Impacts of Mn, Fe, and Oxidative Stressors on MnSOD Activation by AtMTM1 and AtMTM2 in Arabidopsis

It has been reported that the mitochondrial carrier family proteins of AtMTM1 and AtMTM2 are necessary for manganese superoxide dismutase (MnSOD) activation in Arabidopsis, and are responsive to methyl viologen (MV)-induced oxidative stress. In this study, we showed that MnSOD activity was enhanced specifically by Mn treatments. By using AtMnSOD-overexpressing and AtMnSOD-knockdown mutant plants treated with the widely used oxidative stressors including MV, NaCl, H2O2, and tert-butyl hydroperoxide (t-BH), we revealed that Arabidopsis MnSOD was crucial for root-growth control and superoxide scavenging ability. In addition, it has been reported that E. coli MnSOD activity is inhibited by Fe and that MTM1-mutated yeast cells exhibit elevated Fe content and decreased MnSOD activity, which can be restored by the Fe2+-specific chelator, bathophenanthroline disulfonate (BPS). However, we showed that BPS inhibited MnSOD activity in AtMTM1 and AtMTM2 single- and double-mutant protoplasts, implying that altered Fe homeostasis affected MnSOD activation through AtMTM1 and AtMTM2. Notably, we used inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis to reveal an abnormal Fe/Mn ratio in the roots and shoots of AtMTM1 and AtMTM2 mutants under MV stress, indicating the importance of AtMTM1 in roots and AtMTM2 in shoots for maintaining Fe/Mn balance.


Introduction
Superoxide dismutases (SODs) are distributed in the cytoplasm, chloroplasts, and mitochondria of prokaryotic and eukaryotic cells [1][2][3]. They are classified as CuZnSOD, FeSOD, MnSOD, or NiSOD according to the transition metal cofactor ions at the active site [4][5][6]. Cellular superoxide (O 2 •− ) is mainly generated from electron transport chain complexes, and SODs catalyse the dismutation of O 2 •− to O 2 and H 2 O 2 . Toxic H 2 O 2 is converted to H 2 O mainly by catalase, ascorbate peroxidase, and glutathione peroxidase [7]; thus, SODs cooperate with other enzymes to relieve oxidative stress [8][9][10][11]. It has been reported that AtMnSOD-overexpressing plants exhibit increased catalase and peroxidase activities, with decreased malondialdehyde content after NaCl treatment, and maintain a higher germination rate in the presence of oxidative stressors, such as methyl viologen (MV) and H 2 O 2 [12,13]. Plants harboured with antisense AtMnSOD show decreased MnSOD protein levels, with altered tricarboxylic acid cycle enzyme levels and root growth after treatment with NaCl, sorbitol, Fe, and MV [14]. In this study, we established both AtMn-SOD-overexpressing (MnSOD-OE) and AtMnSOD-knockdown (msd1) plants to confirm the cofactor specificity and post-translational regulation of MnSOD under conditions of oxidative stress. In addition, antagonism between Fe and Mn has been reported in tomato and rice plants, in which Fe suppresses Mn levels and vice versa [15][16][17]. Therefore, we also investigated the effect of metal ion treatments on MnSOD activity in Arabidopsis.
The MnSOD dimer in E. coli is localised in the cytosol and is regulated by repressors and Fe ion concentrations. Fe suppresses the biosynthesis of MnSOD at the transcriptional (MnSOD-OE; MnSOD-apoprotein-overexpressing plants) and characterised the AtMnSOD T-DNA insertion (msd1) mutants (Supplementary Figures S1 and S2).
The highest expression line of MnSOD-OE plants with approximately two-fold MnSOD mRNA expression levels was used in the following study; however, MnSOD activity and protein levels in MnSOD-OE plants were not significantly affected. MnSOD-OE plants showed a late-flowering phenotype compared to wild-type (Col) plants (Supplementary Figure S1). msd1 plants were characterized by genotyping and RT-qPCR. We confirmed that msd1 is a heterologous T-DNA insertional knockdown mutant. The lowest expression line with a 20% reduction in MnSOD transcript level was used in the following study. MnSOD activity and protein levels in the msd1 mutant were lower, while the msd1 mutant showed an early flowering phenotype compared to Col plants (Supplementary Figure S2). Moreover, we are unable to screen the homologous msd1 plants, implying the lethal effect of the null mutant for plant germination.

