Structural determination of mugineic acid, an iron(III)-chelating substance secreted from graminaceous plants for efficient iron uptake

Abstract

Iron is an essential element for organisms, but its solubility in soil is often extremely low. Previously, plants were considered to take up iron only after its reduction to ferrous ions. Takagi reported that oat and rice secrete chelating substances that solubilize ferric iron in the rhizosphere for efficient iron uptake. In 1978, Takemoto et al. reported the chemical structure of an iron-chelating compound secreted from barley roots, designated as mugineic acid. Mugineic acid and its derivatives, collectively known as mugineic acid family phytosiderophores (MAs), chelate ferric iron using octahedral hexacoordination. The specific iron uptake system by MAs in graminaceous plants was later classified by Römheld and Marschner as Strategy II, in contrast to Strategy I for reduction-based iron uptake by non-graminaceous plants. Further studies on MAs by Japanese researchers led to the identification of their biosynthetic pathways, corresponding enzymes and encoding genes, their regulation mechanisms, and the production of iron deficiency-tolerant and iron-rich crops.

1. Identification of chelation-based iron uptake in plants

Iron (Fe) is an essential micronutrient for most organisms, including all animals and plants. Although Fe is abundant in soils, it is sparingly soluble in aerobic conditions at neutral or alkaline conditions such as in calcareous soils, which include about 30％ of cultivated areas around the world.¹⁾ Plants growing in such soils often suffer from Fe deficiency due to the extremely low solubility and bioavailability of Fe.²⁾ Historically, studies on Fe uptake mechanisms in plants in low Fe availability conditions were mainly focused on dicotyledonous species, which take up Fe after reduction of ferric Fe [Fe(III)] to ferrous ions (Fe^2＋) on the root surface.^3),4) On the other hand, some bacteria are known to biosynthesize efficient Fe(III) chelators, called siderophores, for the solubilization of Fe in the environment and subsequent uptake of Fe(III)-chelate complexes.^5)-7) In 1976, Dr. Sei-ichi Takagi reported that root-washings from hydroponically cultured oat (Avena sativa L. var. Zenshin) and rice (Oryza sativa L. var. Fujiminori), both belonging to graminaceous species, contained specific compounds that solubilized and chelated hydrated Fe(III) oxide effectively.⁸⁾ The release of the compounds was induced in Fe-deficient conditions, especially in oat, which is much more tolerant of Fe deficiency than rice.⁸⁾ Dr. Takagi further reported chemical characterization of the chelating compounds as an amphoteric, “heat-stable, acid-hydrolyzable non-macromolecule of extraordinarily high polarity”.⁸⁾ This finding indicated the presence of a novel Fe uptake system in plants using Fe(III) chelation instead of reduction, similar to the siderophore system in bacteria, which was previously considered to be absent in plants.

2. Chemical structure and nomenclature of mugineic acid and its derivatives

In 1978, Dr. Tsunematsu Takemoto and coauthors⁹⁾ reported the structure of an Fe-chelating compound isolated from root-washings of barley (Hordeum vulgare L. cv. Minorimugi¹⁰⁾) and designated this compound as mugineic acid.⁹⁾ “Mugi” derives from the Japanese word indicating the specific group of plants including wheat, barley, and rye, and “ne” means roots in Japanese. Thus, mugineic acid means a specific acid secreted from the roots of some graminaceous plants. Japanese-derived nomenclature of mugineic acid, often abbreviated as MA, was then applied worldwide in English, together with the specific Fe(III)-acquisition system in graminaceous plants first reported by Takagi.⁸⁾

