SummaryIron superoxide dismutases (FeSODs; FSDs) are primary antioxidant enzymes in Arabidopsis thaliana chloroplasts. The stromal FSD1 conferred the only detectable FeSOD activity, whereas the thylakoid membrane-and nucleoid-co-localized FSD2 and FSD3 double mutant showed arrested chloroplast development.FeSOD requires cofactor Fe for its activity, but its mechanism of activation is unclear. We used reversed-phase high-performance liquid chromatography (HPLC), gel filtration chromatography, LC-MS/MS, protoplast transient expression and virus-induced gene silencing (VIGS) analyses to identify and characterize a factor involved in FeSOD activation.We identified the chloroplast-localized co-chaperonin CHAPERONIN 20 (CPN20) as a mediator of FeSOD activation by direct interaction. The relationship between CPN20 and FeSOD was confirmed by in vitro experiments showing that CPN20 alone could enhance FSD1, FSD2 and FSD3 activity. The in vivo results showed that CPN20-overexpressing mutants and mutants with defective co-chaperonin activity increased FSD1 activity, without changing the chaperonin CPN60 protein level, and VIGS-induced downregulation of CPN20 also led to decreased FeSOD activity.Our findings reveal that CPN20 can mediate FeSOD activation in chloroplasts, a role independent of its known function in the chaperonin system.

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. We confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMTM1 as they can revive yeast MnSOD activity in yMTM1-mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2-double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1, AtMTM2, and AtMnSOD respond positively to methyl viologen (MV) and metal stress. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. Transient expression of chloroplast-destined AtMnSOD revealed that an evolutionarily conserved activation mechanism, like the chloroplastic-localized MnSOD in some algae, still exists in Arabidopsis chloroplasts. This study strengthens the proposition that AtMTM1 and AtMTM2 participate in the AtMnSOD activation and ion homeostasis.