Characterization of Ferric Reductase Oxidase (FRO) Genes in Populus tremula L.

Many plant species suffer from iron chlorosis, causing millions of dollars loss yearly. Meanwhile, iron is an essential nutrient for human beings and iron deficiency leads to the major human nutritional disorder of anemia, particularly in populations of children and women. Iron uptake and transport in plants are controlled by a group of genes mainly in Ferric Reductase Oxidase (FRO) and Iron-Regulated Transporter (IRT) families. Genes in the FRO family encode the plasma membrane-bound Fe (III)-chelate reductase that reduces insoluble Fe (III) to soluble Fe (II), making Fe available for plant absorption. The function of FRO in Fe metabolism in plants has been verified in many herbaceous species including Arabidopsis, rice, tobacco, and soybean. In this research, the activity of FRO was compared between an iron-deficiency tolerant (PtG) and an iron-deficiency susceptible (PtY) trees of Populus tremula. In PtY, FRO activity in both leaf and root tissues was higher than in PtG under iron-deficient conditions. When Fe (III)-EDTA was applied to PtY, FRO activity increased in root tissues, but decreased in leaf tissues. In PtG, the activity of FRO was not notably changed regardless of iron status. Expression of PtFRO genes cloned from Populus tremula in response to iron deficiency in PtG and PtY was investigated using quantitative real-time PCR (qPCR). Primers for qPCR were designed based on three putative FRO genes annotated in the genome sequence of Populus trichocarpa. The putative amino acid analysis revealed that the three genes contain three signature domains of ferric chelate reductase gene (Ferric_reduct, FAD_binding_8, and NAD_binding_6 domains). The three genes were named as PtFRO3, PtFRO4, and PtFRO7 according to the similarity to FRO genes in Arabidopsis. Sequence analysis showed that PtFRO3, PtFRO4, and PtFRO7 are 96%-99% similar to the corresponding gene sequences in Populus trichocarpa. Expression of PtFRO3 gradually increased under iron deficiency in leaves and decreased after Fe was resupplied. In PtY, expression of PtFRO4 and PtFRO7 decreased under iron deficiency followed by a progressive increase. In PtG, expression of PtFRO4 first increased then decreased, while expression of PtFRO7 gradually increased under iron deficiency. Similar to PtFRO3, resupply of Fe also decreased the expression of both PtFRO4 and PtFRO7. The research will help understand the role of FRO genes in iron metabolisms and address iron chlorosis in woody species.