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. 2016 Dec 12;28(12):2952–2973. doi: 10.1105/tpc.16.00347

Figure 2.

Figure 2.

ABA-Responsive Stomatal Movement Was Restored by the Overexpression of the N-Terminal UND in pub22 pub23 Mutant Plants.

(A) Schematic representation of full-length PUB22 cDNA and PUB22 and UNDPUB18-PUB22 proteins. Solid lines indicate 5ʹ- and 3ʹ-untranslated regions, and the gray bar represents the coding region. The UNDPUB18 motif, U-box domain, and armadillo (ARM) repeats are indicated.

(B) In vitro self-ubiquitination of PUB22 and UNDPUB18-PUB22. The E3 Ub ligase activities of bacterially expressed MBP-PUB22 and MBP-UNDPUB18-PUB22 recombinant proteins and their single amino acid substitution variants (MBP-PUB22V24I and MBP-UNDPUB18-PUB22V293I) were examined with or without E1 (UBA1) and crude extract (10 μg total proteins) of ABA (100 μM)-treated pub18 pub19 pub22 pub23 leaves, as a source of E2 enzyme, along with Flag-tagged Ub (Flag-Ub), MG132 (80 μM), and protease inhibitor cocktail. Reaction products were analyzed by SDS-PAGE, followed by immunoblotting with anti-MBP and anti-Flag antibodies. The level of α-tubulin detected by anti-α-tubulin antibody was used as an equal loading control of input crude extract. The presence of an equal amount of Flag-Ub in each lane was confirmed by anti-Flag antibody. Arrows indicate the migration of the unmodified PUB proteins.

(C) RT-PCR analysis of wild-type, pub22 pub23 double mutant, and pub22 pub23/35S:PUB22-GFP (transgenic lines #1 and #2) and pub22 pub23/35S:Flag-UNDPUB18-PUB22 (transgenic lines #1 and #2) complementation plants. Four independent biological replicate were performed. The UBC10 gene was used as a loading control.

(D) Immunoblot analysis of pub22 pub23 double mutant and pub22 pub23/35S:PUB22-GFP (lines #1 and #2) and pub22 pub23/35S:Flag-UNDPUB18-PUB22 (lines #1 and #2) complementation plants. Ectopic expression of PUB22-GFP and Flag-UNDPUB18-PUB22 proteins was detected using anti-GFP and anti-Flag antibodies, respectively. The level of α-tubulin was used as an equal loading control.

(E) Stomatal movements of wild-type, pub22 pub23 double mutant, and pub22 pub23/35S:PUB22-GFP (lines #1 and #2) and pub22 pub23/35S:Flag-UNDPUB18-PUB22 (lines #1 and #2) complementation plants in response to different concentrations (0, 1, and 10 μM) of ABA. Thirty stomatal apertures per treatment were measured, and three replicates were performed for each experiment. Error bars represent ±se (n = 90; **P < 0.005, one-way ANOVA). Bars = 10 μm.