Figure 1.

Deletion of the N-Terminal UND of PUB18 Impairs ABA-Mediated Stomatal Movements, but Does Not Affect the Drought-Induced Stomatal Closure in Arabidopsis.

(A) Schematic structure of full-length PUB18 cDNA and PUB18 and ∆UND-PUB18 proteins. Solid lines indicate 5ʹ- and 3ʹ-untranslated regions. The gray bar in the upper diagram represents the coding region. The N-terminal UND motif, U-box domain, and armadillo (ARM) repeats are indicated.

(B) In vitro self-ubiquitination assay of PUB18 and ∆UND-PUB18. Recombinant MBP-PUB18, MBP-PUB18^V305I, MBP-∆UND-PUB18, and MBP-∆UND-PUB18^V36I proteins were incubated with Flag-tagged Ub (Flag-Ub) at 30°C for 2 h in the presence or absence of E1 (UBA1) and crude extract (10 μg total proteins) prepared from ABA (100 μM)-treated pub18 pub19 pub22 pub23 quadruple mutant leaves, as a source of E2 enzyme, along with MG132 (80 μM) and protease inhibitor cocktail. Reaction products were resolved using SDS-PAGE and subjected to immunoblot analysis 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, pub18 pub19 double mutant, and pub18 pub19/35S:HA-PUB18 (transgenic lines #1 and #2) and pub18 pub19/35S:Flag-∆UND-PUB18 (transgenic lines #1 and #2) complementation plants. Each experiment was performed with three independent biological replicates. The Ubiquitin Conjugating Enzyme10 (UBC10) gene was used as a loading control.

(D) Immunoblot analysis of pub18 pub19 double mutant and pub18 pub19/35S:HA-PUB18 (lines #1 and #2) and pub18 pub19/35S:Flag-∆UND-PUB18 (lines #1 and #2) complementation plants. Expression levels of HA-PUB18 and Flag-∆UND-PUB18 proteins were determined using anti-HA and anti-Flag antibodies, respectively. The level of α-tubulin was an equal loading control.

(E) Stomatal movements of wild-type, pub18 pub19 double mutant, and pub18 pub19/35S:HA-PUB18 (lines #1 and #2) and pub18 pub19/35S:Flag-∆UND-PUB18 (lines #1 and #2) complementation plants in response to ABA. Light-grown mature rosette leaves were immersed in stomatal opening solution for 2 h and transferred to the solution containing different concentrations (0, 1, and 10 μM) of ABA for 2 h. Stomata were imaged using bright-field microscopy. At least 30 stomatal apertures in each epidermal strip were measured per replicate. Three replicates were performed for each experiment. Error bars represent ±se (n = 90; **P < 0.005, one-way ANOVA). Bars = 10 μm.

(F) Stomatal movements of wild-type, pub18 pub19 double mutant, and pub18 pub19/35S:HA-PUB18 (lines #1 and #2) and pub18 pub19/35S:Flag-∆UND-PUB18 (lines #1 and #2) complementation plants in response to osmotic stress imposed by various concentrations (0, 0.2, and 0.4 M) of mannitol. Error bars represent ±se (n = 90; *P < 0.05, **P < 0.005, one-way ANOVA). Bars = 10 μm.