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.