Effect of Mn and Fe on Transgenic AtMnSOD-Overexpressing Plants
MnSOD activity in Col and MnSOD-OE plants was analysed. Fourteen-day-old seedlings were treated with 1 mM of the metal ions, Mn, Fe, CuSO 4 (Cu), ZnSO 4 (Zn), Mn and Fe, Mn and Cu, or Mn and Zn, for 16 h and analyzed by in-gel SOD activity assay ( Figure 1). We observed that MnSOD activity was increased after Mn treatment, but was decreased after Fe, Cu, and Zn treatments in Col plants. Treatments
The highest expression line of MnSOD-OE plants with approximately two-fold MnSOD mRNA expression levels was used in the following study; however, MnSOD activity and protein levels in MnSOD-OE plants were not significantly affected. MnSOD-OE plants showed a late-flowering phenotype compared to wild-type (Col) plants (Supplementary Figure S1). msd1 plants were characterized by genotyping and RT-qPCR. We confirmed that msd1 is a heterologous T-DNA insertional knockdown mutant. The lowest expression line with a 20% reduction in MnSOD transcript level was used in the following study. MnSOD activity and protein levels in the msd1 mutant were lower, while the msd1 mutant showed an early flowering phenotype compared to Col plants (Supplementary Figure S2). Moreover, we are unable to screen the homologous msd1 plants, implying the lethal effect of the null mutant for plant germination.

Effect of Mn and Fe on Transgenic AtMnSOD-Overexpressing Plants
MnSOD activity in Col and MnSOD-OE plants was analysed. Fourteen-day-old seedlings were treated with 1 mM of the metal ions, Mn, Fe, CuSO4 (Cu), ZnSO4 (Zn), Mn and Fe, Mn and Cu, or Mn and Zn, for 16 h and analyzed by in-gel SOD activity assay ( Figure  1). We observed that MnSOD activity was increased after Mn treatment, but was decreased after Fe, Cu, and Zn treatments in Col plants. Treatments   In-gel SOD activity assay (top) and immunoblotting with α-MnSOD and α-Actin antibodies (bottom) were conducted. Actin was used as a loading control. MnSOD activity and protein levels were measured relative to those in control. Data represent one of three independent repeats.

Post-Translational Regulation of MnSOD through Oxidative Stressors
AtMTM1 is elevated in response to MV, but not H 2 O 2 , when seedlings are grown on plates containing these stressors [31], and we have reported that AtMTM1, AtMTM2, and MnSOD gene expression levels increase after MV treatment [30]. In this study, 14-day-old Col seedlings in 1/2 MS liquid medium were treated with the widely used oxidative stressors including 5 µM MV, 150 mM NaCl, 10 mM H 2 O 2 , or 1 mM tert-butyl hydroperoxide (t-BH) with agitation for 2 to 8 h.
RT-qPCR analysis showed that AtMTM1, AtMTM2, and MnSOD gene expression levels were elevated as early as 2 h after all oxidative stress treatments (Figure 2), and we applied MV or NaCl as the representative stressors in the following studies. The mitochondrial oxidation-responsive gene, AOX1A, was used as a reference, as previously reported [30]. AtMTM1 and AtMTM2 expression levels in control without stressors are shown in Supplementary Figure S3. Our results implied that oxidative stressors specifically induced AtMTM1 and AtMTM2. ors including 5 μM MV, 150 mM NaCl, 10 mM H2O2, or 1 mM tert-butyl hydroperoxide (t-BH) with agitation for 2 to 8 h.
RT-qPCR analysis showed that AtMTM1, AtMTM2, and MnSOD gene expression levels were elevated as early as 2 h after all oxidative stress treatments (Figure 2), and we applied MV or NaCl as the representative stressors in the following studies. The mitochondrial oxidation-responsive gene, AOX1A, was used as a reference, as previously reported [30]. AtMTM1 and AtMTM2 expression levels in control without stressors are shown in Supplementary Figure S3. Our results implied that oxidative stressors specifically induced AtMTM1 and AtMTM2.
We showed that MnSOD activity was similar between Col and MnSOD-OE plants, and was lower in msd1 plants without oxidative stressors (Supplementary Figures S1 and S2). To clarify the physiological role of MnSOD under oxidative stress, we applied two representative oxidative stressors of MV and NaCl. We incubated 14-day-old seedlings of Col, MnSOD-OE, and msd1 in 1/2 MS liquid medium containing 5 μM MV or 150 mM NaCl with agitation for 24 h, and measured MnSOD activity and protein levels ( Figure 3). MnSOD activity was markedly induced in MnSOD-OE and was slightly induced in msd1 plants by MV stressor, and MnSOD activity was still lower in msd1 plants than in Col plants under stress ( Figure 3A,B); however, MnSOD protein levels were not significantly different. Similar expression patterns were observed in Col, MnSOD-OE, and msd1 seedlings under NaCl stress ( Figure 3C   We showed that MnSOD activity was similar between Col and MnSOD-OE plants, and was lower in msd1 plants without oxidative stressors (Supplementary Figures S1 and S2). To clarify the physiological role of MnSOD under oxidative stress, we applied two representative oxidative stressors of MV and NaCl. We incubated 14-day-old seedlings of Col, MnSOD-OE, and msd1 in 1/2 MS liquid medium containing 5 µM MV or 150 mM NaCl with agitation for 24 h, and measured MnSOD activity and protein levels ( Figure 3). MnSOD activity was markedly induced in MnSOD-OE and was slightly induced in msd1 plants by MV stressor, and MnSOD activity was still lower in msd1 plants than in Col plants under stress ( Figure 3A,B); however, MnSOD protein levels were not significantly different. Similar expression patterns were observed in Col, MnSOD-OE, and msd1 seedlings under NaCl stress ( Figure 3C,D). These results indicated the post-translational regulation of MnSOD under oxidative stress in Arabidopsis.

