Arabidopsis PUB22 and PUB23 Are Homologous U-Box E3 Ubiquitin Ligases That Play Combinatory Roles in Response to Drought Stress

Identification of Two Homologous U-Box Genes, PUB22 and PUB23, in Arabidopsis

Previous work showed that a hot pepper (Capsicum annuum) U-box gene, Ca PUB1, was rapidly induced by dehydration and that 35S:Ca PUB1 transgenic Arabidopsis plants exhibited markedly increased sensitivity to water stress (Cho et al., 2006). These results raised the possibility that a U-box E3 Ub ligase participates in the plant response to drought stress. A database search revealed that Ca PUB1 was most closely related to the Arabidopsis PUB22 (At3g52450) and PUB23 (At2g35930) U-box proteins, with 51 and 52% amino acid identities, respectively. Because the cellular functions of PUB22 and PUB23 were not known, we decided to characterize the PUB22 and PUB23 genes at the molecular and genetic levels. PUB22 and PUB23 are predicted to have molecular masses of 48.8 and 45.8 kD, respectively, with 75% amino acid identity to each other (Figure 1A ). However, they share a relatively low degree of sequence identity with other Arabidopsis U-box proteins (30 to 34% identical to PUB24 [At3g11840], 29 to 32% to PUB255 [At3g19380], and 28 to 31% to PUB26 [At1g49780]) (Figure 1B). This is consistent with the notion that U-box genes comprise a large gene family with subsets of genes having distinct cellular functions (Azevedo et al., 2001; Andersen et al., 2004; Mudgil et al., 2004). In addition, PUB22 and PUB23 are 12 to 29% identical to cell death– and biotic defense–related U-box proteins from tobacco (Nicotiana tabacum; Nt CMPG1/ACRE74), tomato (Solanum lycopersicum; Sl CMPG1), parsley (Petroselinum crispum; Pc CMPG1), and rice (Oryza sativa; Os SPL11) (Kirsch et al., 2001; Zeng et al., 2004; Gonzalez-Lamothe et al., 2006; Yang et al., 2006) (Figure 1C). As is the case for other U-box proteins, Arabidopsis PUB22 and PUB23 both contain a single U-box motif in their N-terminal regions. The U-box domains of PUB22 and PUB23 are 90% identical to each other and have 52 to 60% identity with the tobacco, tomato, parsley, and rice U-box proteins mentioned above.

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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.

PUB22 and PUB23 Encode U-Box E3 Ub Ligases

Several lines of evidence indicate that U-box-containing proteins function as E3 Ub ligases (Azevedo et al., 2001; Hatakeyama and Nakayama, 2003; Andersen et al., 2004; Mudgil et al., 2004). To determine whether PUB22 and PUB23 have E3 Ub ligase activity, in vitro self-ubiquitination assays were performed. We produced full-length PUB22 and PUB23 in Escherichia coli as fusion proteins with maltose binding protein (MBP) and subsequently obtained affinity-purified MBP-PUB22 and MBP-PUB23. The fusion proteins were incubated at 30°C in the presence or absence of Ub, ATP, E1 (Arabidopsis UBA1), and E2 (Arabidopsis UBC8) for 1 h and subjected to immunoblot analysis with anti-MBP or anti-Ub antibody. Figure 2A shows that both MBP-PUB22 and MBP-PUB23 were seen as high-molecular-mass ladders characteristic of ubiquitination, whereas there was no ubiquitinated signal in the absence of Ub, ATP, E1, or E2. These results imply that bacterially expressed PUB22 and PUB23 have E3 Ub ligase enzyme activity.

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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).

PUB22 and PUB23 Are Coordinately Induced in Response to Abiotic Stresses

Because PUB22 and PUB23 were initially identified as homologs of abiotic stress–induced hot pepper U-box Ca PUB1 (Cho et al., 2006), we considered the possibility that the expression of PUB22 and PUB23 is modulated by abiotic stresses in Arabidopsis. To address this possibility, their mRNA accumulation profiles were monitored under various stress conditions by RT-PCR. As shown in Figure 2B, in 10-d-old light-grown seedlings, the PUB22 and PUB23 transcripts were rapidly induced by drought after only 30 min. In addition, both genes were markedly upregulated after 6 to 12 h of cold treatment (4°C). We noticed that the induction of PUB23 by water and cold stress was considerably stronger than that of PUB22. High salinity (300 mM) was also able to activate PUB22 and PUB23 within 1 h (Figure 2B). In this case, the magnitude of induction of the two genes was similar. On the other hand, the expression of PUB22 and PUB23 was not influenced by abscisic acid (Figure 2B). Overall, the results of our RNA expression studies are consistent with the hypothesis that both PUB22 and PUB23 function during abiotic stress responses in Arabidopsis.

