INTRODUCTION
Harmful reactive oxygen species (ROS) contain non-free radicals and free
radicals that are crucially regulated by the scavenger superoxide
dismutases (SODs) during plant development and metabolism. Superoxide
radicals are generated by metabolic processes of respiratory and
photosynthetic electron transport chains in mitochondria and
chloroplasts, and under high light, heat, drought, salt, and oxidative
stress (Apel & Hirt, 2004; Fridovich, 1975). Superoxide radicals are
membrane-impermeable and rapidly damage nearby cell components, thus SOD
enzymes catalyze the dismutation of the toxic superoxide radicals to
oxygen and hydrogen peroxide, and defend against ROS (Halliwell, 1994).
SODs are metalloenzymes, and the corresponding cofactors are transition
metal ions (Cu, Fe, Mn, or Ni) that accept or donate an electron during
the dismutation process. Specially, SOD enzyme activation requires a
metallochaperone or transporter that captures and loads the metal ion
into the target SOD apoprotein. Most eukaryotes harbor both MnSOD and
CuZnSOD. Prokaryotes and plants also contain FeSOD. NiSOD is present inStreptomyces (Alscher, Erturk, & Heath, 2002; Choudhury et al.,
1999; Fink & Scandalios, 2002). Arabidopsis MnSOD (AtMSD1) locates in
mitochondria (Bowler, Camp, Montagu, & Inzé, 1994), FeSODs contain
chloroplastic FSD1, FSD2, and FSD3. The stromal FSD1 conferred the
mainly detectable FeSOD activity (Kuo, Huang, & Jinn, 2013a; Kuo et
al., 2013b). CuZnSODs contain cytosolic CSD1, chloroplastic CSD2, and
peroxisomal CSD3 (Kliebenstein, Monde, & Last, 1998).
The post-translational incorporation of Mn cofactor into yeast MnSOD
(ySOD2) apoprotein involves the Mn trafficking transporter for
mitochondrial MnSOD (yMTM1), which belongs to the mitochondrial carrier
family (MCF) (Luk, Carroll, Baker, & Culotta, 2003). MnSOD locates in
the organelle of mitochondria in yeast and Arabidopsis, and also at the
thylakoid membrane in some species of green and blue-green algae
(Kanematsu & Asada, 1979; Kanematsu, Okayasu, & Kurogi, 2012; Okada,
Kanematsu, & Asada, 1979; Regelsberger et al., 2002). To date, the
Mn-related chaperone in mitochondria or chloroplast has not been fully
elucidated. The chloroplast chaperonin 20 (CPN20) functions as the Fe
chaperone for FeSOD activation (Kuo et al., 2013a; Kuo et al., 2013b).
Yeast copper chaperone for SOD1 (CCS) incorporates Cu cofactor into
CuZnSOD apoprotein (ySOD1), and this CCS-dependent pathway also occurs
in human and Arabidopsis (Casareno, Waggoner, & Gitlin, 1998; Chu et
al., 2005; Culotta, Yang, & O’Halloran, 2006; Rae, Torres, Pufahl, &
O’Halloran, 2001). The CCS-independent CuZnSOD activation pathway was
found in Arabidopsis cytoplasm and peroxisome, and glutathione was
involved (Carroll et al., 2004; Huang, Kuo, & Jinn, 2012a; Huang, Kuo,
Weiss, & Jinn, 2012b).
Arabidopsis mitochondria contain the highly conserved MnSOD that
congregates as a homotetramer, and each monomer harbors only one Mn
cofactor (Pilon, Ravet, & Tapken, 2011; Sevilla, López-Gorgé, & del
Río, 1982). Among all species of SOD, MnSOD is responsible for
protecting the respiratory machinery in the mitochondrial matrix (Bowler
et al., 1994; Fridovich, 1975). MnSOD expression is more constant
than FeSOD and CuZnSOD under high light, ozone, and UV-B
exposure (Kliebenstein et al., 1998). Overexpressing MnSOD could
increase stress tolerance in transgenic tobacco and Arabidopsis (Slooten
et al., 1995; Van Camp et al., 1994; Wang, Ying, Chen, & Wang, 2004).
By contrast, decreased MnSOD expression may cause root growth
inhibition and affect mitochondrial redox homeostasis in Arabidopsis
(Morgan et al., 2008). On the other hand, CuZnSOD and FeSOD enzymes
protect chloroplasts against superoxide radicals, and their activities
are easily affected by Cu level via Cu transporters of chloroplastic
envelop-localized P-type ATPase PAA1 and thylakoid membrane-localized
PAA2. Specially, FSD1 and CSD2 genes are affected by
stromal Cu delivery with a reciprocal regulation (Abdel-Ghany et al.,
2005a; Abdel-Ghany, Muller-Moule, Niyogi, Pilon, & Shikanai, 2005b).