The chemical structure of MA,⁹⁾ as shown in Fig. 1, contains three carboxy groups, two amino-derived groups, and two hydroxy groups, which is consistent with the chemical properties of the “chelating compounds” described by Takagi.⁸⁾ Later, structures of eight derivatives of MA were reported, such as avenic acid from oat,¹¹⁾ 2′-deoxymugineic acid (DMA) from wheat,¹²⁾ 3-epihydroxymugineic acid (epiHMA) from barley,^12),13) 3-hydroxymugineic acid (HMA) from rye, and 3-epihydroxy-2′-deoxymugineic acid (epiHDMA) from a perennial grass Lolium perenne cv. Tove (Fig. 1).¹⁴⁾ These derivatives conserve similar Fe(III)-chelating capacity and function in Fe uptake in graminaceous plants, and thus are collectively called mugineic acid family phytosiderophores (MAs).¹⁵⁾ Structural analyses revealed that MAs chelate Fe(III) by forming octahedral hexacoordination using three carboxy groups, two amino-derived groups, and one hydroxy group.^16)-18) Types and amounts of MAs secreted from graminaceous plants differ significantly depending on the species, and they are correlated with the tolerance of the species to low Fe availability.^19)-21)

Fig. 1

Chemical structures and biosynthetic pathway of mugineic acid and its derivatives in graminaceous plants (modified from Kobayashi et al. 2010).⁶⁶⁾ Key molecules referred in the main text are highlighted. Blue arrows and neighboring blue letters indicate the biosynthetic reactions and their corresponding enzymes, respectively. Currently, four other members of the mugineic acid family phytosiderophores have been identified.

On the other hand, another compound structurally similar to MA, called nicotianamine (NA) (Fig. 1), had been reported as a component of tobacco (Nicotiana tabacum L.) leaf.²²⁾ Later studies revealed that NA is a biosynthetic precursor of MAs in graminaceous plants (Fig. 1; refer to session 3). In addition, it functions as a potent chelator of ferrous and ferric Fe as well as other divalent metals including zinc (Zn), copper, manganese, cobalt and nickel, and facilitate internal transport of these metals inside the plant body in both graminaceous and non-graminaceous plants.^23),24)

3. Identification of molecular components of mugineic acid-based iron uptake

The identification of the specific Fe uptake system in graminaceous plants using MAs was greeted with surprise by plant nutritionists around the world. In 1986, Drs. Volker Römheld and Horst Marschner in Germany reported precise confirmation of the Fe uptake mechanisms in various plant species, and proposed that the Fe uptake systems of plants in low Fe availability are largely divided into two groups: Strategy I by non-graminaceous plants, and Strategy II by graminaceous plants.¹⁹⁾ Strategy I, also called the reduction strategy, depends on the induction of ferric-chelate reductase on the root surface that reduces Fe(III) in the rhizosphere. The Fe^2＋ ions generated are subsequently taken up. This system is aided by the excretion of protons and organic substances such as phenolics for the solubilization and reduction of Fe(III). On the other hand, Strategy II, also called the chelation strategy, relies on the production and secretion of MAs. The MAs secreted efficiently solubilize Fe(III) in the rhizosphere, and the generated Fe(III)-MA complexes are subsequently taken up into the roots without reduction. This classification by Römheld and Marschner¹⁹⁾ is totally consistent with recent knowledge on molecular components such as genes, proteins and other substances later identified and characterized,^25),26) except that recent studies revealed that Strategy I and Strategy II are not as mutually exclusive as supposed previously; i.e. some graminaceous plants take up Fe^2＋ ions in addition to Fe(III)-MAs,²⁷⁾ while some dicot species utilize Fe(III)-MAs in addition to Fe^2＋ uptake.²⁸⁾

The biosynthetic pathway of MAs was identified by Japanese groups (Fig. 1).^29)-33) Among these, the group with Dr. Satoshi Mori and the present authors subsequently identified most of the corresponding enzymes and the genes encoding these. We found that MAs are biosynthesized from L-methionine via NA in the roots of graminaceous plants.^29),30) A key enzyme in this pathway, nicotianamine synthase (NAS), catalyzes the trimerization of S-adenosyl-L-methionine and the formation of an azetidine ring to form NA.³⁴⁾ This enzyme is present not only in graminaceous plants but also in all plants analyzed so far, providing NA for internal metal translocation.^23),24) In graminaceous plants, another key enzyme, nicotianamine aminotransferase (NAAT), converts NA to 3′′-keto acid,^23),35) which is subsequently reduced to DMA by deoxymugineic acid synthase (DMAS).³⁶⁾ DMA is present in all graminaceous species analyzed so far and is subjected to further hydroxylation by iron-deficiency-specific clone no. 2 (IDS2) and iron-deficiency-specific clone no. 3 (IDS3) dioxygenases or other derivatization to produce other MAs depending on the species.^37)-40)