PUB22 and PUB23 Are Predominantly Localized in Cytosolic Fractions

To investigate the cellular localization of PUB22 and PUB23, we conducted an in vivo targeting experiment using fusions of PUB22 or PUB23 with synthetic green fluorescent protein (sGFP) as a fluorescent marker in a transient transfection assay. The sGFP gene was fused to the 5′ end of the PUB22 and PUB23 coding regions in-frame under the control of the cauliflower mosaic virus 35S promoter. The resulting constructs were introduced into Arabidopsis protoplasts by polyethylene glycol treatment (Kim et al., 2004). Localization of the fusion protein was then determined by visualization with a confocal microscope. As shown in Figure 3 , fluorescence associated with both PUB22 and PUB23 was exclusively localized to the cytosol, suggesting that they are cytosolic proteins. Rice telomere repeat binding factor1 (Os TRBF1) (Byun et al., 2008) was used as a specificity control. As found previously, Os TRBF1 was localized in the nuclei (Figure 3, bottom row).

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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.

Overexpression of PUB22 and PUB23 Increases Sensitivity to Salt and Water Stress

To explore the in vivo functions of PUB22 and PUB23, we employed overexpression and reverse genetic approaches. First, we generated transgenic Arabidopsis plants in which PUB22 and PUB23 were constitutively expressed under the control of the cauliflower mosaic virus 35S promoter (Figure 4A ). Figure 5A shows the morphological comparison of 35S:PUB22 and 35S:PUB23 plants with wild-type plants in the early stage of development. We observed that the roots of both PUB22- and PUB23-overexpressors were 1.4- to 1.7-fold longer than control roots under standard growth conditions (see Supplemental Figure 1 online). Elongation of control roots was generally unaffected by 50 mM NaCl, but root growth of both transgenic lines was significantly impaired by this mild salinity (Figure 5A). In the presence of 100 mM NaCl, growth of wild-type roots was ∼40% of normal, while elongation of transgenic roots was greatly reduced to 13 to 18% of the control (Figure 5A). Thus, we conclude that 35S:PUB22 and 35S:PUB23 lines are more sensitive to salt stress.

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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.

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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.

We next went on to estimate the capacity of wild-type and 35S:PUB22 and 35S:PUB23 plants to respond to water deficit. Four-week-old plants were subjected to a dehydration treatment that consisted of withholding water for 9 d. During the drought stress period, nearly all plants withered, regardless of genotype (Figure 5B). After rewatering for 3 d, 26 of 44 wild-type plants were able to survive and continued to grow (59% survival). By contrast, a majority of PUB22- and PUB23-overexpressors did not recover from the stress and died. The survival ratios of 35S:PUB22 and 35S:PUB23 plants were 10 to 12% and 3 to 5%, respectively (Figure 5B). Interestingly, the mRNA levels of RD22 and RD29a, two typical drought-induced genes in Arabidopsis (Liu et al., 1998), were downregulated in transgenic lines relative to the wild-type plants in the drought condition (Figure 5D). These data show that 35S:PUB22 and 35S:PUB23 transgenic plants are highly sensitive to water stress. Collectively, our phenotypic analysis indicates that both PUB22- and PUB23-overexpressors are more sensitive to high salinity and water deficit than wild-type plants. On the other hand, 35S:PUB22 and 35S:PUB23 lines showed similar phenotypes compared with wild-type plants in germination rate and stomatal closure in the presence or absence of exogenously applied abscisic acid (see Supplemental Figures 2 and 3 online).

