Figure 4: PUB12/13 are required for ABI1 degradation in plant cells.

(a) ABI1 level is higher in the pub12 pub13 double mutant than in the wild type. The total proteins extracted from wild-type plants or the pub12 pub13 mutant treated with or without 50 μM ABA for 6 h were used for immunoblotting analysis with anti-ABI1 antibody. Total proteins from the pub12 pub13 mutant were diluted from 100 to 50 μg, and short and long exposure time were used in order for comparison. ACTIN was used as a loading control. (b) Relative expression of ABI1 in the wild type (Col), the pub12 pub13 mutant and abi1-1 (Col). Total RNAs extracted from 7-day-old seedlings treated with 50 μM ABA for 1 h were used for real-time RT–PCR. (c) Comparison of degradation between the pub12 pub13 mutant and the wild type. The 7-day-old seedlings were treated with 100 μM CHX for different times. At each time point, total proteins were extracted and used for immunoblotting analysis with anti-ABI1 antibody. ACTIN was used as a loading control. (d) Quantitative analysis of the signal intensity in c. The abundance of ABI1 at the 0 h was set to 1 as a reference for calculating relative abundance of various time point. Error bars are means±s.e.m. (n=3 independent experiments). (e) PUB13-mediated ABI1 degradation requires ABA as well as ABI1 interaction with PYR1. The protoplasts from a transgenic line overexpressing PYR1-Flag or the wild type seedling were co-expressed with 10 μg of pro35S:ABI1-Myc and different amounts of pro35S:PUB13-Flag plasmids (0 to 200 μg), or together with 10 μg of Pro35S-PYR1P88S (only for wild type protoplasts) for 16 h and treated with 10 μM ABA (left, right) or without ABA (middle) for 4 h before immunoblotting analysis was performed using anti-Myc antibody or anti-Flag antibody. PYR1-Flag was used as the loading control. (f) ABI1 is ubiquitinated in plants. Ubiquitinated proteins were enriched from P62-agarose matrix that was incubated with total proteins isolated from two independent transgenic plants stably expressing ABI1-Myc or from wild-type plants. Plant materials were pretreated with 50 μM ABA for 12 h and 50 μM MG132 for 6 h before immunoblotting analysis was performed using anti-Myc antibody.