Role of MnSOD in the Control of Primary Root Growth during Oxidative Stress
A previous study of the Arabidopsis MnSOD-knockdown mutant oiwa showed that it is a female gametophytic mutant with defective embryo sac development and fertilization, and that the mutation affects reactive oxygen species homeostasis in the mitochondria and cytosol [34,35]. In this study, we focused on the role of MnSOD in early root growth In-gel SOD activity assay (top) and immunoblotting with α-MnSOD and α-Actin antibodies (bottom) were conducted. Actin was used as a loading control. MnSOD activity and protein levels were measured relative to those in Col control. Data represent one of three independent repeats.

Role of MnSOD in the Control of Primary Root Growth during Oxidative Stress
A previous study of the Arabidopsis MnSOD-knockdown mutant oiwa showed that it is a female gametophytic mutant with defective embryo sac development and fertilization, and that the mutation affects reactive oxygen species homeostasis in the mitochondria and cytosol [34,35]. In this study, we focused on the role of MnSOD in early root growth under stress conditions. We applied different stressors in 1/2 MS plates and measured the primary root lengths of Col, MnSOD-OE, and msd1 seedlings ( Figure 4).
When seedlings were grown on plates containing 5 nM MV or 50 mM NaCl ( Figure  4A), root growth was inhibited in all seedlings. In addition, 7-day-old MnSOD-OE seedlings had shorter roots and msd1 had longer roots compared to the roots of Col plants.
Five-day-old seedlings with similar root lengths were then transferred from 1/2 MS plates to high-stringency plates containing 10 nM MV, 50 mM NaCl, 500 μM H2O2, or 250 μM t-BH for 3 days ( Figure 4B). Root growth was slightly inhibited by MV, NaCl, and H2O2, and was markedly inhibited by t-BH. In addition, both MnSOD-OE and msd1 seedlings had longer roots in the presence of MV, H2O2, and t-BH, and this may be restricted to the shorter period of treatment. Taken together, we noticed that MnSOD responded to stress conditions during early primary root growth.  Five-day-old seedlings with similar root lengths were transferred from 1/2 MS medium to high-stringency plates containing 10 nM MV, 50 mM NaCl, 500 μM H2O2, or 250 μM t-BH for 3 days, and the root lengths were measured. Data are mean ± SD of three independent repeats. n = 30 seedlings. * Significant at p < 0.05 compared with the Col.