In E. coli , MnSOD and FeSOD share a well-conserved protein folds
and have conserved residues for metal binding (Pugh & Fridovich, 1985;
Wintjens et al., 2004). Incorrect metal insertion altered SOD reduction
potential, and the enzyme had trouble scavenging superoxide radicals
(Miller, 2012; Vance & Miller, 2001). Mn and Fe are interchangeable
between MnSOD and FeSOD, and cause metal ion misincorporation, but
neither Fe-substituted MnSOD nor Mn-substituted FeSOD is active (Ganini,
Petrovich, Edwards, & Mason, 2015; Meier, Barra, Bossa, Calabrese, &
Rotilio, 1982).
In yeast, ySOD2 protein cannot be activated in the cytosol. Mn cofactor
is inserted into the newly synthesized ySOD2 polypeptides via the
mitochondrial carrier protein yMTM1, thus the insertion of Mn into ySOD2
apoprotein is connected to the importing process in mitochondria (Luk,
Yang, Jensen, Bourbonnais, & Culotta, 2005). In yMTM1 -mutant
(ymtm1Δ ) cells, inactivated ySOD2 is associated with the
disrupted mitochondrial Fe homeostasis. When the Fe-S cluster biogenesis
pathway was blocked, Fe for Fe-S cluster diverted to ySOD2 and caused Fe
misincorporation, but the role of yMTM1 in Fe-S cluster maturation has
not been verified (Naranuntarat, Jensen, Panicni, Penner-Hahn, &
Culotta, 2009; M. Yang et al., 2006). In Arabidopsis, mitochondrial
carrier protein AtMTM1 (At4g27940) has 32% identity with yMTM1, and can
recover ySOD2 activity in ymtm1Δ cells (Su et al., 2007).
Mn transporters for MnSOD activation are associated with Mn homeostasis
in yeast and Arabidopsis. Mn homeostasis in yeast involves Mn
transporters SMF1 and SMF2 in the natural resistance-associated
macrophage protein (NRAMP) family (Chen et al., 1999; Luk & Culotta,
2001). SMF1 is located at the plasma membrane of cell surface; SMF2
localizes in intracellular vesicles and functions for Mn trafficking
among intracellular locations (Portnoy, Liu, & Culotta, 2000).SMF1 mutation did not affect ySOD2 activity, but SMF2disruption caused cell-wide Mn starvation and loss of ySOD2 activity
(Luk & Culotta, 2001). Arabidopsis NRAMP family members NRAMP3 and
NRAMP4 have homology with yeast SMF1 and SMF2, and are involved in the
Mn-containing oxygen-evolving complex (OEC). OEC catalyzes the
water-splitting reaction that produces oxygen and provides electrons for
the photosynthetic electron transport chain (Goussias, Boussac, &
Rutherford, 2002; Nickelsen & Rengstl, 2013). Disruption of
vacuole-localized NRAMP3 and NRAMP4 decreased PSII amount
in chloroplasts, but did not affect MnSOD activity in mitochondria
(Allen, Kropat, Tottey, Del Campo, & Merchant, 2007; Lanquar et al.,
2010).
The system of Mn transportation in plants involves families of NRAMP,
Zn-regulated transporter and Fe-regulated transporter-like protein
(ZIP), yellow stripe-like protein (YSL), cation exchanger (CAX), calcium
cation exchanger (CCX), cation diffusion facilitator/metal tolerance
protein (CDF/MTP), vacuolar Fe transporter (VIT), and P-type ATPase
(Socha & Guerinot, 2014). MCF family member MTM1 is directly connected
to MnSOD activation via transporting Mn cofactor into the MnSOD
apoprotein. The essential Mn cofactor is also required for other
metalloproteins such as oxalate oxidase, RNA polymerase, malic enzyme,
and isocitrate dehydrogenase (Bashir, Rasheed, Kobayashi, Seki, &
Nishizawa, 2016; Marschner, 1995; Rostami & Ahangar, 2013; Witholt,
Gwiazda, & Smith, 2000).
The MCF is a large family with 35 members in yeast and more than 50
genes in plants and humans, which is evolutionarily conserved for
transporting the specific substrates and cofactors (Haferkamp &
Schmitz-Esser, 2012). MCF members have three homologous domains which
fold into two transmembrane α-helices, and contain the conserved
mitochondrial energy transfer signature (METS),
P-x-[DE]-x-[LIVAT]-[RK]-x-[LRH]-[LIVMFY]-[QGAIVM],
on the matrix side (Millar & Heazlewood, 2003; Robinson & Kunji,
2006). Study of the evolution of MCF revealed a clustered phylogeny ofAtMTM1 and AtMTM2 , and their amino acid sequences have
high homology (Palmieri, Pierri, Grassi, Nunes-Nesi, & Fernie, 2011).
In this research, we clarified the similar complementary effect of
AtMTM1 and AtMTM2 for ySOD2 activation in ymtm1Δ cells, and
revealed their regulations under methyl viologen (MV)-mediated oxidative
stress. We confirmed that an evolutionarily conserved mechanism for
MnSOD activation is remained in Arabidopsis chloroplast as in some
species of algae. In addition, we strengthened the impact of AtMTM1 and
AtMTM2 on Mn and Fe ion homeostasis.