Organic Chemistry Research on the Mechanistic Elucidation of Iron Acquisition in Barley

Abstract

An organic chemistry approach to the mechanistic elucidation of iron acquisition in graminaceous plants is introduced here. To elucidate this detailed mechanism using phytosiderophores, the efficient synthesis of 2′-deoxymugineic acid (DMA), a phytosiderophore of rice, was established. The synthetic DMA was confirmed to have similar iron transport activity to that of natural mugineic acid (MA). It was also revealed that the addition of synthetic DMA, along with iron, to a rice hydroponic solution enabled the rice to grow well even under an alkaline condition, and DMA clearly showed its high potential as a fertilizer to improve food production. On the other hand, the 2′-hydroxy group of MA was confirmed to serve as a point of introduction for labeling, allowing the synthesis of various mugineic acid derivatives as molecular probes. The incorporation of fluorescent mugineic acid into cells allowed them to be clearly observed by fluorescence confocal analysis, and this provided the first direct experimental evidence of transporter-mediated internalization of mugineic acid into cells.

1. INTRODUCTION

Infertile soils, unfavorable for agricultural use, cover two-thirds of world’s land mass, more than half of which is categorized as alkaline soil.^1,2) In alkaline soils, plants are exposed to iron deficiency stress due to the poor solubility of iron(III) salt such as iron(III) hydroxide, even though high concentrations of iron are contained on the surface of the earth. As iron is an essential element for plant growth processes, such as chlorophyll biosynthesis, plants struggle to grow under the condition of low availability of iron ions in alkaline soils. To overcome this problem, graminaceous plants have developed a unique adaptive strategy characterized by the synthesis and secretion of an iron-chelator phytosiderophore, and the uptake of iron through a specific transporter³⁾ (Fig. 1). Mugineic acid (MA) was first identified as a phytosiderophore in barley by Takemoto et al.⁴⁾ Since the initial disclosure of MA, various analogues of MA have been isolated and identified from graminaceous species and cultivars⁵⁾; they all form water-soluble 1 : 1 complexes with iron(III). Due to their important role in plant physiology, the iron uptake system of graminaceous plants through the phytosiderophore–iron(III) complex has been intensively studied over the past 40 years.⁶⁾ For example, although the stereostructure of mugineic acid–iron(III) complex is still unknown, its cobalt and copper complexes have been elucidated by X-ray analysis to suggest the proposed structure of an iron(III) complex.⁷⁾ The biosynthetic pathway of mugineic acids has also been revealed.^8,9) However, mechanistic details of this iron uptake have not yet been fully elucidated. Thus, we focused our attention on this characteristic system of graminaceous plants, considering that elucidation of the detailed molecular mechanism of their iron acquisition would help to improve food production in alkaline soils.

Fig. 1. Model of the Specific Uptake System for Iron(Fe(III))–Mugineic Acid (MA) Complexes in Barley.

When barley is exposed to iron deficiency stress, MA is biosynthesized from methionine in plants and is secreted from the roots. The released MA forms a complex with insoluble trivalent iron salts such as Fe(OH)₃, and the resulting iron complex is incorporated into cells through a specific transporter, HvYS1. (Color figure can be accessed in the online version.)

2. IDENTIFICATION OF THE MA–Fe(III) TRANSPORTER GENE HvYS1

In our initial research to elucidate the mechanism of acquiring iron at the molecular level, we identified and characterized the MA-Fe(III) transporter, HvYS1, in barley, which is the graminaceous species most tolerant to iron deficiency.¹⁰⁾ The cDNA of HvYS1 is 2430 bp long, and its putative polypeptide contains 678 amino acids. The HvYS1 gene is predicted to encode a plasma membrane protein, and a BLAST search shows that HvYS1 belongs to the oligopeptide transporter (OPT) family, which is reported based on the study of several organisms, including bacteria, archaea, fungi and plants.¹¹⁾ HvYS1 shows high homology with ZmYS1, which is the first protein to have been identified as a phytosiderophore (PS)–iron(III) transporter in maize,¹²⁾ with 72.7% identity and 95.0% similarity (Fig. 2). In particular, all predicted transmembrane regions of the two proteins have high similarities. The expression pattern of the HvYS1 gene in barley showed that this gene was mainly expressed in the roots.¹⁰⁾ Furthermore, the expression was enhanced 50-fold in Fe-deficient roots compared to Fe-sufficient roots. These results suggest that HvYS1 is a transporter involved primarily in iron acquisition from soil in barley roots. Having identified and obtained the HvYS1 gene, various experiments such as the evaluation of transport activity, the development of transgenic plants, and the functional analysis of iron transport became possible.

