Fig. 3: Together with CPP1, DAY regulates the stability of protochlorophyllide oxidoreductase (POR) through its holdase chaperone activity.
From: Chaperone-like protein DAY plays critical roles in photomorphogenesis
a Reduction of DAY causes POR instability in N. benthamiana. Virus-induced gene silencing (VIGS) of N. benthamiana DAY (NbDAY) was performed. Then the VIGS plants (TRV:NbDAY) were agroinfiltrated with the POR C-GFP construct, and leaf mesophyll cells were examined by CLSM. POR C is a major POR isoform in light-grown Arabidopsis. NbDAY silencing caused a significant reduction in chlorophyll autofluorescence and POR C-GFP fluorescence. Scale bar, 20 µm. b DAY has holdase activity. Absorbance at 340 nm (A340) was measured at 1 min intervals to quantify turbidity. Thermal aggregation of malate dehydrogenase (2 µM) was measured at 43 °C over 16 min with 0–6 µM of MBP-∆cTP DAY or, as controls, 4 µM MBP-∆cTP/∆JL DAY (del JL) and 10 µM BSA. ∆cTP, deletion of cTP; ∆JL, deletion of the DnaJ- like domain (JL). c, d DAY interacts with POR C and CPP1 in chloroplasts. BiFC assays between DAY and POR C (c) or CPP1 (d). YFP signals were examined by CLSM in protoplasts after agroinfiltration. MAGNESIUM-PROTOPORPHYRIN IX METHYL TRANSFERASE (MgPMT), which is involved in chlorophyll biosynthesis, was used as a negative control. Scale bar, 10 µm (c) or 20 µm (d). e DAY directly interacts with CPP1 in vitro. Lysates of E. coli cells expressing GST or GST-CPP1 were incubated with MBP or MBP-∆cTP DAY (MBP-∆cTP) immobilized on amylose agarose resins. The resulting eluates were analyzed by immunoblotting using anti-GST antibody. The amount of MBP and MBP-∆cTP used in the pull-down assay were visualized by Coomassie staining.