O2 •− and H2O2 Accumulation and Distribution in AtMnSOD-Overexpressing Plants under Stress
The O2 •− and H2O2 metabolism levels via MnSOD in Arabidopsis are unclear; thus, we used a typical oxidative stressor MV in this study, and treated mature 21-day-old seedlings of Col and MnSOD-OE with 5 μM MV stress for 3 days. The O2 •− and H2O2 accumulation was measured by NBT and DAB staining, respectively ( Figure 5). Relative O2 •− ac- When seedlings were grown on plates containing 5 nM MV or 50 mM NaCl ( Figure 4A), root growth was inhibited in all seedlings. In addition, 7-day-old MnSOD-OE seedlings had shorter roots and msd1 had longer roots compared to the roots of Col plants.
Five-day-old seedlings with similar root lengths were then transferred from 1/2 MS plates to high-stringency plates containing 10 nM MV, 150 mM NaCl, 500 µM H 2 O 2 , or 250 µM t-BH for 3 days ( Figure 4B). Root growth was slightly inhibited by MV, NaCl, and H 2 O 2 , and was markedly inhibited by t-BH. In addition, both MnSOD-OE and msd1 seedlings had longer roots in the presence of MV, H 2 O 2 , and t-BH, and this may be restricted to the shorter period of treatment. Taken together, we noticed that MnSOD responded to stress conditions during early primary root growth.  Five-day-old seedlings with similar root lengths were transferred from 1/2 MS medium to high-stringency plates containing 10 nM MV, 50 mM NaCl, 500 μM H2O2, or 250 μM t-BH for 3 days, and the root lengths were measured. Data are mean ± SD of three independent repeats. n = 30 seedlings. * Significant at p < 0.05 compared with the Col.

O2 •− and H2O2 Accumulation and Distribution in AtMnSOD-Overexpressing Plants under Stress
The O2 •− and H2O2 metabolism levels via MnSOD in Arabidopsis are unclear; thus, we used a typical oxidative stressor MV in this study, and treated mature 21-day-old seedlings of Col and MnSOD-OE with 5 μM MV stress for 3 days. The O2 •− and H2O2 accumulation was measured by NBT and DAB staining, respectively ( Figure 5). Relative O2 •− accumulation was increased under MV treatment in Col, but decreased significantly in MnSOD-OE plants ( Figure 5A). Relative H2O2 accumulation was increased in both Col and MnSOD-OE seedlings under MV treatment, but MnSOD-OE seedlings exhibited lower H2O2 levels ( Figure 5B). These results indicated that mature MnSOD-OE plants retain higher superoxide scavenging ability with decreased O2 •− and H2O2 amounts. accumulation were analyzed by NBT and DAB staining (top), respectively. Each accumulation in a plant was measured relative to Col (bottom). Data are mean ± SD of three independent repeats. * Significant at p < 0.05 compared with the Col.

Root-Length Phenotype of AtMTM1-and AtMTM2-Mutated Seedlings Analysed by Extra Mn Supply
The importance of the Mn carrier proteins, AtMTM1 and AtMTM2, for mitochondrial MnSOD activation has been reported using the miRNA-mediated AtMTM1-knockdown mutant (mtm1), the T-DNA insertional AtMTM2-knockout mutant (mtm2), and mtm1 mtm2double mutants [30]. We have reported that the defective root-length phenotypes of mtm1 and mtm2 single and double mutant seedlings grown in 1/2 MS plates are restored through MnCl 2 (Mn) treatment, and AtMTM1 and AtMTM2 are involved in the root-length control with divergent effects [30]. To further examine the effect of defective Mn and increased Mn supply on MnSOD activity in mtm1, mtm2, and mtm1 mtm2-double mutant plants, we monitored the primary root lengths on basal medium plates without Mn (Mn-deficient) or with Mn at 14 µM (normal Mn), 700 µM (50-fold increase), or 1050 µM (75-fold increase) for 6 days ( Figure 6).
Col plants and all mutant lines showed similar root lengths under Mn-deficient conditions. A normal Mn supply resulted in abnormal root length in the single mutants; mtm1 and mtm2 plants showed shorter and longer root-length phenotypes, respectively. A 50fold increase in Mn supply inhibited root growth, but mtm2 and mtm1 mtm2-double mutant plants retained longer root lengths compared to Col plants. A 75-fold increase in Mn supply inhibited root growth in all seedlings. Based on the toxicity of increased Mn concentration and its inhibition of root growth in Col seedlings, the altered root lengths of mutant plants indicated that AtMTM1 participates more in the control of Mn homeostasis than AtMTM2. Overall, these results confirmed that AtMTM1 and AtMTM2 coordinate Mn homeostasis with divergence.

