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. 2021 Nov 3;118(45):e2103633118. doi: 10.1073/pnas.2103633118

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Copyright © 2021 the Author(s). Published by PNAS.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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Fig. 4. — UBP12 and UBP13 increase PIF7 stability. (A) PIF7 protein levels in WT (Col-0) and UBP12-OE background under WL and SL conditions. At 7 d old, 35S::PIF7-FLASH and UBP12-OE/35S::PIF7-FLASH seedlings grown under WL were moved to shade or remained under WL with or without 50 µM MG132 for 4 h, and then samples were harvested for protein extraction. P-PIF7, phosphorylated form of PIF7. (B) PIF7 protein levels in WT (Col-0) and ubp12-2w/13–3 background under WL and SL conditions. 35S::PIF7-MYC and ubp12-2w/13–3/35S::PIF7-MYC plants were treated as indicated in A. (C) PIF7 protein levels in WT (Col-0), 35S::PIF7-FLASH, UBP12-OE/35S::PIF7-FLASH, and UBP12(C208S)-OE/35S::PIF7-FLASH under WL and SL. At 7 d old, WL-grown seedlings were transferred to SL or left under WL for 4 h before being collected for protein extraction. (D) The polyubiquitination status of PIF7 in various genotypes. Seedlings were grown under WL for 10 d and then were treated with 50 µM MG132 under SL or WL for 4 h. Protein extracts were immunoprecipitated (IP) by using Ni-NTA nickel beads (Qiagen). IP products were analyzed by Western blotting using anti-Ubiquitin and anti-MYC antibodies. (Bottom) Shorter exposure of Middle. Molecular weight standards are indicated. (E and F) Hypocotyl phenotypes (E) and measurements of hypocotyl lengths (F) of WT (Col-0), ubp12-2W/13–3, 35S::PIF7-MYC#14, and ubp12-2w/13–3/35S::PIF7-MYC#14 grown under WL and SL. (Scale bar: 1 cm.) The values shown are mean ± SD (n ≥ 20). *P < 0.05 and **P < 0.01; based on Student’s t test. (G and H) The expression of shade-induced genes under WL and SL in plants indicated in E and F. The values shown are mean ± SD. *P < 0.05 and **P < 0.01; based on Student’s t test.