In Fe-deficient barley roots, the secretion of MAs follows a distinct diurnal rhythm, which peaks just after sunrise and ceases within a few hours.¹⁰⁾ MAs are considered to be synthesized and accumulated until sunrise in particular vesicles near the root surface.^41)-43) The secretion of MAs is mediated by a membrane-bound specific efflux transporter TOM1,⁴⁴⁾ which is thought to occur immediately after the MAs exit from the vesicles through an unknown mechanism.²⁶⁾ Subsequent uptake of Fe(III)-MAs is mediated by membrane-bound YS1/YSL transporters.^45)-47) In barley roots, the secretion of MAs and the uptake of Fe(III)-MAs occur mainly in the apical zones of primary and secondary roots, as well as in newly formed primary roots.^48),49) In rice roots, the secretion of DMA and the uptake of Fe(III)-DMA are thought to occur across the whole root surface in Fe deficiency conditions, based on the expression of the corresponding genes.^{25),26),44),46)}

Identification of these biosynthetic genes of MAs and related transporter genes enabled us to manipulate them using transgenic approaches. Rice is highly susceptible to low Fe availability due to its low capacity to secrete DMA among MAs.²¹⁾ We introduced barley genomic fragments containing biosynthetic genes for MAs with their promoters into rice. These transgenic rice plants exhibited enhanced tolerance to low Fe availability in calcareous soils.^50),51) Overexpression of NAS genes also enhanced the accumulation of Fe and Zn in rice grains, contributing to improved mineral nutrition for human foods.^52),53)

We also noted that the expression of most of the genes for Strategy II Fe uptake is strongly induced in Fe-deficient conditions at the transcript level.^{25),26),34)-36),39),40),44)-47)} Subsequent studies by our group and others led to the identification of various regulators of Fe deficiency responses of these genes.^25),26),54) Such Fe deficiency responses in roots are thought to be regulated by both systemic signals of Fe nutritional status transmitted from the shoots and local signals of Fe availability in the apoplast or rhizosphere.^54)-56) Integration of these signals might be favorable for efficient uptake of Fe from the soils with uneven Fe distribution. Rice roots are reported to grow toward areas with high ammonium concentrations, but this growth phenomenon does not occur toward areas of high Fe concentration.⁵⁷⁾ We and other groups identified candidate Fe sensing molecules that modulate Fe deficiency responses.^54)-56),58) Among these, HRZ ubiquitin ligases bind to Fe and Zn and negatively regulate the expression of Fe deficiency-inducible genes for Fe uptake and translocation.⁵⁸⁾ Knocking-down HRZ genes or enhanced expression of transcription factors for inducing Fe deficiency responses also led to the production of rice lines tolerant to low Fe availability and the accumulation of high Fe and Zn levels in grains.^58)-60)

