Fig. 3: PUB30/31 ubiquitinate and regulate the protein abundance of ERECTA.
From: Direct attenuation of Arabidopsis ERECTA signalling by a pair of U-box E3 ligases
a, Reduced in vivo ERECTA ubiquitination in pub30 pub31 mutant. Immunoprecipitation (IP) was performed using anti-FLAG antibody on solubilized microsomal fraction protein extracts from ERECTA-FLAG plants in ‘WT’ or pub30 pub31 background. Immunoblots (IB) were probed with anti-ubiquitin and anti-FLAG antibody, respectively. b, Quantitative analysis of ERECTA ubiquitination profiles. Error bars represent s.d. (n = 3). Asterisks indicate statistical significance using two-tailed paired Student’s t-test (P = 0.0039). c, ERECTA protein accumulation in WT and pub30 pub31, in the absence and presence of the endocytosis inhibitor Tyr A23. Data normalized by anti-actin. d, Quantification of ERECTA abundance (ERECTA/actin). Error bars represent s.d. (n = 3). Asterisks indicate statistical significance using two-tailed paired Student’s t-test (P = 0.0012). e, PUB30 or PUB31 mediates ERECTA ubiquitination in vivo. Arabidopsis protoplasts were cotransfected with ERECTA-HA, FLAG-UBQ and a control vector or PUB30-MYC or PUB31-MYC. Five micromolar EPFL6 was used for treatment for 1 h. After immunoprecipitation using anti-FLAG beads, the ubiquitinated ERECTA (Ubn-ERECTA) was probed with anti-HA antibody. The total ubiquitinated proteins were probed by anti-FLAG antibody and PUB30 or PUB31 proteins were probed by anti-MYC antibody. The inputs of ERECTA were probed with anti-HA antibody. f, Representative pedicels of mature siliques of pub30 pub31, proPUB30::PUB30W97A; pub30 pub31, proPUB31::PUB31W93A; pub30 pub31 and WT plants. Scale bar, 1 cm. g, Confocal microscopy of 8-day-old abaxial cotyledon epidermis of pub30 pub31, proPUB30::PUB30W97A; pub30 pub31, proPUB31::PUB31W93A; pub30 pub31 and WT plants. Scale bar, 25 μm. h, Morphometric analysis of pedicel length from each genotype. Mature pedicels (n = 15) from 6-week-old plants were measured. One-way ANOVA followed by Tukey’s HSD test was performed and classified their phenotypes into categories (a and b). For P values see Extended Dataset 1. i, Quantitative analysis. Stomatal index of the cotyledon abaxial epidermis from 8-day-old seedlings of respective genotypes (n = 7). One-way ANOVA followed by Tukey’s HSD test was performed and classified their phenotypes into categories (a and b). For P values see Supplementary Data.
