Creation of a reporter plant of Mn toxicity

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Tomato plants (Lycopersicon esculentum L.cv Ponderosa), grown for 10 days with complete nutrient after germination, were subjected to Mn^<2+> deficiency for the following 10 days. During the Mn^<2+> deficiency, the roots lost most of their Mn, but this was accompanied by the emergence of high-affinity Mn^<2+> uptake. A metal-binding peptide was isolated from chymotrypsin-digested membrane proteins of Mn-depleted root tissues. A cDNA for the metal-binding protein was amplified by PCR with mRNAs from Mn-depleted tomato roots. A full-length cDNA (Mdip1) was shown to encode a precursor protein having a 27-amino acid leader sequence at the NH_2-terminus. The leader extension targeted GUS to cell surface of transgenic tobacco with chimeric transgene of GUS and the gene encoding N-terminal extension of Mdip1. The metal-binding site is located in the NH_2-terminal region of the 197-amino acid-long mature protein. A northern blot analysis revealed that the expression of Mdip1 was enhanced by Mn deficiency. Transformation of tobacco plant with Mdip1 cDNA conferred an oxalate oxidase activity, indicating that Mdip1 encodes an oxalate oxidase which was synthesized as a response to low bioavailability of soil Mn. Resulting H_2O_2 from oxalate oxidation might be a reductant for insoluble MnO_2 upon Mn^<2+> acquisition.