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. 2006 Nov-Dec;1(6):290–292. doi: 10.4161/psb.1.6.3590

Deoxymugineic Acid Synthase

A Gene Important for Fe-Acquisition and Homeostasis

Khurram Bashir 1, Naoko K Nishizawa 1,2,✉
PMCID: PMC2634242  PMID: 19704569

Abstract

Plants can be classified in two groups based on Iron (Fe) acquiring strategy. Graminaceous plants (Strategy II) acquire iron through mugineic acid family phytosiderophores (MAs). All MAs are synthesized from L-Met, sharing the same pathway from L-Met to 2′-deoxymugineic acid (DMA) and the subsequent steps may differ depending on plant specie and cultivar. DMA is synthesized through the reduction of a 3″-keto intermediate by deoxymugineic acid synthase (DMAS). Previously, all the genes involved in the synthesis of DMA have been cloned with the exception of DMAS. Recently we have reported the isolation of DMAS genes from rice, wheat, maize and barley. The DMAS belongs to aldo-keto reductase superfamily (AKR). The expression of each of the above DMAS genes is upregulated under Fe-deficient conditions in root tissue, and that of OsDMAS1 and TaDMAS1 are upregulated in shoot tissue. It seems that the expression of DMAS is not regulated at posttranscriptional level. Analysis of OsDMAS1 promoter-GUS transgenic rice suggested that DMA may have role in Fe homeostasis in rice. The cloning of DMAS genes is an important step to develop transgenic rice with increased biosynthesis and section of DMA and ultimately resistant to Fe-deficiency in calcareous soils.

Key Words: deoxymugineic acid, iron, Aldo-Keto reductase, MAs

Iron (Fe) is essential for various cellular events such as respiration and photosynthetic electron transport. Fe is also essential for the synthesis of heme and chlorophyll and low chlorophyll content (chlorosis) of young leaves is the most obvious visible symptom of Fe-deficiency. As Fe is sparingly soluble in neutral to alkaline soils,1 plants have developed sophisticated and tightly regulated mechanisms to acquire Fe from the soil, which can be grouped into two strategies.2 Graminaceous plants (Strategy II plants) solubilize soil Fe by secreting mugineic acid family phytosiderophores (MAs), from their roots.3,4 The resulting Fe(III)-MA complexes are then reabsorbed into the roots through a specific transporter. The production and secretion of MAs markedly increases in response to Fe-deficiency, and tolerance to Fe-deficiency is strongly correlated with the quantity and quality of the MAs secreted. For example, rice, wheat, and maize secrete only 2′-deoxymugineic acid (DMA) in relatively low amounts and are thus susceptible to low Fe availability. In contrast, barley secretes large amounts of many types of MAs, and is therefore more tolerant to low Fe availability.5,6

The biosynthetic pathway for MAs has been characterized.7–10 All MAs are synthesized from L-Met, sharing the same pathway from L-Met to DMA and the subsequent steps may differ depending on the plant specie and cultivar (Fig. 1). We adopted different strategies to clone DMAS. Finally, OsDMAS1 was identified as a member of aldo-keto reductase superfamily (AKR) and its orthologs from barley, wheat and maize were cloned.11 With the isolation of graminaceous DMAS, all the genes of MAs biosynthetic pathway have been isolated, including S-adenosylmethionine synthetase (SAMS),12 nicotianamine synthase (NAS),13 nicotianamine aminotransferase (NAAT),14 IDS2, and IDS3.15–16 Moreover, we have proved that 3″-keto acid is an intermediate in the pathway from NA to DMA.

Figure 1.

Figure 1

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Biosynthetic pathway of mugineic acid family phytosiderophores. Three molecules of S-adenosyl methionine are combined by NA synthase (NAS) to form nicotianamine (NA). The amino group of NA is transferred by NA aminotransferase (NAAT), and the resultant 3″-keto intermediate is reduced to 2′-deoxymugineic acid (DMA) by deoxymugineic acid synthase (DMAS). The subsequent steps differ with the plant species and cultivar.

