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. 2014 Jun 20;26(6):2441–2456. doi: 10.1105/tpc.113.121657

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© 2014 American Society of Plant Biologists. All rights reserved.

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Figure 2. — PIL1 Protein Is Degraded in a COP1-Dependent Manner and Accumulates upon Light Irradiation.

(A) and (B) PIL1 is stabilized by the 26S proteasome inhibitor MG132.

(A) Total protein extracted from two independent lines of the 35S_pro:PIL1-FLAG/WT (#19 and #22) transgenic plants grown in darkness for 4 d and then treated with MG132 or DMSO for 6 h was subjected to immunoblot analysis with anti-FLAG and anti-ACTIN antibodies, respectively.

(B) Root cells of 35S_pro:PIL1-GFP#5/WT seedlings grown under conditions of (A) were analyzed by fluorescence microscopy. Bars = 20 μm.

(C) and (D) PIL1 accumulated in the cop1 mutant background.

(C) Total protein extracted from 4-d-old dark-grown 35S_pro:PIL1-FLAG#19/WT and 35S_pro:PIL1-FLAG#19/cop1-4 seedlings was subjected to immunoblot analysis with anti-FLAG and anti-ACTIN antibodies, respectively.

(D) Root cells of the 35S_pro:PIL1-GFP#5/WT and 35S_pro:PIL1-GFP#5/cop1 seedlings grown under conditions of (C) were analyzed by fluorescence microscopy. Bars = 20 μm.

(E) Immunoblot showing PIL1-FLAG in 35S_pro:PIL1-FLAG#19/WT seedlings grown in continuous indicated conditions for 4 d.

(F) PIL1 accumulated by exposure to the indicated light conditions. Total protein extracted from the 35S_pro:PIL1-FLAG#19/WT seedlings grown in darkness for 4 d and then transferred to the indicated light conditions for the indicated periods of time was subjected to immunoblot analysis.

(G) Immunoblot analysis of PIL1-FLAG in PIL1_pro:PIL1-FLAG#15/pil1 seedlings grown in continuous darkness for 4 d and then exposed to red light for the indicated time periods. PIL1/PIL1 represents the ratio of PIL1 protein to PIL1 mRNA (from Supplemental Figure 4) under the indicated light conditions.

(E) to (G) Total protein was subjected to immunoblot analysis with anti-FLAG and anti-ACTIN antibodies. PIL1/ACT indicates the relative band intensities of PIL1-FLAG normalized to ACTIN and is presented relative to that in darkness set at unity. DK, darkness; BL, blue light; RL, red light; FRL, far-red light.