MnSOD Activity in AtMTM1-and AtMTM2-Mutated Protoplasts after Fe Chelation
Our previous study showed that AtMTM1 and AtMTM2 are necessary for AtMnSOD activation; AtMTM1-mutant plants have lower Fe levels in the roots, but AtMTM2-mutant plants have similar Fe levels in the shoots compared to wild-type plants [30]. To delineate the role of Fe in MnSOD activation in mtm1, mtm2, and mtm1 mtm2-double mutants, we applied the Fe 2+ -specific chelator, BPS, to mesophyll protoplasts at 100 to 1000 μM for 16 h (Figure 7).
The mtm1 mtm2-double mutant protoplasts showed lower MnSOD activity than Col protoplasts without treatment, which is consistent with the results of a previous study Col plants and all mutant lines showed similar root lengths under Mn-deficient conditions. A normal Mn supply resulted in abnormal root length in the single mutants; mtm1 and mtm2 plants showed shorter and longer root-length phenotypes, respectively. A 50-fold increase in Mn supply inhibited root growth, but mtm2 and mtm1 mtm2-double mutant plants retained longer root lengths compared to Col plants. A 75-fold increase in Mn supply inhibited root growth in all seedlings. Based on the toxicity of increased Mn concentration and its inhibition of root growth in Col seedlings, the altered root lengths of mutant plants indicated that AtMTM1 participates more in the control of Mn homeostasis than AtMTM2. Overall, these results confirmed that AtMTM1 and AtMTM2 coordinate Mn homeostasis with divergence.

MnSOD Activity in AtMTM1-and AtMTM2-Mutated Protoplasts after Fe Chelation
Our previous study showed that AtMTM1 and AtMTM2 are necessary for AtMnSOD activation; AtMTM1-mutant plants have lower Fe levels in the roots, but AtMTM2-mutant plants have similar Fe levels in the shoots compared to wild-type plants [30]. To delineate the role of Fe in MnSOD activation in mtm1, mtm2, and mtm1 mtm2-double mutants, we applied the Fe 2+ -specific chelator, BPS, to mesophyll protoplasts at 100 to 1000 µM for 16 h (Figure 7).
Plants 2022, 11, x 8 of 14 [30]. In this study, we revealed that mtm1 and mtm2 protoplasts exhibited slightly decreased MnSOD activity compared to Col protoplasts. The 100 and 500 μM BPS treatments slightly inhibited MnSOD activity in Col protoplasts, and the 1000 μM BPS treatment markedly decreased MnSOD activity. By comparing relative MnSOD activity within each treatment, mtm1 mutant protoplasts exhibited decreased MnSOD activity after 500 μM BPS treatment, and mtm2 and mtm1 mtm2-double mutant protoplasts showed significantly decreased MnSOD activity from 100 μM BPS, implying that AtMTM2 was more sensitive to BPS treatment than AtMTM1. Overall, these results indicated that the altered Fe homeostasis is involved in AtMTM1 and AtMTM2-mediated MnSOD activity.

Fe/Mn Ratio in AtMTM1-and AtMTM2-Mutant Seedlings Treated with Methyl Viologen (MV)
We further elucidated the balance between Mn and Fe in mtm1, mtm2, and mtm1 mtm2-double mutants using inductively coupled plasma-optical emission spectrometry  The mtm1 mtm2-double mutant protoplasts showed lower MnSOD activity than Col protoplasts without treatment, which is consistent with the results of a previous study [30]. In this study, we revealed that mtm1 and mtm2 protoplasts exhibited slightly decreased MnSOD activity compared to Col protoplasts. The 100 and 500 µM BPS treatments slightly inhibited MnSOD activity in Col protoplasts, and the 1000 µM BPS treatment markedly decreased MnSOD activity. By comparing relative MnSOD activity within each treatment, mtm1 mutant protoplasts exhibited decreased MnSOD activity after 500 µM BPS treatment, and mtm2 and mtm1 mtm2-double mutant protoplasts showed significantly decreased MnSOD activity from 100 µM BPS, implying that AtMTM2 was more sensitive to BPS treatment than AtMTM1. Overall, these results indicated that the altered Fe homeostasis is involved in AtMTM1 and AtMTM2-mediated MnSOD activity.