T-DNA Insertion Mutants of PUB22 and PUB23 Are Highly Resistant to Severe Drought Stress

To further define the in vivo functions of PUB22 and PUB23, we analyzed mutants carrying T-DNA insertions in the PUB22 and PUB23 genes. The pub22 and pub23 mutants have a T-DNA insertion after nucleotide 1347 on chromosome 3 (line SALK_072621) and after nucleotide 1106 on chromosome 2 (line SALK_063470), respectively (Figure 4B). Subsequently, we established a homozygous double knockout mutant for the PUB22 and PUB23 genes by crossing pub22 and pub23 (Figure 4C). Because the T-DNA insertion is near the C termini of PUB22 and PUB23, T-DNA disruption of the PUB22 and PUB23 genes was further verified by RT-PCR using three different sets of primers. As shown in Figure 4D, the mutant seedlings contained a negligible amount of full-length PUB mRNAs. In addition, a low level of partial mRNAs was detected with a primer set that amplified a region upstream of the insertion site in PUB22 and PUB23. This indicates that, although there are some aberrant or truncated transcripts, pub22 pub23 lacks full-length and functional mRNAs for both PUB22 and PUB23.

In contrast with what we observed in PUB22- and PUB23-overexpressors, pub22 pub23 mutant seedlings did not differ in appearance from wild-type seedlings (Figure 5A; see Supplemental Figure 1 online). In addition, the pub22 pub23 double mutant did not display any detectable phenotype in response to 50 to 100 mM NaCl compared with the wild-type seedlings (Figure 5A). However, pub22 pub23 plants exhibited strongly elevated tolerance to severe water deficit, as opposed to 35S:PUB22 and 35S:PUB23 transgenic plants, which were hypersensitive to dehydration.

Four-week-old wild-type, pub22, pub23, and pub22 pub23 plants were not watered for 12 d and then rehydrated for 3 d. Before rehydrating, all of the wild-type plants were severely wilted, and <7% survived after watering (Figure 5C). During this severe drought period, 28 to 34% of pub22 and pub23 mutants survived and resumed their growth to maturity when rewatered afterward. The survival ratio of pub22 pub23 double mutant plants was elevated over that of the single mutants, to ∼70% (Figure 5C). The enhanced tolerance of the double mutant line to severe drought stress indicates combinatory roles of PUB22 and PUB23. The transcript levels of RD22 and RD29a were also significantly upregulated in pub22 pub23 relative to the wild-type plant (Figure 5D), which is in good agreement with the view that RD22 and RD29a are important for the response to water stress (Liu et al., 1998). Because overexpression of PUB22 or PUB23 increased sensitivity to dehydration, and mutations in these genes elevated tolerance to the stress, these results could be interpreted as evidence that the amount of functional PUB22 and PUB23 is inversely correlated with the degree of tolerance to water stress. Thus, we conclude that the U-box E3 UB ligases PUB22 and PUB23 are negative regulators that play important roles in a subset of physiological responses to counteract drought stress in Arabidopsis. The pub22, pub23, and pub22 pub23 mutants were indistinguishable from wild-type plants in germination rate and in stomatal closure with or without abscisic acid (see Supplemental Figures 2 and 3 online).

PUB22 and PUB23 Interact with RPN12a, a Non-ATPase Subunit of the 26S Proteasome Complex

To investigate whether PUB22 and PUB23 affect the drought stress response via ubiquitination of target proteins, we first used yeast two-hybrid screening. In this experiment, the capacity of the yeast strain AH109 to grow in the absence of His was used as a marker for the interaction between PUB22 and putative target proteins. As a result of this screening, we found that His auxotrophy was restored when PUB22 was cotransformed with RPN12a, encoding a non-ATPase subunit of the 26S proteasome complex (GenBank accession number AY230846) (Smalle et al., 2002) (Figure 6A ). This suggests an interaction between PUB22 and RPN12a in yeast cells. As a specificity control, we tested the association between RPN12a and PUB22^1-160 and PUB22^155-435 mutant proteins. As shown in Figure 6A, the PUB22^155-435 derivative interacted with RPN12a, while the PUB22^1-160 derivative, which is mainly composed of the N-terminal U-box domain, failed to bind RPN12a, indicating that the ability to interact with RPN12a resides in the C-terminal region of PUB22.

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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.

To corroborate the interaction between PUB22 and RPN12a, we performed an in vitro pull-down assay. PUB22 and RPN12a were expressed in E. coli and purified as MBP and hemagglutinin (HA) fusion proteins, respectively. The fusion proteins were coincubated with an amylose affinity matrix, followed by extensive washing. The bound protein was then eluted from the amylose resin by 10 mM maltose and immunoblotted with anti-MBP and anti-HA antibodies. Figure 6B shows that HA-RPN12a was pulled down from the amylose affinity resin by MBP-PUB22, while HA-RPN12a alone did not bind the resin. Consistent with the results of the yeast two-hybrid assay, PUB22^155-435, which lacks the U-box region, was able to interact with RPN12a. In parallel, we repeated yeast two-hybrid and in vitro pull-down experiments using PUB23. As shown in Figures 6A and 6B, PUB23 also interacted with RPN12a.

