Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northernblot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.

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We proposed that an Fe-deficiency-induced gene, Ids3 (Iron deficiency specific clone no. 3), from barley (Hordeum vulgare L.) roots encodes a dioxygenase that catalyzes the hydroxylation step from 2'-deoxymugineic acid (DMA) to mugineic acid (MA). To prove this hypothesis, we introduced the Ids3 gene into rice (Oryza sativa L.), which lacks Ids3 homologues and secretes DMA, but not MA. Transgenic rice plants, carrying either Ids3 cDNA or a barley genomic DNA fragment (20 kb) containing Ids3, were obtained using Agrobacterium-mediated transformation. Ids3 cDNA under the control of the cauliflower mosaic virus 35S promoter was constitutively expressed in both the roots and the leaves of the transgenic rice, regardless of Fe nutrition status. In contrast, in the roots of transformants carrying a barley genomic fragment, transcripts of Ids3 were markedly increased in response to Fe deficiency. Slight expression of Ids3 was also observed in the leaves of the Fe-deficient plants. Western blot analysis confirmed the induction of Ids3 in response to Fe deficiency in the roots of the transformants carrying a genomic fragment. These expression patterns indicate that the 5'-flanking region of Ids3 works as a strong Fe-deficiency-inducible promoter in rice, as well as in barley. Both kinds of transgenic rice secreted MA in addition to DMA under Fe-deficient conditions, but wild-type rice secreted only DMA. This is in vivo evidence that IDS3 is the "MA synthase" that converts DMA to MA.

2'-Deoxymugineic acid (DMA), one of mugineic acid-family phytosiderophores (MAs), was synthesized in vitro both from Lmethionine and from nicotianamine (NA) with a cell-free system derived from root tips of iron-deficient barley (Hordeum vulgare L.). The reactions producing DMA from NA needed an amino group acceptor (i.e. 2-oxoglutarate, pyruvate, or oxalacetic acid) and a reductant (i.e. NADH or NADPH). The activity of the enzymes to produce NA from L-methionine was the highest at about pH 9.This biosynthetic activity was markedly induced by iron-deficiency stress. The synthesis of NA from S-adenosyl-L-methionine was more efficient than from L-methionine. From the results with the cell-free system reported here, we propose a revised biosynthetic pathway of MAs.Plants with 'Strategy 2' for iron acquisition (16) excrete iron-chelators, called phytosiderophores, from the roots to solubilize the external insoluble Fe3", and the amount of the secreted phytosiderophores increases under iron deficiency stress. MAs2 are the only examples of the phytosiderophores known so far (21). Their chemical properties (9,14,20,23) and their physiological significance (4,7,8,11,12,15,22) have already been established.The biosynthetic pathway of MAs is presently considered to be that three molecules of L-methionine are combined to form NA, which is then converted to DMA by deamination and hydroxylation at the 3"-carbon, and subsequent series of hydroxylation of DMA lead to other members of MAs. This pathway has been supported by the incorporation of L-['4C] methionine or L-['3C]methionine into MAs in vivo (5, 10) and the inhibition of deamination in vivo (6, 18). To characterize the biosynthesis furthermore, we recently developed a cellfree system for the biosynthesis of NA from L-methionine, using crude protein fraction from the roots of iron-deficient barley (18). For a larger scale preparation of the sample, we 'Partly supported by Grant-in-Aid (No. 62219004 and No. 62303014) from the Ministry of Education, Science and Culture, Japan 2Abbreviations: MAs, mugineic acid-family phytosiderophores; DMA, 2'-deoxymugineic acid; NA, nicotianamine; SAM, S-adenosyl-L-methionine; Taps, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid also reported the cell-free system from suspension culture of Nicotiana megalosiphon (19).On the above-mentioned hypothetical pathway of the biosynthesis of MAs, NA is a key substance, not only because its structure is much similar to MAs but also because the biosynthetic steps from NA to MAs can be thought to be evolutionally acquired by the 'strategy 2' plants, or lost by the 'strategy 1' plants: NA is widely distributed among the plants of both strategy 1 and 2, but MAs can be found only in some graminaceous plants (1,3,17). Nevertheless, there has been no direct evidence that NA is converted to DMA. The principal reason for this lack of data may be that there have been no systems to produce DMA in vitro.We thus improved our previous cell-free system in the following order. First, the cel...

We examined the effects of intake of Korean foxtail millet protein (FMP) on plasma levels of lipid, glucose, insulin, and adiponectin in genetically type 2 diabetic KK-A y mice. When mice were fed a normal FMP diet or a high-fat-high-sucrose diet containing FMP for 3 weeks, in both experiments plasma concentrations of high-density lipoprotein cholesterol (HDL-cholesterol) and adiponectin increased remarkably in comparison with a casein diet group, whereas concentrations of insulin decreased greatly and that of plasma glucose was comparable to that in the casein diet group. Considering the role of adiponectin, insulin, and HDL-cholesterol in diabetes, atherosclerosis, and obesity, it appears likely that FMP may improve insulin sensitivity and cholesterol metabolism through an increase in adiponectin concentration. Therefore, FMP would serve as another beneficial food component in obesity-related diseases such as type 2 diabetes and cardiovascular diseases.