Fe/Mn Ratio in AtMTM1-and AtMTM2-Mutant Seedlings Treated with Methyl Viologen (MV)
We further elucidated the balance between Mn and Fe in mtm1, mtm2, and mtm1 mtm2double mutants using inductively coupled plasma-optical emission spectrometry (ICP-OES). To reveal the Fe/Mn ratio in root and shoot tissues, we used MV as a representative stressor, and applied higher MV dosage with long-term treatment in this study, since AtMTM1 and AtMTM2 was altered slightly in short-term MV treatment as shown in Figure 2. Fourteen-day-old seedlings were incubated in 1/2 MS liquid medium containing 10 µM MV with agitation for 3 days (Figure 8). Before treatment, the Fe/Mn ratio was lower in the roots of mtm1, but higher in the roots of mtm2 and mtm1 mtm2-double mutants compared to the ratio in Col seedlings. In addition, the Fe/Mn ratios were similar in the shoots of all seedlings. However, MV-induced stress caused a decrease in the Fe/Mn ratio in the roots of mtm1 seedlings ( Figure 8A) and an increase in the Fe/Mn ratio in the shoots of mtm2 seedlings compared to the ratios in Col seedlings ( Figure 8B). Taken together, these findings indicated that AtMTM1 and AtMTM2 are involved in Fe/Mn balance in the roots and shoots, respectively. decreased MnSOD activity from 100 μM BPS, implying that AtMTM2 was more sensitive to BPS treatment than AtMTM1. Overall, these results indicated that the altered Fe homeostasis is involved in AtMTM1 and AtMTM2-mediated MnSOD activity. Figure 7. MnSOD activity of Col, mtm1, mtm2, and mtm1 mtm2-double mutant protoplasts after Fe 2+specific chelator (BPS) treatment. Protoplasts were treated without or with 100, 500, or 1000 μM BPS for 16 h. MnSOD activity in protoplasts was measured relative to Col without BPS treatment. Data are mean ± SD of three independent repeats. * Significant at p < 0.05 compared with the Col.

Fe/Mn Ratio in AtMTM1-and AtMTM2-Mutant Seedlings Treated with Methyl Viologen (MV)
We further elucidated the balance between Mn and Fe in mtm1, mtm2, and mtm1 mtm2-double mutants using inductively coupled plasma-optical emission spectrometry (ICP-OES). To reveal the Fe/Mn ratio in root and shoot tissues, we used MV as a representative stressor, and applied higher MV dosage with long-term treatment in this study, since AtMTM1 and AtMTM2 was altered slightly in short-term MV treatment as shown in Figure 2. Fourteen-day-old seedlings were incubated in 1/2 MS liquid medium containing 10 μM MV with agitation for 3 days (Figure 8). Before treatment, the Fe/Mn ratio was lower in the roots of mtm1, but higher in the roots of mtm2 and mtm1 mtm2-double mutants compared to the ratio in Col seedlings. In addition, the Fe/Mn ratios were similar in the shoots of all seedlings. However, MV-induced stress caused a decrease in the Fe/Mn ratio in the roots of mtm1 seedlings ( Figure 8A) and an increase in the Fe/Mn ratio in the shoots of mtm2 seedlings compared to the ratios in Col seedlings ( Figure 8B). Taken together, these findings indicated that AtMTM1 and AtMTM2 are involved in Fe/Mn balance in the roots and shoots, respectively.