To complement the above experiments, we then performed an in vivo coimmunoprecipitation experiment. Transgenic Arabidopsis plants that contained the 35S:6XMyc-PUB22 construct were generated. An extract was prepared from cells of wild-type or T3 transgenic seedlings and immunoprecipitated with anti-Myc antibody followed by immunoblotting using anti-RPN12a or anti-Myc antibody. As shown in Figure 6C, RPN12a was effectively coprecipitated with anti-Myc antibody in 35S:6XMyc-PUB22 extract but not in wild-type seedlings. This indicates the interaction between PUB22 and RPN12a in Arabidopsis. Overall, the results in Figure 6 are consistent with the hypothesis that RPN12a is a target protein for ubiquitination by PUB22 and PUB23.

Ubiquitination of RPN12a by PUB22 and PUB23 in Vitro and in Vivo

To further substantiate the interaction of PUB22 and PUB23 with RPN12a, we performed an in vitro ubiquitination assay. Recombinant HA-RPN12a protein was incubated at 30°C in the presence or absence of Ub, ATP, E1, E2, and MBP-PUB22 for 1 h and then subjected to immunoblotting using anti-HA antibody. A high-molecular-mass ladder was clearly detected when HA-RPN12a was incubated with MBP-PUB22 (Figure 7A ), indicating the presence of multiple forms of HA-RPN12a differing in the number of ubiquitin tags attached. The exclusion of E1, E2, or MBP-PUB22 from the reaction abolished production of this ladder. Furthermore, the PUB22 V24I mutant, in which a conserved Val residue in the U-box domain was changed to Ile, failed to ubiquitinate HA-RPN12a. We obtained identical results using PUB23 and the PUB23 V29I mutant as shown in Figure 7A. To confirm the ubiquitination of HA-RPN12a, the ubiquitination reaction mixtures were coincubated with anti-HA antibody and protein A–Sepharose and washed extensively. The bound proteins were eluted and analyzed using anti-HA or anti-Ub antibody. The results clearly show that a high-molecular-mass ladder is ubiquitinated RPN12a (Figure 7B).

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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.

We next conducted in vivo ubiquitination assays. In these experiments, the 35S:HA-RPN12a construct was stably transformed into pub22 pub23 knockout mutant plants and PUB22-overexpressing transgenic plants. Light-grown 10-d-old 35S:HA-RPN12a/pub22 pub23 and 35S:HA-RPN12a/35S:PUB22 transgenic T3 seedlings were obtained and subsequently incubated for 2 h with 10 μM MG132, an inhibitor of the 26S proteasome complex, to stabilize ubiquitinated proteins. Total protein was prepared from treated tissues and used for immunoblotting with anti-HA or anti-actin antibody. Figure 7C reveals the production of high-molecular-mass smear ladders when HA-RPN12a was expressed in the 35S:PUB22 transgenic plants. By contrast, ubiquitination was hardly detectable when HA-RPN12a was expressed in the pub22 pub23 double knockout mutant plant. The crude extracts of wild-type and transgenic plants were then coincubated with anti-HA antibody and protein A–Sepharose. After extensive washing, the bound proteins were eluted and analyzed using anti-HA, anti-RPN12a, or anti-Ub antibody. As demonstrated in Figure 7D, a high-molecular-mass ladder is indeed ubiquitinated RPN12a in Arabidopsis. Collectively, these data further support the view that HA-RPN12a is ubiquitinated by PUB22 in Arabidopsis.

Cytosolic RPN12a Is Associated with a Smaller Protein Complex in Addition to 19S RP in Water-Stressed Wild-Type and PUB-Overexpressing Plants

