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

**Background:** Mitochondria are major iron sinks in plant cells, requiring iron for Fe-S cluster and heme biosynthesis. Iron import into mitochondria involves ferric reductase oxidases and transporters of the mitochondrial carrier family (MCF), including mitoferrins (MITs). While mitoferrins have been characterized in yeast, humans, rice, Arabidopsis, and potato, their role in cucumber—the third most-produced vegetable worldwide—was unknown. This study aimed to identify and functionally characterize mitoferrin-encoding genes in cucumber (Cucumis sativus).

**Methods:** Two cucumber genes, CsMIT1 and CsMIT2, were identified using Arabidopsis MIT1 (At2g30160) and MIT2 (At1g07030) cDNA sequences as queries against cucumber genomic contigs. Expression profiles were analyzed by real-time PCR in roots, hypocotyls, cotyledons, and first leaves of two-week-old seedlings grown under standard conditions, as well as under short-term (24 h) and long-term (two weeks) iron deficiency (30 µM BPS) or iron excess. Subcellular localization was assessed by expressing CsMIT1-GFP and CsMIT2-GFP fusion proteins in Arabidopsis thaliana protoplasts and yeast cells, with MitoTracker Red counterstaining and confocal microscopy. Functional complementation was tested in the Saccharomyces cerevisiae Δmrs3Δmrs4 double mutant (defective in mitochondrial iron import) grown under Fe-limited conditions (100 µM BPS). Substrate specificity was evaluated using yeast mutants sensitive to Cu, Cd, Co, and Zn. Cytosolic iron levels were measured via gentisate 1,2-dioxygenase (c-GDO) activity, and mitochondrial iron content was determined by flame atomic absorption spectrometry (AAS) following HNO3 digestion.

**Key Results:** CsMIT1 and CsMIT2 encode proteins of 331 and 311 amino acids (35.74 and 33.17 kDa), respectively, each with six transmembrane helices and conserved MCF motifs. Both transcripts were detected in all vegetative organs. CsMIT1 expression was approximately two-fold higher in cotyledons and leaves than in roots and hypocotyls. CsMIT2 expression was highest in cotyledons (approximately seven-fold higher than in roots, two-fold higher than in hypocotyls/leaves), with root levels more than three times lower than in hypocotyls and leaves. Under short-term (24 h) iron deficiency, both CsMIT1 and CsMIT2 mRNA levels decreased in leaves. Under short-term iron excess, CsMIT2 transcripts moderately increased in leaves. Under long-term iron deficiency, CsMIT1 mRNA levels increased in roots, while CsMIT2 was unaffected. CsMIT1-GFP and CsMIT2-GFP colocalized with MitoTracker Red in both Arabidopsis protoplasts and yeast cells. Expression of either CsMIT1 or CsMIT2 restored growth of the Δmrs3Δmrs4 mutant under Fe-limited conditions; co-expression of both did not further enhance growth. Neither protein restored growth of heavy metal-sensitive yeast mutants (Cu, Cd, Co, Zn). Cytosolic iron levels were two times higher in Δmrs3Δmrs4 cells with empty vector than in wild-type, while expression of CsMIT1-GFP or CsMIT2-GFP reduced cytosolic iron to wild-type levels. Mitochondrial iron levels were two times lower in Δmrs3Δmrs4 cells with empty vector than in wild-type, and expression of CsMIT1-GFP or CsMIT2-GFP restored mitochondrial iron to wild-type levels.

**Clinical Implications:** While this is a plant biology study without direct clinical applications, the findings have agricultural significance. Understanding mitochondrial iron transport in cucumber—a major global crop—may inform strategies to improve iron biofortification and crop resilience to iron-deficient or iron-excess soils. The organ-specific and iron-responsive regulation of CsMIT1 and CsMIT2 suggests potential targets for breeding or genetic engineering to optimize iron homeostasis under varying environmental conditions, which could improve crop yield and nutritional quality.