Fig. 2. Comparison of Amino Acids Sequences of HvYS1 and ZmYS1

The fourteen transmembrane regions of HvYS1 (Gene Accession No. AB214183) or ZmYS1 (Gene Accession No. AF186234) predicted by TMHMM Ver. 2 (http://www.cbs.dtu.dk/services/TMHMM/) are shown by blue lines. The yellow box indicates the chimera region for HvYS1-ZmYS1 chimera transport activity. (Color figure can be accessed in the online version.)

3. PRACTICAL RESEARCH USING MUGINEIC ACIDS

Although barley has excellent alkaline tolerance, not all graminaceous plants have an equivalent ability. For example, rice and corn can hardly grow in alkaline soils. This is attributed to the fact that the secretion ability of 2′-deoxymugineic acid (DMA), a phytosiderophore of rice, is much lower than that of barley (Fig. 3 left). Therefore, Professor Nishizawa and colleagues reported that by introducing the mugineic acid biosynthesis gene of barley into rice, rice obtained an MA secretory capacity and exhibited excellent alkaline tolerance, equivalent to that of barley.¹³⁾ Inspired by this pioneering report, we considered that an external addition of DMA–Fe(III) complex as a fertilizer may enable rice and corn to grow even in alkaline soil. (Fig. 3, right) In addition, it is expected that an alkaline tolerance exceeding that of barley can be obtained by adjusting the amount of DMA–Fe(III) added. Since this approach utilizes inherent transporters of graminaceous plants, it does not require gene recombination; it is possible to conduct this investigation using ordinary plants. However, MA has not been commercially available, and DMA is very expensive ($ 2000/2.5 mg). Thus, it was impractical to consider the application of DMA as fertilizer, or even to affordably supply the minimum amount necessary for mechanistic studies. In response to this problem, we attempted to establish a stable method for supplying mugineic acids by chemical synthesis.

Fig. 3. Application of DMA to the Cultivation of Rice in Alkaline Soil

The low tolerance of rice to an alkaline environment is due to their poor secretion of DMA. Thus, the external addition of synthetic DMA is expected to allow rice to recover from iron deficiency stress. DMA, 2′-deoxymugineic acid. (Color figure can be accessed in the online version.)

MA and DMA are characterized by a structure in which L-azetidine-2-carboxylic acid, aspartic acid, and malic acid unit are reductively connected, respectively. Although many synthetic studies of MA and DMA have been reported so far,^14,15) we have developed the following practical synthetic method of DMA production based on a concept that limits 1) the use of amino acid protecting groups as far as possible, and 2) the isolation and purification of each synthetic intermediate.

The synthesis began with commercially available N-tert-butoxycarbonyl(Boc)-L-allylglycine 1. After ozone oxidation of 1 in methanol leading to 2, the reaction mixture was directly treated with unprotected L-azetidine-2-carboxylic acid 3 and NaBH₃CN to afford 4. Evaporation of methanol, followed by treatment with dry HCl in EtOH, caused the removal of the Boc group and esterification of 2-carboxylic acid simultaneously to give 5. After the evaporation of HCl and EtOH, reductive amination of 5 with 6 in methanol gave a protected DMA 7. Since 7 becomes possible to extract with an organic solvent, major byproducts such as unprotected amino acids were readily separated at this stage. After purification by short-pass silica gel column chromatography, deprotection afforded DMA 2 in good yield¹⁶⁾ (Chart 1).

Chart 1. Practical Synthesis of DMA

Having prepared sufficient quantities of DMA, the actual addition of DMA to the rice medium became possible. Normal rice without genetic modification was grown in an acidic medium at pH 5.8 (left in Fig. 4). Although rice could not grow in the medium without iron ion (No. 1), it was found that rice grows well by adding iron ions to the medium (to No. 2 (3 µM) and to No. 3 (30 µM)). On the other hand, in an alkaline medium at pH 8 (right in Fig. 4), rice could not grow soley by adding iron ions (No. 2, 3). However, when synthetic DMA was added to the medium, together with iron ions, it was found that the DMA allows the rice to grow well, even in an alkaline medium (No. 5, 6). This result shows that ordinary rice can absorb iron ions even under alkaline conditions by the external addition of DMA–Fe(III) complex. In addition, we recently revealed that DMA plays a significant role in iron delivery inside plants, and in nitrogen fixation.¹⁷⁾ Therefore, if mugineic acids can be supplied as fertilizer, farming in alkaline soil is expected to be realized as a simple approach without using genetically modified crops.