The 26S proteasome complex is composed of a 20S catalytic core particle and two 19S RPs, the base and the lid (Baumeister et al., 1998). RPN12a, together with other non-ATPase subunits, forms the lid subcomplex, which functions in substrate recognition and processing before Ub-tagged proteins enter into the core particle. The results in Figures 6 and 7 suggest that RPN12a is a substrate of PUB22 and PUB23. This raises two tantalizing possibilities. First, perhaps RPN12a is ubiquitinated and degraded via the 26S proteasome pathway. In this case, one might expect to find lower amounts of RPN12a in PUB-overexpressing plants and higher amounts in pub22 pub23 mutant plants. To examine this possibility, we performed immunoblot analysis. Because both PUB22 and PUB23 are exclusively localized in the cytosolic fraction in Arabidopsis protoplasts (Figure 3), total proteins prepared from 14-d-old wild-type, pub22 pub23, and PUB-overexpressing seedlings were separated into nuclear and cytoplasmic fractions. These were relatively free of cross contamination, as judged by the absence of cross reaction with antibodies to cytosolic and nuclear proteins, actin, and RNA polymerase II, respectively (Figure 8A ). Immunoblotting using anti-RPN12a antibody revealed that the cellular amounts of RPN12a in wild-type, pub22 pub23, and PUB-overexpressing plants are comparable in both nuclear and cytosolic fractions (Figure 8B). In addition, there were no detectable changes in RPN12a mRNA levels in these transgenic and knockout mutant plants (see Supplemental Figure 4 online).

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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.

Alternatively, ubiquitination of RPN12a might alter its pattern of interactions with other components of the 19S RP. Gel filtration chromatography was then used to size-fractionate cytosolic and nuclear proteins, and elution of RPN12a was monitored by immunoblotting. In wild-type seedlings, the majority of cytosolic RPN12a eluted in a protein complex with an estimated molecular mass of ∼800 to 900 kD (Figure 8C), which is in accordance with the reported molecular size of the 19S RP (Peng et al., 2001). Intriguingly, in water-stressed wild-type seedlings, in which PUB22 and PUB23 are upregulated, cytosolic RPN12a eluted in broader fractions ranging between 900 and 200 kD. This shift of RPN12a toward smaller molecular mass complexes was also clearly detected in both 35S:PUB22 and 35:PUB23 plants, but we did not observe such a shift in pub22 pub23 mutant seedlings (Figure 8C). By contrast, nuclear RPN12a was exclusively eluted in a protein complex of ∼800 to 900 kD in all plants examined (Figure 8D). Thus, these results may reflect the possibility that a significant amount of RPN12a is ubiquitinated by PUB22 and PUB23 in the cytoplasm of water-stressed and PUB-overexpressing plants, and furthermore, that the Ub-tagged RPN12a is dissociated from the 19S RP.

Plant Materials and Growth Conditions

Arabidopsis thaliana ecotype Columbia was used throughout this study. Arabidopsis plants were grown, transformed, and treated as described previously (Cho et al., 2006; Lee et al., 2006). The PUB22 and PUB23 genes were cloned into vector pBI121 (ABRC stock number CD3-388). The pub22 and pub23 T-DNA insertion mutants (line SALK_072621 and SALK_063470) were obtained from the ABRC (http://www.Arabidopsis.org). The pub22 pub23 double mutants were generated by crossing pub22 with pub23 as described (Joo et al., 2006). Primers used in the genotyping and RT-PCR of homozygous mutants are listed in Table 1 .

Phylogenetic Analysis

Alignment of protein sequences was performed with ClustalW in MEGA4 software (Tamura et al., 2007). The evolutionary relationship was inferred using the neighbor-joining method (Saitou and Nei, 1987). The optimal tree had the summed branch length of 4.37412444, and bootstrapping was performed with 20,000 replicates. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in units of the number of amino acid substitutions per site.

RT-PCR

Arabidopsis seedlings were subjected to various abiotic stresses as described previously (Zeba et al., 2006). Total RNA was extracted from the treated tissues using the Easy-BLUE total RNA extraction kit following the manufacturer's manual (Intron Biotechnology). The first-strand cDNA synthesis and RT-PCR were performed as described previously (Joo et al., 2006). The first-strand cDNA, synthesized from 10 μg of total RNA, was amplified by PCR using oligonucleotide primers specific for the PUB22 and PUB23 genes (Table 1). PCR was performed in a total volume of 25 μL containing 1 μL of the first-strand cDNA reaction products, 1 μM primers, 10 mM Tris (pH 8.0), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 200 μM deoxynucleotides, and 2.5 units of high-fidelity Ex-Taq polymerase (Takara). Twenty thermal cycles were performed, each consisting of 45 s at 95°C, 1 min at 60°C, and 90 s at 72°C in an automatic thermal cycler (Perkin-Elmer/Cetus). The RT-PCR products were separated in 1% agarose by electrophoresis. The relative intensities of bands for PUB22, PUB23, RD29a, and RAB18, compared with those of the respective 18s rRNA signals, were determined using MultiGauge version 3.1 software (Fuji Film) and normalized to 1.00 for the untreated wild-type sample.

