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

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.


nature research | reporting summary
April 2020 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Methods n/a Involved in the study

ChIP-seq
Flow cytometry

MRI-based neuroimaging
The sample size used for most pot experiments (n =3) provide adequate power to show statistical significance in Tukey's HSD (Honestly significant significance). In our experiments, we used excessive numbers of seeds at germination (5 times or more of the numbers required for the experiments), and then selected proper seedlings showing similar growth (3 plants / pot) for transplanting. We measured the average value of the 3 new leaves for each plant in the pots. The sample size of pot experiments of supplemental Figure 8 was 2, because of low availability of synthesized material and the objective which was to show tendency of efficacy as a supplementary result. The sample size of pot experiment for analysis of metal concentration ( Figure 6) was 8-10 for sufficient power to show statistical significance in Tukey's HSD (Honestly significant significance). The sample size of field experiment (Figure 8) was from 16 to 48, which was sufficient for Tukey's statistical analysis (HSD). For this experiment, 10 times of the required amount of the seeds were germinated, and then proper seedlings showing similar growth (3 plants / pot) were selected at transplanting. We measured the average value of the 3 new leaves for each plant in field. Transporter analysis (Figure 3b, c) was performed in 4 replications for sufficient power to show statistical significance. Soil solution was measured 3 times by ICP-AES in the soil assay test (Supplemental table 2). The qPCR analysis (supplemental figure 7) was performed in three times using cDNA from 3 plants together in each treatment. Cytotoxicities were measured 3 times by LDH assay and ATP assay (supplementary fig 11) for sufficient power to show statistical significance (Dunnett's test). Sample size of chemical synthesis and synthetic procedures were described in supplementary information with details including TLC analysis so that synthesis can be easily reproduced.
We have excluded outliers in Figure 6 (n = 1 for PDMA, Fe-PDMA and Fe-EDTA, and n = 2 for Zn-PDMA, out of n = 10) because of extraordinarily low Fe concentrations due to possible technical problem during acid digestion for ICP measurement.
All replication attempts were successful. All the experiments were performed independently.
Most pot experiments (Figures 2, 4, 5, 6, and Supplemental Fig 8) were performed in growth chamber and pots were set randomly. The large pot experiment (Supplemental Figure 9) was performed in greenhouse and pots were set randomly. The design of pilot filed experiment (Figure 8) was randomized locationally, 16 hills (3 plants / hill) were transplanted into one block (1m x 1m), and 10 blocks was set randomly in each treatment. There was no extra opportunity to apply randomization.
No blinding was performed. Our experimental groups needed to share information such as the purity of synthetic compounds, the amount synthesized, the growth experimental conditions, in vitro assay conditions, and so on.