4. An analog of mugineic acid as an efficient iron-supplying agent

The properties of MAs for Fe acquisition in graminaceous roots prompted us to utilize them as Fe-supplying agents. Due to the high synthetic cost of MAs and their rapid degradation by soil microorganisms,⁶¹⁾ Drs. Motofumi Suzuki, Kosuke Namba and coauthors produced an analog of DMA, designated as proline-2′-deoxymugineic acid (PDMA) using improved chemical synthesis, as an alternative Fe-supplying agent (Fig. 2).⁶²⁾ Application of Fe(III)-PDMA solution to calcareous soils effectively remedies Fe chlorosis, a typical symptom of Fe deficiency, in rice and maize.^62),63) Application of free PDMA without metal chelation showed similar effects, indicating the efficacy of PDMA to solubilize soil Fe in alkaline conditions to supply Fe for graminaceous plants.⁶²⁾ The effect of PDMA was sustained for about 2 weeks after a single application, showing longer sustainability than DMA, but PDMA is also biodegradable, making it suitable for field application.⁶²⁾ Structural analysis of the barley HvYS1 transporter, which takes up Fe(III)-DMA in the Strategy II system, showed similar transport mechanisms between Fe(III)-DMA and Fe(III)-PDMA.¹⁸⁾ In addition, Fe(III)-PDMA is also effective for non-graminaceous crops such as cucumber, pumpkin, and field-grown peanuts.^64),65) Fe(III)-PDMA is highly reducible by cucumber roots, indicating the possibility of its capacity to supply Fe also by Strategy I.⁶⁴⁾

Fig. 2

(Color online) Structures of 2′-deoxymugineic acid and its analog proline-2′-deoxymugineic acid as a novel iron-supplying agent.

5. Conclusions

Identification of the chemical structure of MA by Takemoto et al.⁹⁾ in addition to its nomenclature, provided a molecular basis for the original identification of the novel Fe uptake system of plants by Takagi et al.⁸⁾ These findings created a paradigm shift in plant Fe nutrition, leading to the proposal of Strategy I and Strategy II by Römheld and Marschner,¹⁹⁾ and becoming common knowledge in the plant nutrition field. Further extensive studies on MAs by our group and others paved the way for understanding of the molecular mechanisms of Fe homeostasis in plants, leading to the development of Fe-efficient crops with increased bioproduction in alkaline soils as well as generating Fe- and Zn-rich crops for the improvement of human mineral nutrition.

Acknowledgements

We thank Dr. Satoshi Mori (NPO-WINEP) for helpful suggestions.

Notes

Contributed by Naoko K. NISHIZAWA, M.J.A.; Edited by Akira ISOGAI, M.J.A.

Correspondence should be addressed to: T. Kobayashi and N. K. Nishizawa, Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa 921-8836, Japan (e-mail: [e-mail] [T. K.]; [e-mail] [N. K. N.]).

Footnotes

This paper commemorates the 100th anniversary of this journal and introduces the following paper previously published in this journal. Takemoto, T., Nomoto, K., Fushiya, S., Ouchi, R., Kusano, G., Hikino, H., Takagi, S., Matsuura, Y. and Kakudo, M. (1978) Structure of mugineic acid, a new amino acid possessing an iron-chelating activity from roots washings of water-cultured Hordeum vulgare L. Proc. Jpn. Acad. Ser. B 54 (8), 469-473 (https://doi.org/10.2183/pjab.54.469).

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• 64)  Ueno, D., Ito, Y., Ohnishi, M., Miyake, C., Sohtome, T. and Suzuki, M. (2021) A synthetic phytosiderophore analog, proline-2′-deoxymugineic acid, is efficiently utilized by dicots. Plant Soil 469, 123-134.
• 65)  Wang, T., Wang, N., Lu, Q., Lang, S., Wang, K., Niu, L. et al. (2023) The active Fe chelator proline-2′-deoxymugineic acid enhances peanut yield by improving soil Fe availability and plant Fe status. Plant Cell Environ. 46, 239-250.
• 66)  Kobayashi, T., Nakanishi, H. and Nishizawa, N.K. (2010) Recent insights into iron homeostasis and their application in graminaceous crops. Proc. Jpn. Acad. Ser. B. 86, 900-913.

