Arabidopsis PUB22 and PUB23 are homologous U-Box E3 ubiquitin ligases that play combinatory roles in response to drought stress

Abstract

Ubiquitination is involved in diverse cellular processes in higher plants. In this report, we describe Arabidopsis thaliana PUB22 and PUB23, two homologous U-box-containing E3 ubiquitin (Ub) ligases. The PUB22 and PUB23 genes were rapidly and coordinately induced by abiotic stresses but not by abscisic acid. PUB22- and PUB23-overexpressing transgenic plants were hypersensitive to drought stress. By contrast, loss-of-function pub22 and pub23 mutant plants were significantly more drought-tolerant, and a pub22 pub23 double mutant displayed even greater drought tolerance. These results indicate that PUB22 and PUB23 function as negative regulators in the water stress response. Yeast two-hybrid, in vitro pull-down, and in vivo coimmunoprecipitation experiments revealed that PUB22 and PUB23 physically interacted with RPN12a, a subunit of the 19S regulatory particle (RP) in the 26S proteasome. Bacterially expressed RPN12a was effectively ubiquitinated in a PUB-dependent fashion. RPN12a was highly ubiquitinated in 35S:PUB22 plants, but not in pub22 pub23 double mutant plants, consistent with RPN12a being a substrate of PUB22 and PUB23 in vivo. In water-stressed wild-type and PUB-overexpressing plants, a significant amount of RPN12a was dissociated from the 19S RP and appeared to be associated with small-molecular-mass protein complexes in cytosolic fractions, where PUB22 and PUB23 are localized. Overall, our results suggest that PUB22 and PUB23 coordinately control a drought signaling pathway by ubiquitinating cytosolic RPN12a in Arabidopsis.

Figure 1.

Sequence Analysis of Arabidopsis PUB22 and PUB23. (A) Schematic structures of PUB22 and PUB23. Open bars indicate coding regions, whereas shaded bars depict the U-box motif. (B) Comparison of the derived amino acid sequences of selected Arabidopsis U-box family members and hot pepper Ca PUB1. Amino acid residues that are conserved in at least four of the six sequences are shaded. Amino acids that are identical in all six proteins are highlighted in black. (C) Phylogenetic analysis of selected U-box family members from Arabidopsis, parsley, rice, tobacco, tomato, and hot pepper. The dendrogram was conducted in MEGA4 software with the neighbor-joining method. The optimal tree with the summed branch length of 4.37412444 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

Figure 2.

In Vitro Self-Ubiquitination and RT-PCR Analyses. (A) Recombinant MBP-PUB22 and MBP-PUB23 fusion proteins were incubated at 30°C for 1 h in the presence or absence of E1 (UBA1), E2 (UBC8), ATP, and Ub. Samples were resolved by 8% SDS-PAGE and subjected to immunoblot analysis with anti-MBP (top panels) or anti-Ub (bottom panels) antibody. (B) Light-grown 10-d-old Arabidopsis seedlings were subjected to cold temperature (6 to 12 h at 4°C), drought (0.5 to 1 h), high salinity (1 to 2 h with 300 mM NaCl), or abscisic acid (1.5 to 3 h with 100 μM). In all panels, 0 represents controls. Induction profiles of PUB22, PUB23, RD29, and RAB18 were examined by RT-PCR. 18s rRNA was used as a loading control. The magnitude of relative induction was quantified using MultiGauge version 3.1 software (Fuji Film) as described in Methods. The values are means ± sd (n = 3).

Figure 3.

Subcellular Localization of Arabidopsis PUB22 and PUB23 Proteins. The 35S:GFP, 35S:GFP-PUB22, and 35S:GFP-PUB23 constructs were transformed into protoplasts prepared from Arabidopsis seedlings, and expression of the introduced genes was viewed after 16 h by confocal microscopy (LSM META 510; Carl Zeiss) under dark-field or light-field conditions. Os TRBF1, which is localized in the nuclei, is shown as a specificity control.

Figure 4.

Molecular Characterization of the PUB22- and PUB23-Overexpressing Transgenic Plants and the pub22 and pub23 Mutant Lines. (A) RT-PCR analysis of wild-type and 35S:PUB22 (lines 7 and 17) and 35S:PUB23 (lines 9 and 10) transgenic plants. The Ub gene was used as a loading control. (B) Schematic representation of the pub22 and pub23 alleles, with the T-DNA insertions shown as inverted triangles. Shaded bars indicate coding regions, while open bars show the 5′ and 3′ untranslated regions (UTR). There are no introns in PUB22 and PUB23. Gene-specific (forward and reverse) and T-DNA–specific (LBa-1) primers used in the genotyping and RT-PCR are shown with arrows. (C) Genotyping of the pub22, pub23, and pub22 pub23 mutant plants. A set of gene-specific and T-DNA–specific primers used for genomic PCR are indicated at right. (D) RT-PCR analysis of PUB22, PUB23, and Ub10 mRNAs in wild-type, pub22, pub23, and pub22 pub23 plants. In this experiment, three different primer sets were used for PUB22 and PUB23, as indicated at right. Primers used in genotyping PCR and RT-PCR are listed in Table 1.

Figure 5.

