Os03g0296800

The rice gene Os03g0296800 was reported as mit in 2011. MIT belongs to the MSC family of proteins.

Annotated Information

Function

• MIT is a mitochondrial Fe transporter essential for rice growth and development.

• MIT knock out mutation caused a growth defect during seed development as revealed by the analysis of mit-1 heterozygous plants examined for the segregation of homozygous and heterozygous plants in the progeny. These analyses further revealed that, although to a lesser extent, MIT also has a role in germination.

• In the MIT mutation plant, changes in mitochondrial Fe accumulation could affect the Fe-S cluster synthesis in mitochondria. The reduction in the chlorophyll soil-plant analysis development (SPAD) value may be due to problems in mitochondrial Fe-S cluster synthesis that indirectly affect the chloroplasts. It has already been shown that knockout plants for the mitochondrial-synthesized Fe-S cluster exporter exhibit chlorosis23.

Mutation

• As the homozygous knockout of MIT in mit-1 plants proved lethal, the researchers characterized MIT-knockdown (mit-2) plants (Fig. 1). Hydroponically grown mit-2 plants were smaller than similarly grown WT plants (Fig. 1a; Supplementary Fig. S2). PCR analysis confirmed the integration of the T-DNA 604 bp upstream of the start codon and the homozygous status of mit-2 plants (Supplementary Fig. S2a,b). Moreover, quantitative RT-PCR analysis confirmed that the expression of MIT in the mit-2 plants was reduced compared with WT plants (Fig. 1b). There was a significant reduction in root and shoot dry weight as well as in the root and shoot length, leaf width and chlorophyll content (Supplementary Fig. S2c–h). Hydroponically grown mit-2 plants accumulated 51% more Fe than WT plants in the shoots (Fig. 1c). The accumulation of Mn was also changed (Fig. 1d) whereas no change in the accumulation of Cu and Zinc (Zn) was observed (Supplementary Fig. S2i,j).

• Mitochondria isolated from shoot tissue of mit-2 plants accumulated less Fe compared with WT mitochondria. Mitochondrial Fe concentration of mit-2 plants was 49% less, compared with mitochondria isolated from WT plants (Fig. 1f). Changes in Mn and Cu accumulation were also observed (Fig. 1g; Supplementary Fig. S2k). The expression of aconitase genes was not changed in mit-2 plants, whereas the total and mitochondrial aconitase activity decreased in the mit-2 plants compared with WT plants. In shoot tissue, the total aconitase activity of mit-2 was 39% less compared with WT plants, whereas a reduction of 42% was observed for the mit-2 aconitase activity from isolated mitochondria (Fig. 1e,h).

• The growth of the mit-2 plants in soil was also significantly impaired compared withWT plants. The average number of tillers in the mit-2 plants was 8 compared with 20 in the WT plants; in addition, the mutants were compromised in terms of plant height (Supplementary Fig. S2m,n). Flowering was delayed in the mit-2 plants, and fertility was also significantly lower, reducing the yield by 59% compared with that in WT plants (Fig. 1i–k). Although the growth of soil-grown mit-2 heterozygous plants was impaired compared with that of WT plants, it was superior to that of mit-2 homozygous plants for all these characteristics (Fig. 1i–k; Supplementary Fig. S2m,n), confirming that the phenotype was specific to the mit-2 plants.

• As mitochondrial activity is important for cell division, the researchers generated calli from WT and mit-2 plants; the size and weight of the calli was significantly reduced in the mit-2 calli compared with the WT (Supplementary Fig. S2o).

Figure.1 Characterization of mit-2 plants. .