Discussion
The plant mitochondrial carrier family (MCF) contains approximately 60 proteins that coordinate metabolic and ionic homeostasis between the cytosol and mitochondria [36][37][38][39]. The mitochondrial MCF proteins, AtMTM1 and AtMTM2, are Mn-specific carrier proteins involved in MnSOD activation in Arabidopsis [30,31]. In a previous study, yeast cytosollocalised MnSOD was inactive, but its activity could be restored by treatment with a high concentration of Mn [27], and mitochondrial MnSOD was markedly enhanced by Mn treatment. Moreover, wild-type yeast cells treated with Fe show increased mitochondrial Fe levels and MnSOD protein levels, but retain normal MnSOD activity [29]. In this study, MnSOD activity was largely increased under higher concentrations of Mn treatment (Figure 1). This result indicated the Mn cofactor specificity of Arabidopsis MnSOD activation and the increased apoprotein levels in MnSOD-OE plants occurred through Mn treatment. It is worthy to adjust metal concentration based on the normal range in 1/2 MS media. In addition, we observed antagonisms between Mn and other metals, including Fe, Cu, and Zn, for MnSOD activity in Arabidopsis, which agrees with the results of an earlier study on the effect of Mn and Fe on MnSOD activity in E. coli [19].
It has been reported that AtMTM1 is an oxidation-responsive gene [31,40]. In this study, we showed that both AtMTM1 and AtMTM2 gene expression levels respond to MV, NaCl, H 2 O 2 , and t-BH-induced oxidative stress in Col plants (Figure 2), and that elevated AtMTM2 gene expression levels can be detected earlier than elevated AtMTM1 levels. In addition, MnSOD-OE plants exhibited significantly enhanced MnSOD activity, but msd1 plants showed slightly decreased MnSOD activity compared to MnSOD activity in Col plants under MV and NaCl treatments (Figure 3), indicating the post-translational regulation of MnSOD. It is worthy to investigate the post-translation regulation such as phosphorylation or ubiquitination, in order to elucidate the mechanism of MnSOD activation. In a previous study, AtMnSOD antisense plants displayed shorter root lengths under the high-stringency oxidative stress conditions of 0.5 µM MV [14]. The AtMnSOD mutant, oiwa-1, shows defective embryo sac formation, but the auxin gradient is not altered in this mutant [34,35]. Plants with AtMnSOD overexpression driven by the seedspecific promoter, At2S3, have higher germination rates after 10 µM MV and 10 mM H 2 O 2 treatment [13]. In this study, we examined the root growth phenotype of 7-day-old MnSOD-OE and msd1 seedlings on plates containing 5 nM MV or 50 mM NaCl (Figure 4). The different root lengths reflected the participation of MnSOD in early root growth. However, after transferring 5-day-old seedlings from 1/2 MS to higher-stringency conditions for 3 days, both MnSOD-OE and msd1 seedlings showed longer roots. Thus, it is worth monitoring the root length for a longer period. Since MnSOD is the only reported enzyme that has the ability to catalyse superoxide (O 2 •− ) in mitochondria, we quantified O 2

•−
and H 2 O 2 levels in mature (21-day-old) MnSOD-OE seedlings, and demonstrated that the overexpression of MnSOD enhanced the superoxide scavenging ability and maintained the cellular levels of H 2 O 2 ( Figure 5). Overall, we demonstrated that MnSOD is crucial for the control of early root growth under stress conditions and that it scavenges superoxide radicals during plant development. Moreover, it is worthy to elucidate MnSOD-mediated oxidative stress and the accompanied shorter primary root length, as well as the phenotypes of branch roots and root hairs.
In this study, we used basal medium, with and without Mn treatment, and monitored the root length of AtMTM1 and AtMTM2 single and double mutants ( Figure 6). The results reflected the different sensitivities of AtMTM1 and AtMTM2 to Mn, as previously described [30]. These experiments revealed that mtm1 had a greater ability than mtm2 to restore root length in the presence of Mn, indicating the importance of AtMTM1 in root growth. It is worthy to investigate the relationship of the primary root phenotype between msd1 seedlings and mtm1 mtm2-double mutant, since both plants shared the similar longer primary root length. In a previous study, we used mtm1 mtm2-double mutant protoplasts to show that the mitochondrial carrier proteins, AtMTM1 and AtMTM2, are crucial for MnSOD activation [30]. In this study, we found that AtMTM1 and AtMTM2-single mutant protoplasts exhibited slightly lower MnSOD activity than control protoplasts without treatment. These findings increased the evidence for the physiological roles of these carrier proteins, and indicated that AtMTM1 and AtMTM2 have different sensitivities for MnSOD activation. Moreover, we applied an Fe chelator and found that MnSOD activity was lower in mtm2 than in mtm1 plants, and it clearly decreased in mtm1 mtm2-double mutant protoplasts (Figure 7). These results indicated that AtMTM1 and AtMTM2-mediated MnSOD activation is affected by Fe homeostasis, and we suggested that the disrupted systems by Fe homeostasis include MnSOD apoprotein synthesis in the cytosol, Mn binding to MnSOD via mitochondrial AtMTM1 and AtMTM2, or tetrameric MnSOD activation in the mitochondrial matrix.
It has been reported that the yeast MnSOD promoter region contains heme and stress-related regulatory sites, and that MnSOD transcription is regulated by heme [41]. The associations between mitochondrial MnSOD levels; Fe levels; the expression levels of the Fe/S cluster biogenesis genes, MRS3, MRS4, SSQ1, GRX5, and YFH1, have been investigated [29,42], and elevated mitochondrial Fe levels do not correlate well with MnSOD activity in yeast. Since mitochondrial heme synthesis and Fe-S cluster biogenesis involve Fe utilisation and Fe homeostasis [33,43], we suggest that Arabidopsis MnSOD transportation or activation via AtMTM1 and AtMTM2 are affected by unknown Fe-S proteins. Moreover, a previous study of the Fe/Mn ratio in plants revealed an antagonistic relationship between Mn and Fe levels in root absorption and translocation from roots to shoots [16,44]. In this study, we observed that the Fe/Mn ratio was altered in the roots of mtm1 plants and the shoots of mtm2 plants under MV stress (Figure 8), implying physiological roles of AtMTM1 and AtMTM2 in Fe/Mn balance. It is possible that AtMTM1 or AtMTM2 are also involved in Fe regulation, and it is worth monitoring the root length of mtm1 and mtm2 plants grown on the basal medium with Fe added.

