FIGURE 4.

Protease activity of CtpA for AtpD1 processing from four oxygenic photosynthetic organisms. (A) Characterizations of the phenotypes (left) and pD1 processing status (right) in transgenic atctpa plants expressing AtCtpA-HA, PpCtpA-HA, CrCtpA-HA, and SeCtpA-HA under different light conditions. Plants were gown in soil under continuous light as follows: NL, normal light (80 μmol/s/m²) for 3 weeks; FL, normal light for 1 week, then fluctuating light for 2 weeks (the light intensity changes are shown in Supplementary Figure 3A); HL, normal light for 2 weeks and then high light (350 μmol/s/m²) for 1 week. Scale bars denote 1 cm. The pD1 processing in these transgenic plants was assessed by immunoblotting with α-HA (for ectopic expressed CtpA-HAs) and α-pD1 tail (for native pD1). Two plants were examined for each transgenic line. The filters stained with Ponceau S (Pon. S) were used as loading controls. Asterisks indicate non-specific bands. (B) In vitro protease activities of the PpCtpA and CrCtpA were tested with recombinant AtpD1 substrate. Recombinant AtpD1 was a fusion of GST with the C-terminal 50 aa fragment of Arabidopsis thaliana pD1. Proteolytic reactions were performed in pH 5.0, 7.0, and 9.0, respectively, stopped at the time points of 0, 5, 10, 20, and 60 min, and examined for the pD1 processing statuses by immunoblot against α-pD1 tail (for pD1) and α-D1 (for D1 and pD1) antibodies.