Fig. 6: The phosphorylation status of the NPH3 14-3-3-binding site (S744) is dynamically modulated by the light regime. | Nature Communications

Fig. 6: The phosphorylation status of the NPH3 14-3-3-binding site (S744) is dynamically modulated by the light regime.

From: Light-triggered and phosphorylation-dependent 14-3-3 association with NON-PHOTOTROPIC HYPOCOTYL 3 is required for hypocotyl phototropism

Fig. 6

The closed and open arrowheads indicate the positions of “generally” phosphorylated and dephosphorylated NPH3 proteins, respectively. a Immunoblot analysis and 14-3-3 far-western of anti-GFP immunoprecipitates from N. benthamiana leaves transiently expressing GFP:NPH3 or GFP:NPH3-S744A. Expression was driven by the 35S promoter. Dark-adapted tobacco plants were either maintained in darkness (D) or treated with blue light (BL) (10 μmol m−2 s−1) for 40 min. Immunoprecipitated proteins were separated on 7.5% SDS-PAGE gels. b Immunoblot analysis and 14-3-3 far-western of anti-GFP immunoprecipitates from Arabidopsis nph3-7 expressing GFP:NPH3. Expression was driven by the 35S promoter. Three-day-old etiolated seedlings were treated with cycloheximide (100 μM) for 1 h and either maintained in D, treated with BL (1 μmol m−2 s−1) for the indicated time, or re-transferred to D (1 h) after 30 min of irradiation (R-D). Immunoprecipitated proteins were separated on 7.5% SDS-PAGE gels. The upper panel shows representative confocal microscopy images of hypocotyl cells from the seedlings under the specified conditions. Scale bars, 25 μm. c Immunoblot analysis of total protein extracts from Arabidopsis nph3-7 ectopically expressing GFP:NPH3 (see b). Three-day-old etiolated seedlings were either maintained in D, treated with BL (1 μmol m−2 s−1) for the indicated time or re-transferred to D for the indicated time after 30 min of irradiation (R-D). Proteins were separated on 7.5% SDS-PAGE gels. d Immunoblot analysis of total protein extracts from N. benthamiana leaves transiently co-expressing MAP:mCherry:SAC1 and either GFP:NPH3 or GFP:NPH3-S744A. Expression was driven by the 35S promoter. Dark-adapted tobacco plants were either maintained in D or treated with BL (10 μmol m−2 s−1) for 40 min. Proteins were separated on 7.5% SDS-PAGE gels. All experiments were performed at least three times with similar results.

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