Identification and Functional Analysis of Two Mitoferrins, CsMIT1 and CsMIT2, Participating in Iron Homeostasis in Cucumber

Mitochondria are one of the major iron sinks in plant cells. Mitochondrial iron accumulation involves the action of ferric reductase oxidases (FRO) and carriers located in the inner mitochondrial membrane. It has been suggested that among these transporters, mitoferrins (mitochondrial iron transporters, MITs) belonging to the mitochondrial carrier family (MCF) function as mitochondrial iron importers. In this study, two cucumber proteins, CsMIT1 and CsMIT2, with high homology to Arabidopsis, rice and yeast MITs were identified and characterized. CsMIT1 and CsMIT2 were expressed in all organs of the two-week-old seedlings. Under Fe-limited conditions as well as Fe excess, the mRNA levels of CsMIT1 and CsMIT2 were altered, suggesting their regulation by iron availability. Analyses using Arabidopsis protoplasts confirmed the mitochondrial localization of cucumber mitoferrins. Expression of CsMIT1 and CsMIT2 restored the growth of the Δmrs3Δmrs4 mutant (defective in mitochondrial Fe transport), but not in mutants sensitive to other heavy metals. Moreover, the altered cytosolic and mitochondrial Fe concentrations, observed in the Δmrs3Δmrs4 strain, were recovered almost to the levels of WT yeast by expressing CsMIT1 or CsMIT2. These results indicate that cucumber proteins are involved in the iron transport from the cytoplasm to the mitochondria.