Phenotypes of Wild-Type, 35S:PUB22 and 35S:PUB23 T4 Transgenic, and pub22, pub23, and pub22 pub23 T3 Mutant Plants in Response to Salt and Drought Treatment. (A) Seeds were sown on Murashige and Skoog medium containing 3% (w/v) sucrose and 0.8% (w/v) phytoagar without or with NaCl (50 to 100 mM), and the growth patterns of roots were monitored after 10 d. The values are means ± sd (n = 40). Bar = 1.8 cm. (B) Four-week-old wild-type and PUB22- and PUB23-overexpressing plants were subjected to dehydration treatment for 9 d, followed by rewatering for 3 d. Dehydration tolerance was assayed as the ability of plants to resume growth when returned to normal conditions following water stress. The values are means ± sd (n = 44). (C) Four-week-old wild-type and mutant (pub22, pub23, and pub22 pub23) plants were subjected to drought stress for 12 d, followed by rewatering for 3 d. The values are means ± sd (n = 50). (D) Induction levels of RD22 and RD29a in wild-type, 35S:PUB22 and 35S:PUB23 transgenic, and pub22 pub23 mutant plants upon drought stress. Wild-type, 35S:PUB22, 35S:PUB23, and pub22 pub23 seedlings were grown for 3 weeks on Murashige and Skoog medium containing 3% (w/v) sucrose and 0.8% (w/v) phytoagar under light-grown conditions in a square Petri dish. For dehydration treatment, the lid of the dish was opened under light for 1 h at room temperature, and induction profiles of PUB22, PUB23, RD22, and RD29a genes were examined by RT-PCR. The UBC10 transcript level was used as a loading control.

Figure 6.

Arabidopsis PUB22 and PUB23 Interact with RPN12a. (A) Yeast two-hybrid assay. RPN12a was cloned into pGADT7, and PUB22, PUB23, and deletion mutants (PUB22^1-160 and PUB22^155-435) were cloned into pGBKT7. Yeast AH109 cells were cotransformed with a combination of the indicated plasmids. To test protein–protein interactions, yeast cells were plated onto SD/−His/−Trp/−Leu medium including 10 mM 3-amino-1,2,4,-triazole and allowed to grow for 4 d at 30°C. (B) In vitro pull-down assay. MBP-PUB22, MBP-PUB23, and deletion mutants (MBP-PUB22^1-160 and PUB22^155-435) were incubated with HA-RPN12a and amylose affinity resin. The bound protein was eluted, resolved by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The blot was probed with anti-HA or anti-MBP antibody. (C) In vivo coimmunoprecipitation assay. The whole cell free extracts containing 500 μg of proteins were prepared from wild-type and 35S:6XMyc-PUB22 T3 transgenic seedlings and then incubated with anti-Myc antibody. Immunoprecipitated proteins were detected by immunoblotting using anti-RPN12a or anti-Myc antibody.

Figure 7.

Ubiquitination Assays of RPN12a. (A) and (B) In vitro ubiquitination of RPN12a by PUB22 and PUB23. (A) Recombinant HA-RPN12a protein was incubated in the presence or absence of Ub, ATP, E1, E2, and wild-type (MBP-PUB22 and MBP-PUB23) or mutant (MBP-PUB22^V24I and MBP-PUB23^V29I) proteins for 1 h and subjected to immunoblotting using anti-HA antibody. (B) The ubiquitination reaction mixtures without (−E1) or with (+E1) E1 were coincubated with anti-HA antibody and protein A–Sepharose (20 μL). The bound proteins were eluted, separated by SDS-PAGE, and analyzed using anti-HA or anti-Ub antibody. (C) and (D) In vivo ubiquitination of RPN12a by PUB22. (C) Intact whole seedlings of wild-type and transgenic T3 seedlings (35S:HA-RPN12a/pub22 pub23 and 35S:HA-RPN12a/35S:PUB22) were incubated for 2 h with 10 μM MG132. A total of 30 seedlings for each sample were ground in protein extraction buffer, resolved by SDS-PAGE, and analyzed by immunoblotting with anti-HA and anti-actin antibody. (D) Crude extracts of wild-type and transgenic plants were coincubated with anti-HA antibody and protein A–Sepharose (40 μL). The bound proteins were eluted and analyzed using anti-HA, anti-RPN12a, or anti-Ub antibody as described above.

Figure 8.

Gel Filtration Analysis of RPN12a. (A) Preparation of cytosolic and nuclear proteins. Cytosolic and nuclear protein extracts were isolated from 14-d-old light-grown wild-type, double knockout mutant (pub22 pub23), and transgenic (35S:PUB22 and 35S:PUB23) plants. Proteins (10 μg) in each sample were separated by SDS-PAGE, blotted, and probed with anti-actin or anti-polymerase II antibody. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein stained with Ponceau S is shown as a positive control for the cytosolic fraction. C; cytosolic fraction; N, nuclear fraction. (B) Immunoblot analysis of RPN12a protein. Cytosolic (left) and nuclear (right) protein samples were prepared from 14-d-old light-grown wild-type, double knockout mutant (pub22 pub23), and transgenic (35S:PUB22 and 35S:PUB23) plants. Both cytosolic and nuclear extracts of each plant were subjected to SDS-PAGE, followed by immunoblot analysis using anti-RPN12a, anti-actin, or anti-polymerase II antibody. Rubisco protein stained with Ponceau S is shown as a loading control. Lane 1, wild type; lane 2, pub22 pub23; lane 3, 35S:PUB22; lane 4, 35:PUB23. (C) and (D) Immunoblot analysis of Sephacryl s300 gel filtration fractions obtained from 14-d-old light-grown wild-type, transgenic (35S:PUB22 and 35S:PUB23), and double knockout mutant (pub22 pub23) plants. Cytosolic (C) and nuclear (D) column fractions of each plant were subjected to SDS-PAGE, followed by immunoblot analysis with anti-RPN12a polyclonal antibody. The elution profile of Rubisco protein is shown as a control. The elution positions of marker proteins are indicated above the gel blots.