Figure 5.

AtOR^His and AtOR share a similar interaction with AtPSY and the same effect on regulating AtPSY protein level. A, BiFC of AtOR or AtOR^His interaction with AtPSY. Vectors with N terminus of YFP (nYFP) and C terminus of YFP (cYFP), AtOR-nYFP and AtPSY-cYFP, or AtOR^His-nYFP and AtPSY-cYFP were coexpressed in tobacco leaves. The fluorescence signals were detected with a confocal microscope. Bar = 20 μm. B, Yeast two-hybrid (Y2H) analysis of AtOR and AtOR^His interactions with AtPSY. Combinations of vectors with N terminus of ubiquitin (Nub), C terminus of ubiquitin (Cub), Nub-AtOR, Nub-AtOR^His, or AtPSY-Cub were coexpressed in yeast (Saccharomyces cerevisiae) cells. Yeast was grown on nonselective medium (synthetic defined premix/Uri-Thr-Leu [SD/UTL]) and selective medium (synthetic defined premix/Ala-Thr-His-Leu-Uri-Met [SD/ATHLUM]), with 0.05 mm Met with four series of 10 times dilution. For the β-galactosidase activity assay, yeast was cultured in liquid medium overnight and tested. OD₆₀₀, absorbance at wavelength 600 nm; oNPG, o-nitrophenyl-β-d-galactopyranoside. C, Western blot of AtPSY in the calli of Col, AtOR, AtOR^His, and AtOR^Ala lines. Actin protein level was used as a loading control. Relative PSY protein levels were calculated from quantification of western band signals. *, Significant difference when compared with Col (P ≤ 0.05, n = 3).