Fig. 4. Effect of DMA Application and pH on the Growth of Rice Seedlings in Hydroponic Culture

The growth experiments were conducted in a water solution containing 0 or 30 µM DMA, with a variety of iron concentrations, for 18 d. The addition of DMA enabled rice seedlings to grow normally even under an alkaline condition (entry 5, 6 under pH 8.0). (Color figure can be accessed in the online version.)

Toward a further practical synthesis of DMA, we are investigating the catalytic asymmetric synthesis of L-azetidine-2-carboxylic acid 3, which is the most expensive starting material, and also the development of an inexpensive derivative of DMA. In the catalytic asymmetric synthesis of 3, we have already succeeded in establishing a column-free route from a cheap starting material to 3 in high yield. In the near future, we plan to develop to a field experiment using DMA which we synthesized in large scale.

4. DEVELOPMENT OF MA DERIVATIVES AS MOLECULAR PROBES FOR THE MECHANISTIC ELUCIDATION OF IRON ACQUISITION IN BARLEY

As mentioned at the beginning, we succeeded in isolating and identifying the MA–Fe(III) complex transporter gene HvYS1 from iron deficient barley root. This transporter was found to selectively transport the mugineic acid iron complex; however, the uptake of complexes with other metals was substantially reduced compared to that with iron.¹⁰⁾ By using synthetic DMA and chimeric proteins, the amino acid sequence responsible for the substrate specificity of HvYS1 was elucidated. We determined that residues 350–392 in HvYS1, which include the variable regions in the seventh outer membrane loops (Fig. 2), are essential and sufficient to define the transport specificity.¹⁸⁾ However, the detailed mechanism of iron complex recognition remains unknown. We would like to determine how this outer membrane loop distinguishes the central metal of the complex. Therefore, in this study, we attempted to develop mugineic acid derivatives as molecular probes for tracking the mugineic acid inside plants and thus elucidating the three-dimensional structure and dynamic behavior of the transporters.

In the probe synthesis, it was important to determine which site among many polar functional groups of mugineic acid allowed for the introduction of a labeling group. Actually, the introduction of labeling groups into mugineic acid have been studied, but no successful cases have been reported. The main reason is that the probes lost the ability to form a complex with iron due to structural changes caused by the introduction of the labeling group, making it difficult to pass through the transporter. By contrast, we focused on the hydroxyl group at the 2′-position as a potential site to introduce a labeling group. Therefore, MA and 2′-epi-mugineic acid 10 were also synthesized, and we investigated their ability to transport an iron complex through the HvYS1 transporter expressed in oocytes, along with DMA. MA and 10 were prepared by converting N-benzyloxycarbonyl(Cbz)-L-allylglycine 8 to 9 by allylic oxidation, and then synthesizing it in a similar manner to the synthesis of DMA (Fig. 5a). Measurement of their activities showed that the synthetic MA, epi-MA (10), and DMA had the same iron transport activity as natural MA¹⁷⁾ (Fig. 5b). It was revealed that the 2′-hydroxyl group is not involved in the activity, since neither its presence or absence, nor the opposite configuration of the 2′-hydroxyl group, affects the formation and uptake of the iron complex. Thus, we next investigate the establishment of a method for introducing a labeling group to the 2′-hydroxyl group.

Fig. 5. a) Synthesis of MA and epi-MA; b) Iron Transporting Activities of Synthetic MA–, 2′-epi-MA–, and DMA–Iron(III)

The activity was accessed by two-electrode voltage clamp analysis with Xenopus oocytes. Currents relative to natural MA–iron(III) are shown when the value of natural MA–iron(III) is taken as 100. Error bars: standard deviation (S.D.) (n=5–8).

The previously obtained 9 was converted into protected mugineic acid 11 with a free-hydroxyl group at the 2′-position by the same method. Next, although various efforts were made to introduce a labeling group at the 2′-hydroxyl group of 11, it was difficult to directly incorporate a labeling group. Therefore, the introduction of various substituents was carefully examined, and it was found that only a small and reactive propargyl group was introducible. Fortunately, the click reaction of the propargyl group of the resulting 12 proceeded smoothly. Thus, 12 was treated with copper(I) iodide and 4-bromobenzyl azide in tetrahydrofuran (THF), followed by deprotection under an acidic condition, whereby the desired triazole mugineic acid 13a was obtained in quantitative yield. Similarly, photoaffinity labeled 13b and fluorescent labeled 13c were also synthesized (Chart 2).

