In vivo evidence that Ids3 from Hordeum vulgare encodes a dioxygenase thatconverts 2 '-deoxymugineic acid to mugineic acid in transgenic rice

We proposed that an Fe-deficiency-induced gene, Ids3 (Iron deficiency Speci fic clone no. 3), from barley (Hordeum vulgare L.) roots encodes a dioxygen ase that catalyzes the hydroxylation step from 2 ' -deoxymugineic acid (DMA ) to mugineic acid (MA). To prove this hypothesis, we introduced the Ids3 g ene into rice (Orvza sativa L.), which lacks Ids3 homologues and secretes D MA, but not MA. Transgenic rice plants, carrying either Ids3 cDNA or a barl ey genomic DNA fragment (20 kb) containing Ids3, were obtained using Agroba cterium-mediated transformation. Ids3 cDNA under the control of the caulifl ower mosaic virus 35S promoter was constitutively expressed in both the roo ts and the leaves of the transgenic rice, regardless of Fe nutrition status . In contrast, in the roots of transformants carrying a barley genomic frag ment, transcripts of Ids were markedly increased in response to Fe deficien cy. Slight expression of Ids3 was also observed in the leaves of the Fe-def icient plants. Western blot analysis confirmed the induction of Ids3 in res ponse 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 Fz-deficiency-inducible promoter in rice, as wel l as in barley. Both kinds of transgenic rice secreted MA in addition to DM A 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 .