The DMAS genes belong to AKR and have homology to Papaver somniferum codeinone reductases (AKR4B2-3) and Medicago sativa (AKR4A2) and Glycine max (AKR4A1) chalcone polyketide reductases (Fig. 2). AKR have 15 families (AKR1–AKR15)17 and the criteria for classification of AKR is that members within a family have less than 40% amino acid sequence identity with other families and that members within a subfamily have greater than 60% sequence identity.18 Based on this criterion, DMAS proteins do not fall in existing subfamilies of AKR4.11 The sequence of DMAS was submitted to AKR database maintained at http://www.med.upenn.edu/akr/.18 After introduction of conservative substitutions, the identity with existing members of AKR4B reached up to 65% and ZmDMAS1, OsDMAS1, HvDMAS1 and TaDMAS1 were assigned the numbers from AKR4B5 to AKR4B8 respectively. The substrate-binding domain was identified as described.19 In rice, the putative substrate binding site was identified as A(50)-HY(52–53)-W(85)-HW(116–17)-V(119)-KA(127–28)-G(305)-Y(307)-S(309). The NADPH-binding domain is also conserved, which is a striking feature of the AKRs.19 As members of AKR family are functionally redundant, there are chances that other members of AKR family may show the DMAS activity. The existence of multiple genes for NAS and NAAT in rice20 and barley13–14,21 also supports this hypothesis. Although the closest paralog of OsDMAS1 did not show enzyme activity in our hands, the existence of more than one gene can not be ruled out.

Figure 2.

Figure 2

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Phylogenetic characterization of DMAS genes. Unrooted phylo-genetic tree of the aldo-keto reductase superfamily (AKR4). The details and accession numbers of the AKR proteins are at www.med.upenn.edu/akr/members.html. AK102609 is a homolog of OsDMAS1 that lacks DMAS activity.

An important question is the subcellular localization of DMAS. In silico analysis showed that DMAS may be localized to cytoplasm or microbody. On the other hand, MAs are thought to be synthesized in vesicles derived from rough ER.22 Based on optimum pH (pH 9), NAS23 and NAAT24 are thought to be localized to these vesicles. The optimum pH for enzymatic activity of DMAS (pH 8–9) suggests that the enzyme localizes to some subcellular organelle(s) similar to NAS and NAAT where DMA is synthesized and stored until secretion.

Like all other genes involved in the MA biosynthetic pathway, DMAS genes are upregulated under Fe-deficient conditions in root tissue. Comparison of Northern and Western blot analysis revealed that DMAS is not regulated posttranscriptionally. DMA is detected in Fe-sufficient shoot in rice, which is possibly translocated from roots in a complex with Fe.25 This hypothesis is strengthened by the fact that under Fe-sufficient conditions, OsDMAS1 expression was only observed in the portions of roots that are involved in long-distance transport. Under Fe-deficient conditions, the amount of DMA increases in rice shoots, and the expression of DMAS suggest that DMA is at least partially synthesized in shoot tissue. This DMA is thought to be involved in Fe homeostasis and does not participate in the acquisition of Fe from the soil.

Alkaline soils cover approximately 30% of world's arable land and rice grown on such soils is prone to Fe-deficiency. Our laboratory is engaged in the development of transgenic rice plants that are tolerant of alkaline soils with low Fe availability. Previously a genomic fragment containing two NAAT genes from barley were introduced in rice and the resulting transformants produced four times more grains as compared with control, although the production of DMA was only 1.8 times higher than control.26 The introduction of HvDMAS1, alone or in combination with NAS, NAAT, IDS2 and IDS3, into rice would be a good strategy to develop transgenic rice plants tolerant to Fe-deficiency in calcareous soils. On the other hand the plants lacking or over expressing DMAS will reveal the role of DMA in iron homeostasis.

Acknowledgements

We are thankful to Dr. Takanori Kobayashi for critically reading the manuscript. K. Bashir is a Ph.D. student supported by ministry of Education, Culture, Sports, Science and Technology, Japan.

Abbreviations

AKR

Aldo-Keto reductase superfamily

DMA

2′-deoxymugineic acid

Fe

iron

MAs

mugineic acid family phytosiderophores

Addendum to: Cloning and Characterization of Deoxymugineic Acid Synthase Genes in Graminaceous Plants. Bashir K, Inoue H, Nagasaka S, Takahashi M, Nakanishi H, Mori S, Nishizawa NK. J Biol Chem. 2006;281:32395–32402. doi: 10.1074/jbc.M604133200.

Footnotes

Previously published online as a Plant Signaling & Behavior E-publication: http://www.landesbioscience.com/journals/psb/abstract.php?id=3590

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