Chart 2. Comprehensive Synthesis of Labeled Mugineic Acids through Click Reaction

THF, tetrahydrofuran; TFA, trifluoroacetic acid.

Having prepared various labeled mugineic acids, complex forming ability and iron transporting activity were measured. The complex formation of 13a–c with iron was confirmed by agreement with the theoretical value and the isotope pattern of trivalent iron in the MS spectrum, suggesting that the labelled mugineic acids 13a–c form a 1 : 1 complex with iron. Next, the transport activity of 13–iron complex was measured using oocytes that overexpressed the transporter (HvYS1), and it was revealed that all of the labeled compounds, 13a–c, retained transport activity (Fig. 6). Thus, we are the first to establish a method for introducing a labelling group into mugineic acid.

Fig. 6. Iron(III) Complex Transport Activities of Labeled Mugineic Acids

Fe(III)-complex transport activities of synthetic labeled mugineic acids were measured by two-electrode voltage-clamp analysis in Xenopus oocytes. Currents induced by Fe(III) complexes of the MA derivatives, DMA 13a, 13b, and 13c (each 50 µM) in oocytes, were injected with HvYS1 cRNA (black bars, n=5–11) or water (white bars, n=3–10). Significant differences between injection with HvYS1 cRNA and water, as determined by Tukey’s test, are indicated by an asterisk (*, p<0.01).

Having confirmed the transport activity of coumarin-labelled 13c, fluorescence observation was conducted to examine the cell uptake of 13c. An oocyte overexpressing the transporter (HvYS1) and a control cell were treated with a 50 µM aqueous solution of 13c–iron complex, and after 15 min the oocytes were washed with water. When each cell was observed with a fluorescence microscope, the inside of HvYS1-expressed oocyte cell was clearly visualized in fluorescence mode (Fig. 7d), whereas the inside of the control oocytes was not (Fig. 7b). This experiment indicated that the iron complex of the fluorescent labelled mugineic acid 13c was incorporated through the HvYS1 transporter. Therefore, it was experimentally proved that the MA–iron(III) complex is internalized through the transporter (HvYS1).¹⁹⁾

Fig. 7. Observation of Fluorescence-Labeled MA 13c as Its Fe(III) Complex in Xenopus Oocytes

Oocytes injected with a, b) water as a control, or c, d) HvYS1 cRNA were incubated in ND96 buffer containing 13c–Fe(III) (50 µM) for 15 min at 16°C. Uptake of fluorescence-labeled MA–Fe(III) was monitored using a laser scanning confocal imaging system (Olympus FluoView 1000) in a differential interference contrast (DIC) mode (a and c) or in a laser fluorescence mode (b and d). Scale bar: 200 µm (a–d).

5. CONCLUSION

In this review, our recent efforts to elucidate the mechanism of iron acquisition in graminaceous plant have been introduced. Our initial research for the isolation and identification of a MA–Fe(III) transporter gene advanced the various studies on mugineic acids, and now we have contributed to the organic chemistry approach by introducing a method for evaluating the transport activity of mugineic acid derivatives. The developed mugineic acid derivatives as fluorescent probes and photoaffinity probes will allow us to elucidate the fate of mugineic acid inside a plant and determine the dynamic function of the transporter, respectively, in the near future. Specific labeling of transporters using mugineic acid probes has already been successful, and analysis of the dynamic behavior of transporters in living plants is currently underway in our laboratory. In addition, practical synthesis of DMA was developed for mechanistic studies of the transporter HvYS1, which expands the possibility of DMA as a fertilizer. It was demonstrated that the addition of DMA enables the cultivation of rice, even under alkaline conditions. The world population is estimated to reach 9.8 billion by 2050.²⁰⁾ The Food and Agriculture Organization of the United Nations (FAO) suggests that food production must increase up to 1.5 times to feed the growing world population.²¹⁾ Thus, agriculture using DMA in alkaline soils would present a practical solution to efficiently increase global food production, without severe environmental destruction, in order to avoid a pending food crisis.

Acknowledgments

We thank professors Shoichi Kusumoto (Suntory Foundation for Life Sciences), Mugio Nishizawa (Tokushima Bunri University), Keiji Tanino (Hokkaido University), and Yasufumi Ohfune (Osaka City University) for their support of this research and valuable discussion. This work was partially supported by JSPS KAKENHI, Grant Numbers JP16H01156 in Middle Molecular Strategy, and JP16H03292. K.N. is grateful to the Naito Foundation, the Yamada Foundation, the Kurata Foundation, and the Uehara Memorial Foundation for support through research funds.

Conflict of Interest

The authors declare no conflict of interest.

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