Chloroplast-destined AtMSD1 activation is independent ofAtMTM1 and AtMTM2
To seek other candidates for MnSOD activation, we generated cytosol-destined and chloroplast-destined AtMSD1, and transfected these constructs into the WT and mtm1-i mtm2 protoplasts for comparison(Figures 11, 12) .
The destinations of cytosol-localized AtMSD1 (Δ-TP-AtMSD1) and chloroplast-localized AtMSD1 (Chl-TP-AtMSD1) were confirmed with a reporter YFP and analyzed by confocal microscopy (Figure 11A) . Besides, the exogenous-expressed AtMSD1-Tag and endogenous MnSOD activities could be distinguished by in-gel SOD activity assay and immunoblotting (Figure 11B, lanes 1, 2) . Particularly, the chloroplast-destined AtMSD1 was activated but not the cytosol-localized AtMSD1 (Figure 11B, lanes 3, 4) . Overall, we found the exogenous expressed AtMSD1 activation inside chloroplast was independent of mitochondrial AtMTM1 and AtMTM2, and unclarified chloroplastic factors contributed to chloroplast-destined MSD1 metalation.
We also created a modified AtMSD1 with a 3xFLAG tag, and transfected this construct into the WT and mtm1-i mtm2 -double mutant protoplasts (Figure 12) . The exogenous-expressed AtMSD1-3xFLAG activity and protein were confirmed by in-gel SOD activity assay and immunoblotting (Supplemental Figure S14) , respectively. Notably, the exogenous expressed AtMSD1-3xFLAG activity was lower inmtm1-i mtm2 than the WT with transfection of 10 μg DNA for 16 h(Figure 12, lanes 3, 6) , implying AtMTM1 and AtMTM2 participated in AtMSD1 activation.