Data from: Metal tolerance protein MTP6 affects mitochondrial iron and manganese homeostasis in cucumber

Members of the cation diffusion facilitator family have been identified in all kingdoms of life. They have been divided into three subgroups – Zn-CDF, Fe/Zn-CDF and Mn-CDF – based on their putative specificity to transported metal ions. The plant metal tolerance 6 (MTP6) proteins fall into the Fe/Zn-CDF subgroup, however their function in iron/zinc transport has not been confirmed yet. Here we characterize the MTP6 protein from cucumber. When expressed in yeast and protoplasts isolated from A. thaliana cells, CsMTP6 localized in mitochondria and contributed to the efflux of iron and manganese from the mitochondria. The immunolocalization of CsMTP6 in cucumber membranes confirmed that CsMTP6 is associated with mitochondria. The root expression and protein level of CsMTP6 were significantly up-regulated in conditions of iron deficiency and iron excess but were not affected by Mn availability. These results indicate that plant MTP6 proteins contribute to the distribution of iron and manganese between the cytosol and mitochondria of plant cells and are regulated by Fe to maintain the mitochondrial and cytosolic iron homeostasis under various Fe availability

GDO activity in yeast data

Cytosolic gentisate dioxigenase (c-GDO) activity in the wild type (WT) or yeast mutant cells (mmt1/2) expressing c-GDO or c-GDO and cucumber CsMTP6 protein. c-GDO activity is expressed as nanomoles of substrate converted per minute per mg of protein. The data obtained come from the analysis of four individual yeast transformants

Supplementary Figures and Tables

PDF document with supplementary tables and figures: Supplementary Table S1. Yeast strains used in this work; Supplementary Fig. S1. Organ expression pattern of CsMTP6 in cucumber; Supplementary Fig. S2. ClustalW alignment of the CsMTP6 nucleic acid and CsMTP6 amino acid sequences; Supplementary Fig. S3. Western blot analysis of mitochondria isolated from yeast expressing CsMTP6; Supplementary Fig. S4. Localization of CsMTP6 in Δzrc1, K667 and Δsmf1 cells; Supplementary Fig. S5. Effect of CsMTP6 expression on yeast sensitivity to Zn, Ni and Cd

Metal content in yeast data

Mitochondrial metals (Fe, Cu, Co, Zn, Mn, Cd) content in yeast transformed with empty vector or expressing CsMTP6. The yeast were grown in the presence of 100 microM concentration of each metal for 12 hours prior the preparation of mitochondria. metals content was determined by Atomic Absorption Spectrophotometer (AAS 3300, Perkin Elmer). The data are expressed in nmol x mg-1 protein and come from three separate experiments

CsMTP6 expression data

Organ expression pattern of CsMTP6 in cucumber. Real-time expression analyses of the level of CsMTP6 transcript in the roots, hypocotyls, cotyledons, leaf petioles and leaves of 2-week-old cucumber seedlings growing in standard nutrition media. The CsMTP6 transcript levels relative to the constitutively expressed reference gene CACS were calculated (Lightcycler 480, Roche software) from the arithmetic means of ΔCp values obtained in three independent experiments. each experiment was performed in triplicate

Metal content in protoplasts data

Mitochondrial metals (Fe, Mn) content in protoplasts prepared from Arabidopsis thaliana suspension cells and transformed with empty vector pA7 or with the vector carrying cucumber CsMTP6 gene. Following transformation, protoplasts were incubated with 100 mcroM FeSO4-EDTA or 100 microM MnSO4 with slow agitation. Metal content was determined by AAS.The data are expressed in nmol of metal x mg-1 protein and were obtained in three separate experiment

AtOMA1 Affects the OXPHOS System and Plant Growth in Contrast to Other Newly Identified ATP-Independent Proteases in Arabidopsis Mitochondria

Compared with yeast, our knowledge on members of the ATP-independent plant mitochondrial proteolytic machinery is rather poor. In the present study, using confocal microscopy and immunoblotting, we proved that homologs of yeast Oma1, Atp23, Imp1, Imp2, and Oct1 proteases are localized in Arabidopsis mitochondria. We characterized these components of the ATP-independent proteolytic system as well as the earlier identified protease, AtICP55, with an emphasis on their significance in plant growth and functionality in the OXPHOS system. A functional complementation assay demonstrated that out of all the analyzed proteases, only AtOMA1 and AtICP55 could substitute for a lack of their yeast counterparts. We did not observe any significant developmental or morphological changes in plants lacking the studied proteases, either under optimal growth conditions or after exposure to stress, with the only exception being retarded root growth in oma1-1, thus implying that the absence of a single mitochondrial ATP-independent protease is not critical for Arabidopsis growth and development. We did not find any evidence indicating a clear functional complementation of the missing protease by any other protease at the transcript or protein level. Studies on the impact of the analyzed proteases on mitochondrial bioenergetic function revealed that out of all the studied mutants, only oma1-1 showed differences in activities and amounts of OXPHOS proteins. Among all the OXPHOS disorders found in oma1-1, the complex V deficiency is distinctive because it is mainly associated with decreased catalytic activity and not correlated with complex abundance, which has been observed in the case of supercomplex I + III2 and complex I deficiencies. Altogether, our study indicates that despite the presence of highly conservative homologs, the mitochondrial ATP-independent proteolytic system is not functionally conserved in plants as compared with yeast. Our findings also highlight the importance of AtOMA1 in maintenance of proper function of the OXPHOS system as well as in growth and development of Arabidopsis thaliana