In Vitro Self-Ubiquitination and Immunoblot Analyses

Recombinant MBP-PUB22 and MBP-PUB23 proteins were expressed in Escherichia coli and purified by affinity chromatography using amylose resin (New England Biolabs). Purified fusion proteins (500 ng) were brought to 60 μL with ubiquitination reaction buffer (50 mM Tris-HCl, pH 7.5, 2.5 mM MgCl₂, 0.5 mM DTT, 4 mM ATP, 5 μg of Ub [Sigma-Aldrich], 100 ng of Arabidopsis E1 [UBA1], and 100 ng of Arabidopsis E2 [UBC8]) and incubated at 30°C for 1 h as described previously (Cho et al., 2006). The full-length Arabidopsis UBA1 (U21814) and UBC8 (DQ027022) clones were obtained from the ABRC (http://www.Arabidopsis.org) and ligated into the pProEx Hta vector (Invitrogen). (His)₆-UBA1 and (His)₆-UBC8 fusion proteins were purified by affinity chromatography using nickel-nitrilotriacetic acid agarose superflow resin (Qiagen). Reaction products were separated by SDS-PAGE and subjected to immunoblot analysis using anti-MBP antibody (New England Biolabs) or anti-Ub antibody (Santa Cruz Biotechnology) as described previously (Lee et al., 2006).

Subcellular Localization

The sGFP cDNA clone was fused in-frame to the 5′ end of the full-length PUB22 and PUB23 coding region and ligated into the pBI221 transient expression vector. The sGFP-PUB22 and sGFP-PUB23 fusion constructs were transformed into protoplasts prepared from wild-type Arabidopsis seedlings by polyethylene glycol treatment (Kim et al., 2004). The expression of sGFP-PUB22 and sGFP-PUB23 was monitored at 16 h after transformation. Fluorescence images were viewed with confocal microscopy (LSM META 510; Carl Zeiss). sGFP and rice (Oryza sativa) Os TRBF1, which is localized in the nuclei, were used as specificity controls.

Yeast Two-Hybrid Screening

The yeast strain AH109 (Clontech) was first transformed with pAS2-1-PUB22 as a bait, followed by transformation with a 3-d-old etiolated seedling cDNA library in pACT vector (http://www.Arabidopsis.org). Approximately 3 × 10⁶ yeast transformants were screened on the selective medium SD/−His/−Trp/−Leu with 10 mM 3-amino-1,2,4,-triazole. Positive clones were identified by sequencing. One of the positive clones was RPN12a. To confirm the interaction between PUB22 and RPN12a, full-length PUB22 and RPN12a cDNA, or deletion constructs, were ligated into pGBKT7 and pGADT7 vectors (Clontech; Matchmaker3), respectively. These constructs as well as empty vector controls were transformed into yeast strain AH109. 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.

In Vitro Pull-Down Assay

For the in vitro pull-down assay, the bacterially expressed HA-RPN12a and MBP-PUB22, MBP-PUB23, MBP-PUB22^1-160, and MBP-PUB22^155-435 were coincubated in 30 μL of amylose resin (New England Biolabs), washed extensively, and eluted from the amylose affinity resin using 10 mM maltose as described previously (Cho et al., 2006). The eluted protein was resolved by 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The blot was probed with anti-HA antibody or anti-MBP antibody.

In Vivo Coimmunoprecipitation Experiment

Light-grown 10-d-old seedlings of wild-type and 35S:6XMyc-PUB22 T3 transgenic plants were pretreated for 2 h with 10 μM MG132, an inhibitor of the 26S proteasome. Each sample was ground in liquid nitrogen, and proteins were extracted in immunoprecipitation (IP) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM MgCl₂, and 1× complete protease inhibitor cocktail). For coimmunoprecipitation assay, 500 μg of total proteins was incubated for 2 h at 4°C with 1 μg of monoclonal anti-Myc antibody (Applied Biological Materials), and then 40 μL of protein A–Sepharose (GE Healthcare), previously equilibrated with IP buffer, was added and incubated for 1 h at 4°C. The precipitated samples were washed four times with the IP buffer and eluted with 0.1 M Gly buffer, pH 2.7. Each sample was separated by SDS-PAGE and subjected to immunoblot analysis using anti-Myc antibody (Applied Biological Materials) or anti-RPN12a antibody (Biomol).

