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. 2012 Mar 9;24(3):1114–1126. doi: 10.1105/tpc.111.092130

Figure 7.

Figure 7.

Functional Analyses of the Ser-747 Residue of PHOT2.

(A) Immunoblot analysis of the expression levels of PHOT2 GFP, phot2 Lys-746/A-GFP, and phot2 Ser-747/A-GFP in the transformants. Approximately 30 μg (per well) of microsomal protein from each genotype was resolved in 7.5% SDS-PAGE and subjected to immunoblotting for PHOT2. The blot was then stained with Ponceau S solution to test for equal loading of protein samples.

(b) Phot2 Ser-747/Ala replacement reduces blue light responses of stomatal opening. Abaxial-leaf epidermal strips from Arabidopsis plants about 5 weeks old were tested for their relative sensitivity to blue light for stomatal opening. The phot2 Ser-747/A GFP transformant line exhibited reduced stomatal opening compared with the PHOT2 GFP and phot2 Lys-746/A GFP controls, suggesting that Ser-747 functioned as a major recognition site for 14-3-3 λ. Data represent mean ± se (n ≥ 75) from two different experiments with similar results. Except for the lowest fluence rate, both the PHOT2GFP and PHOT2GFP Lys-746/A transformants had apertures that were significantly greater than those of the PHOT2GFP Ser-747/A transformant (P < 0.05). Different letters denote aperture values for the phot2 Ser-747/A transformant significantly below those of the control. All blue light treatments were performed with background red light at 50 μmol m−2 s−1. All plants are in the double mutant background (phot1-5 phot2-T DNA insertion) and were kept under a 16-h-light/8-h-dark day/night cycle at 22°C.

(C) PHOT2 GFP, phot2 Lys-746/A GFP, and phot2 Ser-747/A GFP transformant lines exhibit sensitivity to blue light equal to that of the phot1-5 control. Representative data (mean ± se; n ≥ 22) from two experiments with similar results are shown.

(D) Arabidopsis transformant lines expressing PHOT2-GFP, phot2 Lys-746/A-GFP, and phot2 Ser-747/A GFP together with a nontransformant line, all ~6 weeks old, were investigated for the leaf-curling phenotype seen in the double mutant background (phot1-5 phot2-T DNA insertion); plants were grown at 22°C under a 16-h-light/8-h-dark day/night cycle. Leaves of all three transformant lines showed full expansion, but not the nontransformant. Bar = 1 cm.

(E) PHOT2-GFP, phot2 Lys-746/A-GFP, and phot2 Ser-747/A-GFP are equally functional in rescuing cotyledon positioning phenotype. No difference in cotyledon position was seen in the 8-d-old transformant seedlings expressing wild-type PHOT2-GFP, phot2 Lys-746/A-GFP, and phot2 Ser-747/A-GFP compared with the phot1-5 control. The phot1-5 phot2-T-DNA nontransgenic control exhibited a downward cotyledon-positioning phenotype. Seedlings were grown under a 16-h-light/8-h-dark day/night cycle at 22°C. Images were all taken 1 h after lights on.