Expression Pattern

• Quantitative RT–PCR revealed that the transcripts of MIT were three times lower in the roots and shoots of plants exposed to Fe-limited conditions compared with plants grown hydroponically in the presence of 100 μM Fe. MIT transcripts were increased in the roots and shoots when the plants were exposed to excess Fe (500 μM Fe; Fig. 2a,b). To further understand its role during germination and seed development, the MIT promoter was used to drive the expression of β-glucuronidase in rice. MIT expression was observed during germination and at all stages of seed development. During germination, MIT expression was specific to the embryo (Fig. 2c–f). Expression was observed in the leaf primordia and coleorhizae 1 day after germination (Fig. 2d). It increased subsequently and, 3 days after germination, was observed in whole embryo (Fig. 2f). Expression was also observed from anthesis through seed development (Fig. 2g, left to right), supporting the hypothesis that MIT has a crucial role in these growth stages. Further the steady state transcripts of MIT were observed during all growth stages of rice plant including root, leaves, stem, anther, pistil, lemma, palea, ovary, embryo and endosperm as revealed by microarray analysis (Supplementary Fig. S1).

Figure.2 Expression analysis of MIT. (a, b) The changes in expression of MIT in response to Fe availability − Fe: 0 μM Fe; + Fe: 100 μM Fe; + + Fe: 500 μM Fe. (a) Root. (b) Shoot. (c–g) MIT promoter driven β-glucuronidase expression during germination (c–f) and seed development (g); (c) 0; (d) 1; (e) 2; (f) 3 days after germination. (g) From left to right, before anthesis 1, 4, 12, 16, 20 and 25 days after anthesis. Scale, 500 μm. The graph shows mean ± s.d.; *P < 0.05, ANO VA, n = 3. .

Evolution

• MIT belongs to the MSC family of proteins. The members of this family localize to the inner mitochondrial membrane, have conserved regions and transport a wide range of substrates, including Fe24,25. The MSC family members transporting Fe into the mitochondria have been characterized in several organisms including yeast (Mrs3-Mrs4 (ref. 7)), mouse26, Drosophila27 and zebrafish28 (mitoferrin). The mitoferrin has been characterized in detail and the interactions of ferrochelatase, mitoferrin-1 and Abcb10 has also been revealed29. MIT has 39, 41 and 42% homology to Mrs3, Mrs4 and mitoferrin, respectively; whereas phylogenetic analysis revealed the absence of any close homologue in rice (Supplementary Fig. S3). The yeast Δmrs3Δmrs4 phenotype is only significant under low-Fe conditions (in the presence of Fe a substantial amount of Fe is imported into mitochondria), indicating that it is not the only MIT, although it is the only high-affinity MIT in yeast30. Besides Fe, Mrs3–Mrs4 also transport copper (Cu)31. In the yeast Δmrs3Δmrs4 double mutant, activity of the yeast vacuolar Fe and Mn transporter Ccc1 increases to avoid Fe toxicity in the cytoplasm, resulting in increased Fe and Mn accumulation in the vacuole and Fe deficiency in the cytoplasm7,8. As a result, the Fe-uptake system is triggered leading to a greater accumulation of these metals in the cells compared with the WT strain.

Subcellular localization

• MIT localizes to mitochondria and complements the growth of Δmrs3Δmrs4. A homology search (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi) confirmed the presence of the conserved regions of mitochondrial solute carrier family (MSC) proteins in MIT (amino acids from 134–166 and 171–223). MIT-green fluorescent protein (GFP) localized to mitochondria, when expressed in tobacco BY-2 cells whereas GFP alone, used as control, localized to the cytoplasm (Fig. 3a–f).

• MIT expressed in Δmrs3Δmrs4 yeast mutants complemented the growth defect of the mutant yeast (Fig. 3g). In Δmrs3Δmrs4 yeast, the expression of Fe uptake genes is upregulated7. The upregulation of FIT2 and FET3 is reverted to wild-type levels upon expression of the rice gene (Fig. 3h,i). Moreover, it also reversed the changes in accumulation of Fe and Cu (Fig. 3j–m). These results suggested a clear role for MIT in Fe homeostasis.

Figure.3 Subcellular localization and yeast complementation assay of MIT. .

Labs working on this gene

• Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan.
• Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino Itakura-machi, Gunma 374-0193, Japan.
• Department of Plant Molecular Systems, Biotech and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Republic of Korea.
• Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa 921-8836, Japan.