In Vitro and in Vivo Ubiquitination Analyses

For the in vitro ubiquitination experiment, recombinant HA-RPN12a protein was incubated at 30°C in the presence or absence of Ub, ATP, E1, E2, and MBP-PUB22 or MBP-PUB23 for 1 h and then subjected to immunoblotting using anti-HA antibody. For a specificity control, the PUB22 V24I and PUB23 V29I mutant proteins, in which a conserved Val residue in the U-box domain was changed to Ile, were used for the ubiquitination assay. To confirm the ubiquitination of HA-RPN12a, the ubiquitination reaction mixtures were coincubated with anti-HA antibody and 20 μL of protein A–Sepharose and then washed three times extensively with IP buffer. The bound proteins were eluted from protein A–Sepharose by 0.1 M Gly buffer, pH 2.7. Samples were separated by SDS-PAGE and analyzed using anti-HA or anti-Ub antibody.

For the in vivo ubiquitination experiment, light-grown 10-d-old intact whole seedlings of wild-type and T3 transgenic plants (35S:HA-RPN12a/pub22 pub23 and 35S:HA-RPN12a/35S:PUB22) were pretreated for 2 h with 10 μM MG132. A total of 30 seedlings for each sample were ground in protein extraction buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, and 1× complete protease inhibitor cocktail (Roche) and then analyzed by immunoblotting using anti-HA antibody. To confirm the ubiquitination of HA-RPN12a, the crude extracts of wild-type and transgenic plants were coincubated with anti-HA antibody and 40 μL of protein A–Sepharose and then washed three times extensively with IP buffer. The bound proteins were eluted and analyzed using anti-HA, anti-RPN12a, or anti-Ub antibody as described above.

Gel Filtration Chromatography

Fourteen-day-old seedlings were homogenized in nuclear grinding buffer consisting of 100 mM MOPS (pH 7.6), 5% Dextran T-40, 2.5% Ficoll, 250 mM sucrose, 10 mM MgCl₂, 2 mM phenylmethylsulfonyl fluoride, and 1× protease inhibitor cocktail (Roche). The extract was then passed over two layers of mesh and centrifuged at 1000g for 10 min to harvest the supernatant (cytosol) and pellet (nuclear). The pellets were washed twice with nuclear grinding buffer plus 0.1% Triton X-100. The nuclear fraction was dissolved in nuclear grinding buffer and sonicated twice for 15 s Cytosolic and nuclear fraction extracts were passed through a 0.2-μm filter before loading onto a Sephacryl s300 gel filtration column (Hiprep 16/60; GE Healthcare). Gel filtration experiments were performed as described (Kim et al., 2001).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: PUB20 (At1g66160), PUB21 (At5g37490), PUB22 (At3g52450), PUB23 (At2g35930), PUB24 (At3g11840), PUB25 (At3g19380), PUB26 (At1g49780), PUB27 (At5g64660), PUB28 (At5g09800), PUB29 (At3g18710), PUB30 (At3g49810), PUB31 (At5g65920), RPN12a (AY230846), UBA1 (U21814), UBC8 (DQ027022), Ca PUB1 (ABA59556), Nt CMPG1/ACRE74 (AAP03884), Sl CMPG1 (AAZ57336), Pc CMPG1 (AAK69402), Os SPL11 (AAT94161), and Os TRBF1 (NP001043442).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Root Growth Patterns of Wild-Type, 35S:PUB22 and 35S:PUB23 Transgenic, and pub22 pub23 Mutant Seedlings.

Supplemental Figure 2. Germination Rates of Wild-Type, 35S:PUB22 and 35S:PUB23 Transgenic, and pub22 pub23 Mutant Seedlings in the Absence or Presence of Abscisic Acid.

Supplemental Figure 3. Stomatal Aperture Measurements of Wild-Type, 35S:PUB22 Transgenic, and pub22 pub23 Mutant Plants in the Absence or Presence of Abscisic Acid.

Supplemental Figure 4. RPN12a mRNA Levels in Wild-Type, 35S:PUB22 and 35S:PUB23 Transgenic, and pub22 pub23 Mutant Plants.

Supplemental Data Set 1. Text File Corresponding to the Alignments Used for Phylogenetic Analysis in Figure 1C.