Root Length Assay in Response to Oxidative Stressors and Mn Treatment
The root lengths of 7-day-old seedlings on 1/2 MS plates containing mild-stringency stressors of 5 nM MV or 50 mM NaCl were measured. In addition, 5-day-old seedlings were transferred from 1/2 MS medium to plates containing higher-stringency stressors of 10 nM MV or 150 mM NaCl for 3 days were conducted.
Moreover, the root lengths of seedlings in the Mn deficiency condition were analyzed by using the basal medium containing 5 mM KNO 3 [54]. Seedlings were grown on plates supplied without MnCl 2 (Mn deficient) or with MnCl 2 at 14 µM (normal Mn), 700 µM (50-fold increase), or 1050 µM (75-fold increase) for 6 days.

Fe/Mn Ratios in Roots and Shoots in Response to MV
The inductively coupled plasma-optical emission spectrometry (ICP-OES) (PerkinElmer OPTIMA 5300) analysis was applied to measure Mn and Fe metal contents in plants [59,60]. Fourteen-day-old seedlings were incubated in 1/2 MS liquid medium containing 10 µM MV with agitation for 3 days, then shoots and roots were separated. An amount of 0.1 g dried samples were used for ICP-OES analysis [30]. Spinach and tomato leaves (NIST SRM-1570a and NIST SRM-1573a) were the references. The output Mn and Fe contents by ICP-OES were converted to Fe/Mn ratio.

Statistical Analysis
All experiments were repeated independently at least three times. Statistical analysis involved Student's t-test and Duncan's multiple range test. p value < 0.05 was considered statistically significant.

PCR Primers and GenBank Accession Numbers
Primers and gene accession numbers are listed in Supplementary Table S1.

Conclusions
This study strengthened the importance of MnSOD activation through its carrier proteins AtMTM1 and AtMYM2 by metal ion treatments and oxidative stressors. We showed that MnSOD activity was specifically enhanced by Mn treatment, and antagonism occurred between Mn and other metals for MnSOD activation. We clarified the post-translational regulation of MnSOD during oxidative stress and demonstrated that MnSOD participates in the control of early root growth and enhances superoxide scavenging efficiency in mature seedlings. It is worthy to connect the altered root-length phenotype and elevated MnSOD enzyme activity. We also revealed that altered Fe homeostasis inhibited MnSOD activity through the carrier proteins AtMTM1 and AtMTM2. Especially, AtMTM1 and AtMTM2 participate in Fe/Mn regulation with tissue specificity. It would be interesting to investigate the mechanism of MnSOD post-translational regulation through phosphorylation site mutation, and to elucidate the substrate affinity of AtMTM1 and AtMTM2 through in vitro studies.