DISCUSSION
Mitochondria matrix communicates with the cytosol via the MCF proteins
that are evolutionarily conserved. Arabidopsis MCF proteins contain
three homologous repeated domains of about 100 amino acids, and each
domain has a characteristic motif (Kunji, 2004; Picault et al., 2004).
MCF proteins exist as homodimers, and the monomer represents one
functional entity (Kunji & Crichton, 2010). These carrier proteins
locate at the mitochondrial inner membrane and catalyze specific
transport of inorganic ions, cofactors, metabolites, and nucleotides
into the mitochondrial matrix (Haferkamp & Schmitz-Esser, 2012).
Phylogenetic analysis showed that the high homology of MCF genesAtMTM1 and AtMTM2 are clustered (Palmieri et al., 2011),
and characterization of yMTM1 functional-like gene AtMTM1was reported (Su et al., 2007). In this study, we characterized its
homolog AtMTM2 , investigated the redundant function of AtMTM1 and
AtMTM2, and looked for other candidates for AtMSD1 activation. For the
physiological roles of AtMTM1 and AtMTM2, we provided evidence of their
mitochondrial localization, interaction with AtMSD1, and the same
complementation ability for ySOD2 activation in ymtm1Δ cells(Figs. 1, 2) . Co-transformation of AtMTM1 increased
AtMSD1 activity, and AtMSD1 enhanced the survival rate in ysod2Δcells under MV stress (Figure 3) . This phenomenon is consistent
with the concept that the mitochondrial MCF protein yMTM1 has a positive
effect on increased ySOD2 activity (Luk et al., 2003). Our data
strengthened the importance of AtMTM1 and AtMTM2 for AtMSD1 activity and
cell growth in yeast and Arabidopsis.
Expression analysis of Arabidopsis MCF proteins revealed that paired
carriers have distinct specificities during plant growth and abiotic
stress (Catoni et al., 2003; Hoyos et al., 2003; Maia et al., 1998;
Watanabe, Nakazono, Tsutsumi, & Hirai, 1999). For the discrepancy ofAtMTM1 and AtMTM2 , we found AtMTM1 andAtMTM2 had enhanced expressions in flower tissue during
development, but AtMTM2 played a dominant role compared withAtMTM1 under MV stress (Figures 4, 5) . The previous
study mentioned MnSOD activity increased under Cu and Cd stresses
(Drążkiewicz, Skórzyńska-Polit, & Krupa, 2007). In this study, we
applied multiple metal-mediated oxidative stresses, not onlyAtMSD1 but AtMTM1 and AtMTM2 gene expressions
increased synchronously (Figure 6) . For comparison ofmtm1-i knock-down and mtm2 knock-out single mutants, onlymtm2 mutant had significantly up-regulated AtMTM1 gene
expression under MV stress, and these two single mutants had a
contrasting effect of root growth compared with the WT in response to MV
stress (Figures 7, 8) . All these results confirmed the
redundant AtMTM1 and AtMTM2 genes have different
capabilities toward stresses.
Notably, the decreased MnSOD protein accompanied by increased FeSOD
protein level was found in MnSOD -antisense Arabidopsis (Morgan et
al., 2008). In our study, the mtm1-i mtm2 -double mutant also had
decreased MnSOD activity with increased FeSOD activity (Figure
9) . We proposed the physiological regulations of AtMTM1 andAtMTM2 stretch across mitochondria and chloroplasts, and may have
an impact on flowering in Arabidopsis. Moreover, the Mn supplement
rescued the root lengths of mtm1-i , mtm2 , and mtm1-i
mtm2 mutants to the WT, and revealed the complementary effect of Mn
transportation (Figure 10) .
To look for other candidates for the post-translational process of MnSOD
activation, we generated modified AtMSD1 constructs of cytosolic and
chloroplastic versions, and only the chloroplast-destined MSD1 can be
activated in WT protoplasts (Figure 11) . We proposed the
organelle-specific Mn transporters or chaperones for
chloroplastic-destined MSD1 activation exist in Arabidopsis. This
phenomenon is similar to the model that Mn insertion is associated with
the mitochondrial importing and the protein folding process (Luk et al.,
2005). Taken together, we concluded the insertion of Mn cofactor via the
membrane transporter is necessary for MnSOD activation in Arabidopsis.
In addition to mitochondria, MnSOD is present in the thylakoids of some
prokaryotic and eukaryotic algae (Kanematsu & Asada, 1979; Okada et
al., 1979; Regelsberger et al., 2002). In this study, we found the
factors for the exogenous-expressed AtMSD1 metalation still exist in
Arabidopsis chloroplasts, implying chloroplastic Mn transporters may
participate in chloroplast-destined AtMSD1 activation that could be
evolutionarily conserved. The chloroplast transporter CMT1 mediated Mn
transportation (Eisenhuta et al., 2018; Zhang et al., 2018), and the
PAM71/CCHA1 regulated Mn and Ca homeostasis in chloroplast (Schneider et
al., 2016; Wang et al., 2016). Both inner envelope-localized CMT1 and
thylakoid membrane-localized PAM71/CCHA1 are crucial for the biogenesis
of Mn cluster Mn4CaO5 during
photosynthesis (Krieger-Liszkay & Thomine, 2018). Taken together, the
roles of CMT1, PAM71/CCHA1, Mn cluster-related factors, and released Mn
from PSII for AtMSD1 activation in chloroplast remain to be
investigated.
Moreover, we found the exogenous-expressed AtMSD1 activity inmtm1-i mtm2 -double mutant was expressed at a lower level compared
with the WT (Figure 12) . Thus, we proposed the defectiveAtMTM1 and AtMTM2 cause the blockage of Mn transportation
and affect AtMSD1 metalation synergistically. Several factors for
mitochondrial MnSOD inactivation have also been studied in yeast. Fe-S
cluster biogenesis genes GRX5 and SSQ1 involved in ySOD2
inactivation by disrupting mitochondrial Fe homeostasis in yeast
(Naranuntarat et al., 2009). In Mn-deficient plants, the altered Mn
content in root or shoot tissue was involved in the abnormal root
growth, altered Fe homeostasis, and gene expressions (Alejandro et al.,
2017; Rodríguez-Celma et al., 2016; Yang et al., 2008). yMTM1
facilitated the insertion of Mn cofactor for ySOD2 activation, butymtm1Δ retained the normal Mn level in mitochondria (Luk et al.,
2003). In our study, we found Mn transportation was defective inmtm1-i , mtm2 , and mtm1-i mtm2 mutants, and both Mn
and Fe contents in root and shoot were responsive to Mn treatment,
especially in mtm1-i mtm2 -double mutant (Figure 13) . We
further proposed AtMTM1 and AtMTM2 transporters regulate Mn and Fe
homeostasis with a reciprocal regulation. For the metal ion affinity,
many metal transporters have multiple metal substrates, but the
substrate specificities of AtMTM1 and AtMTM2 remain to be clarified.
In conclusion, we emphasized the physiological function of Arabidopsis
mitochondrial carrier proteins AtMTM1 and AtMTM2 for MnSOD enzyme
activation, and compared their gene expression profiles during
development and oxidative stress. In addition to AtMTM1 and AtMTM2, we
suggested the MnSOD importing process is associated with other
transporters or chaperones that may exist in both mitochondria and
chloroplasts. We confirmed AtMTM1 and AtMTM2 transporters regulate the
intracellular Mn and Fe redox states, and the involved mechanism may
cross organelles in cells.