Scopoletin 8-hydroxylase a novel enzyme involved in coumarin biosynthesis and iron-deficiency responses in Arabidopsis

Iron (Fe) deficiency is a serious agricultural problem, particularly for alkaline soils. Secretion of coumarins by Arabidopsis thaliana roots is induced under Fe-deficiency. An essential enzyme for the biosynthesis of major Arabidopsis coumarins, scopoletin and its derivatives, is Feruloyl-CoA 6'-Hydroxylase1 (F6'H1), that belongs to a large enzyme family of the 2-oxoglutarate and Fe(II)-dependent dioxygenases. Another member of this family, which is a close homologue of F6'H1, and is encoded by a strongly Fe-responsive gene, At3g12900, is functionally characterized in the presented work. We purified the At3g12900 protein heterologously expressed in Escherichia coli and demonstrated that it is involved in the conversion of scopoletin into fraxetin, via hydroxylation at the C8-position, scopoletin 8-hydroxylase (S8H). Its function in plant cells was confirmed by the transient expression of S8H protein in Nicotiana benthamiana leaves, followed by the metabolite profiling and the biochemical and ionomic characterization of Arabidopsis s8h knockout lines grown under various Fe regimes. Our results indicate that S8H is involved in coumarin biosynthesis, as part of mechanisms used by plants to assimilate Fe.