Figure 5.

ACS Protein Stability Is Regulated by the Level of ETO1/EOL and 14-3-3 Proteins.

(A) The steady state level of HA-EOL2 and HA-ACS5 proteins with increasing HA-14-3-3ω expression. Protoplasts were transformed with plasmids expressing HA-EOL2, HA-ACS5, and increasing levels of HA-14-3-3ω. An empty vector was added as appropriate to ensure equal amounts of plasmid DNA was added to each reaction. Total proteins were analyzed by immunoblotting using an anti-HA antibody or an anti-GFP antibody. The GFP signal is used as a transformation control.

(B) Increased expression of EOL2 protein promotes degradation of ACS5 protein. Protoplasts were transformed with plasmids expressing HA-14-3-3ω, HA-ACS5, and increasing levels of HA-EOL2. An empty vector was added as appropriate to ensure equal amounts of plasmid DNA was added to each reaction. Total proteins were analyzed by immunoblotting using an anti-HA antibody or an anti-GFP antibody.

(C) and (D) Ethylene production from two independent transgenic lines overexpressing EOL2 (EOL2-OX) as etiolated (C) or light-grown (D) seedlings. The inset shows the expression of the HA-EOL2 proteins in the lines. mean ± se; n = 3. Col, Columbia.

(E) and (F) Ethylene production from the wild type (E) or a transgenic line expressing 14-3-3ω-myc from an estradiol-inducible promoter (F). Seedlings were grown in the light in the presence of the indicated level of estradiol for 12 d and then capped for 24 h and the level of ethylene accumulated measured. The inset shows the expression of 14-3-3ω-myc in response to different concentration of estradiol. Significance was determined using analysis of variance and Tukey’s HSD post-hoc test. *P < 0.05 and **P < 0.01. Error bars indicate se; n = 3.