Directed evolution of yeast ferric reductase to produce plants with tolerance to iron deficiency in alkaline soils

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Abstract
One of the most serious problems in agriculture worldwide is low iron (Fe) availability, due to high soil pH. About 30% of arable land is too alkaline for optimal crop production. Non-graminaceous and dicot plants, which use a reduction strategy to uptake Fe, suffer from Fe deficiency under these conditions, because the ferric chalets reductase in the root plasma membrane functions inefficiently at high pH. The refrel (reconstructed yeast ferric reductase) gene was subjected to random mutagenesis to obtain variants with high activity under high pH conditions. A mutant library was screened using a yeast in vivo assay system, and screens at pH 8.0 and 8.5 produced 10 candidates. In vivo ferric reductase activity was analyzed quantitatively. Yeast cells carrying the variant with the highest ferric reductase activity showed 6.0, 8.7, and 38 times greater activity at pH 8.0, 8.5, and 9.0, respectively, than did cells containing the original refrel gene. An amino acid substitution at position 312 was common to most of the high-activity variants. This substitution is believed to play an important role in the increased reductase activity at high pH. Interestingly, this mutation is near a hams-coordinating histidine co on, and the corresponding residue is probably located in the intramembranous region close to the cytoplasm. The variant gene with the highest reductase activity was introduced into tobacco, and transgenic tobacco carrying the gene showed enhanced tolerance to low Fe availability. This result should be useful in the engineering of non-graminaceous and dicot plants tolerant to Fe deficiency in alkaline soils.