Related Articles

• Takemoto, T., Nomoto, K., Fushiya, S., Ouchi, R., Kusano, G., Hikino, H., Takagi, S., Matsuura, Y. and Kakudo, M. (1978) Structure of mugineic acid, a new amino acid possessing an iron-chelating activity from roots washings of water-cultured Hordeum vulgare L. Proc. Jpn. Acad. Ser. B 54 (8), 469-473

Appendices

[From Proc. Jpn. Acad. Ser. B, Vol. 54 No. 8, pp. 469-473 (1978)]

Non-standard abbreviation list

DMA

2′-deoxymugineic acid

DMAS

deoxymugineic acid synthase

epiHDMA

3-epihydroxy-2′-deoxymugineic acid

epiHMA

3-epihydroxymugineic acid

Fe

iron

HMA

3-hydroxymugineic acid

IDS2

iron-deficiency-specific clone no. 2

IDS3

iron-deficiency-specific clone no. 3

MA

mugineic acid

MAs

mugineic acid family phytosiderophores

NA

nicotianamine

NAAT

nicotianamine aminotransferase

NAS

nicotianamine synthase

PDMA

proline-2′-deoxymugineic acid

SAMS

S-adenosyl-L-methionine synthetase

Zn

zinc

Profile

Takanori Kobayashi was born in 1975. He earned his bachelor's degree in the Faculty of Agriculture at the University of Tokyo in 1998. He obtained his master's degree in 2000, and his Ph.~D. (Agricultural Sciences) in 2003 in the Graduate School of Agricultural and Life Sciences at the University of Tokyo. He started to work as a research fellow at the University of Tokyo from 2002, and he has been a specially appointed assistant professor since 2008. He moved to the Research Institute for Bioresources and Biotechnology at Ishikawa Prefectural University as a special researcher (2011-2012 and 2015-2016), and as a specially appointed associate professor (2012-2015). He also worked as a PRESTO researcher at the Japan Science and Technology Agency (2011-2015). He then served as an associate professor at the Research Institute for Bioresources and Biotechnology at Ishikawa Prefectural University in 2016, and he was promoted to full professor at the same institute in 2019. His research area has remained in plant iron nutrition at the molecular level since the beginning of his research career. He identified various molecular components involved in the regulation of iron uptake responses in graminaceous plants, such as cis-acting elements, their interacting transcription factors, and ubiquitin ligases repressing the expression of iron uptake-related genes. His findings paved the way for the identification and characterization of as yet unknown iron sensors and iron nutritional signals in plant cells. He is also engaged in the production of crops with enhanced tolerance to iron deficiency, and mineral-rich crops with enhanced concentrations of iron and zinc in edible parts. He received the Young Scientists' Prize of the Commendation for Science and Technology by the Minister of Education, Culture, Sports, Science and Technology in 2015 for his work on “Research on molecular mechanisms of iron deficiency responses in plants”.

Naoko K. Nishizawa was born in 1945. She earned her bachelor's degree in Agricultural Chemistry, Faculty of Agriculture at the University of Tokyo in 1968 and her Ph. D. in Agricultural Sciences from the University of Tokyo. She started her career with studies on the ultra-structural analysis of crop plants and performed pioneering work on crop uptake of organic compounds and found that rice plants uptake and utilize organic nitrogen as a nutrient source by the mechanism of heterophagy in 1977. This was the first demonstration of the occurrence of a heterophagy mechanism in the plants. She was employed as an assistant professor at the University of Tokyo in 1982. She and Dr. Satoshi Mori started to work on iron acquisition mechanisms in graminaceous plants in collaboration with Prof. Sei-ichi Takagi, who found mugineic acid family phytosiderophores, and clarified the biosynthetic pathway of phytosiderophores. This led to subsequent extensive studies that identified many proteins and genes participating in iron acquisition and their regulatory network, and developed transgenic rice with enhanced tolerance to iron deficiency in calcareous soil. From 1995 to 1996, she was in the Laboratory of Plant Molecular Biology of The Rockefeller University and working on the molecular mechanism of signal transduction pathways in plants. She was promoted to lecturer in 1996 and to full professor in 1997. She moved to the Research Institute for Bioresources and Biotechnology at Ishikawa Prefectural University as a full professor in 2009 and was elected as president in 2019. She was elected as a Fellow of the American Association for the Advancement of Science (AAAS) in 2011. With Dr. Satoshi Mori, she was awarded the Japan Academy Prize in 2014. She is now an elected member of Japan Academy since 2020.