimolar FeSO₄ in water before application. Values are means of three replicates in which each replicate is average of three plants in each pot, and different letters at the same time point indicate significant differences at P < 0.05 by Tukey’s honestly significant difference test (two-sided). P-values for one-way analysis of variance (ANOVA) are follows: 0 days, 0.0001; 3 days, <0.0001; 5 days, <0.0001; 7 days, <0.0001; 11 days, <0.0001. b Rice plants at 3 days after the last application. DMA 2′-deoxymugineic acid, EDTA ethylenediaminetetraacetic acid, EDDHA ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid), DFO desferrioxamine, SPAD soil and plant analyzer development.

Fig. 3. Transport of synthetic DMA analogs.

a Structures of synthetic DMA analogs. b Addition of 50 μM ⁵⁵Fe(III)-DMA and ⁵⁵Fe(III)-proline deoxymugineic acid (PDMA) to HvYS1-expressing Sf9 insect cells (1 × 10⁷) enhanced their Fe(III)-transport activity. In contrast, ⁵⁵Fe(III)-GDMA and ⁵⁵Fe(III)-MGDMA only marginally enhanced the Fe(III)-transport activities of the cells. Each Fe(III) complex contained 10 mol% radioactive ⁵⁵Fe. Sf9 insect cells transfected with empty vector were used as a control. Values are means with standard deviations of the four cell lysate replications (two-tailed t-test for Fe(III)-PDMA in HvYS1). c Fe(III)-transporting activities of synthetic DMA and PDMA in Xenopus oocytes injected with HvYS1, OsYSL15, or ZmYS1 cRNA, or water (negative control) were measured by two-electrode voltage clamp analysis. Values are means with standard deviations of the four replicates (two-tailed t-test for water injection). d Detection of PDMA derivatized using 9-fluorenylmethoxycarbonyl chloride (FMOC) by liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) analysis in the xylem sap of rice plants supplied with Fe-DMA and Fe-PDMA in the nutrient solution. DMA 2′-deoxymugineic acid, PDMA proline-2′-deoxymugineic acid, GDMA glycine-2′-deoxymugineic acid, MGDMA N-methylglycine-2′-deoxymugineic acid, HvYS1 Hordeum Vulgare yellow stripe 1, OsYSL15 Oryza Sativa yellow stripe 1 like transporter 15, ZmYS1 Zea mays yellow stripe 1.

Fig. 4. Effects of PDMA, a synthetic DMA derivative, on rice in calcareous soil.

a SPAD values of the newest leaves after a single application of the Fe chelates at 4 days after transplantation. DMA and PDMA were mixed with equimolar FeSO₄ in water before application. Values are the means of three replicates in which each replicate is average of three plants in each pot. Different letters indicate significant differences at P < 0.05 by Tukey’s honestly significant difference test (two-sided). P-values for one-way analysis of variance (ANOVA) are follows: 0 days, 0.9394; 2 days, 0.2530; 4 days, 0.0008; 7 days, <0.0002; 9 days, <0.0001; 11 days, 0.0002; 14 days, <0.0001. b Rice plants at 14 days after a single application of the Fe chelates. DMA 2′-deoxymugineic acid, PDMA proline-2′-deoxymugineic acid, EDDHA ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid), SPAD soil and plant analyzer development.

Fig. 5. Effects of unchelated PDMA on rice in calcareous soil.

a SPAD values of the newest leaves after a single application of 1–30 µM unchelated PDMA compared with other Fe chelates at 6 days after transplantation. PDMA was also mixed with equimolar FeSO₄ or Fe₂(SO₄)₃. Values are the means of three replicates in which each replicate is average of three plants in each pot. Different letters indicate significant differences at P < 0.05 by Tukey’s honestly significant difference test (two-sided). P-values for one-way analysis of variance (ANOVA) are follows: 0 days, 0.5287; 4 days, <0.0001; 6 days, <0.0001; 8 days, <0.0001; 11 days, <0.0001; 14 days, 0.0017. b Rice plants at 14 days after a single application of the Fe chelates. PDMA proline-2′-deoxymugineic acid, EDDHA ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid), EDTA ethylenediaminetetraacetic acid, SPAD soil and plant analyzer development.

Fig. 6. SPAD values and metal concentrations in the newest leaves of rice plants supplied with chelating agents.

a SPAD values were measured 7 days after a single application at 4 days after transplantation. b–e Metal concentrations in the newest leaves. Ten pots were used in each treatment, and three rice plants were grown in each pot. The three newest leaves in each pot were collected and used for measurement of SPAD values and metal concentrations by inductively coupled plasma optical emission spectrometry. The colors of the plots of each treatment through a–e indicate samples from the same pots. Values are means with standard deviations indicated by error bars (n = 10 for control, Fe-EDDHA and Zn-EDTA, n = 9 for PDMA, Fe-PDMA, and Fe-EDTA, and n = 8 for Zn-PDMA, after excluding outliers of Fe concentration), in which each replicate is either average of three plants in each pot for a, or total metal concentration of the three plants in each pot for b–e. Different letters indicate significant differences at P < 0.05 by Tukey’s honestly significant difference test (two-sided). PDMA proline-2′-deoxymugineic acid, EDDHA ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid), EDTA ethylenediaminetetraacetic acid, SPAD soil and plant analyzer development, D.W. dry weight.

Fig. 7. Synthesis of PDMA.

PDMA·HCl salt 7 was synthesized from allylglycine 1 with an overall yield of 38%. Purification by column chromatography was performed once. Boc tert-butoxycarbonyl, ^tButert-butyl, NaBH(OAc)₃ sodium triacetoxyborohydride, NaBH₃CN sodium cyanoborohydride, AcOH acetic acid.

Fig. 8. Pilot field experiment showing the effects of chelates on rice growth in calcareous soil.

a SPAD values of the newest leaves from 1 to 4 weeks after chelate application, showing the superiority of 30 µM unchelated PDMA and 30 µM Fe-ethylenediamine di(o-hydroxyphenylacetic) acid (EDDHA) in overcoming Fe deficiency. Values are the mean of three replicates in which each replicates is control, 48 (Contol), 32 (30 µM PDMA, 3 µM PDMA, 30 µM Fe-EDDHA), and 16 (30 µM Fe-DTPA). Different letters indicate significant differences at P < 0.05 by Tukey’s honestly significant difference test (two-sided). P-values for one-way analysis of variance (ANOVA) are follows: 1 week <0.0001, 2 weeks <0.0001, 3 weeks <0.0001, 4 weeks <0.0001. b Photographs of rice plants at 2–4 weeks after treatment. PDMA proline-2′-deoxymugineic acid, EDDHA ethylenediamine-N,N’-bis(2-hydroxyphenylacetic acid), DTPA diethylenetriaminepentaacetic acid.