The Plant Cell, Vol. 20: 1899–1914, July 2008, www.plantcell.org ã 2008 American Society of Plant Biologists
Arabidopsis PUB22 and PUB23 Are Homologous U-Box E3
Ubiquitin Ligases That Play Combinatory Roles in Response to
Drought Stress
W
Seok Keun Cho,a Moon Young Ryu,a Charlotte Song,b June M. Kwak,b and Woo Taek Kima,1
a Department
b Department
of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea
of Cellular Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742
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 PUBoverexpressing 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.
INTRODUCTION
Plants are frequently exposed to stressful environmental stimuli,
which can have an enormous impact on plant growth and
development. Water stress resulting from drought or high salinity
is responsible for dramatic reductions in crop yield worldwide
(Boyer, 1982). To avoid or tolerate such detrimental conditions,
plants have developed a variety of defense strategies, often
involving genes regulated by the particular stress. A large and
increasing number of genes regulated by water stress have been
identified recently (Bray, 1997; Zhu, 2002). Nevertheless, the
biological functions of these genes with respect to stress tolerance or sensitivity are still largely unknown in higher plants. It is
pertinent, therefore, to study the functions of stress-related
genes for the development of transgenic crops that have improved tolerance to water deficit.
The ubiquitination pathway mediates a posttranslational modification of cellular proteins, commonly targeting them for destruction by the proteasome. In this process, ubiquitin (Ub), a
76–amino acid protein, becomes conjugated to Lys residues of
target substrate proteins (Moon et al., 2004; Smalle and Vierstra,
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Woo Taek Kim
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.108.060699
2004; Dreher and Callis, 2007). Ub is attached to the target
proteins in three steps: (1) Ub is activated by a Ub-activating
enzyme (E1); (2) activated Ub is then transferred to a Ubconjugating enzyme (E2); and (3) it becomes covalently attached
to the substrate protein by a Ub ligase (E3). The impact of
ubiquitination on a given target protein depends on many factors,
including the level and placement of ubiquitination. Polyubiquitination often promotes rapid degradation by the 26S proteasome
complex, while monoubiquitination or multiubiquitination can
instead alter protein activity, lipidation, subcellular localization,
and interaction with other proteins (Pickart and Eddins, 2004;
Mukhopadhyay and Riezman, 2007). The ubiquitination system
appears to be present in all eukaryotic cells and is implicated in
many cellular processes, including differentiation, cell division,
hormonal responses, and biotic and abiotic stress responses.
The U-box domain is a modified ring finger motif composed of
;75 amino acids. Many U-box–containing proteins function as
E3 Ub ligases (Hatakeyama and Nakayama, 2003). The Arabidopsis thaliana genome contains at least 60 U-box genes (http://
plantsubq.genomics.purdue.edu), suggesting that they have
diverse roles in plants. Indeed, several plant U-box proteins
were recently shown to participate in responses to hormones
(Amador et al., 2001), biotic stress (Kirsch et al., 2001; Zeng et al.,
2004; Gonzalez-Lamothe et al., 2006; Yang et al., 2006), abiotic
stress (Luo et al., 2006), and self-incompatibility (Liu et al., 2007).
In this study, we identified two homologous U-box genes from
Arabidopsis, PUB22 and PUB23, which are rapidly induced in
response to abiotic stresses. We present molecular and genetic
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characterization of the pub22 pub23 double knockout mutant
and 35S:PUB22 and 35S:PUB23 transgenic plants. The results
suggest an intimate link between U-box E3 Ub ligases and water
stress responses. We also demonstrate that PUB22 and PUB23
physically interact with and ubiquitinate RPN12a, a non-ATPase
subunit of the 26S proteasome complex, resulting in partial
dissociation of RPN12a from the 19S regulatory particle (RP).
Overall, our results indicate that PUB22 and PUB23 are negative
regulators of the water stress signaling pathway in Arabidopsis.
obtained affinity-purified MBP-PUB22 and MBP-PUB23. The
fusion proteins were incubated at 308C in the presence or
absence of Ub, ATP, E1 (Arabidopsis UBA1), and E2 (Arabidopsis
UBC8) for 1 h and subjected to immunoblot analysis with antiMBP or anti-Ub antibody. Figure 2A shows that both MBPPUB22 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.
RESULTS
PUB22 and PUB23 Are Coordinately Induced in Response to
Abiotic Stresses
Identification of Two Homologous U-Box Genes, PUB22 and
PUB23, in Arabidopsis
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 RTPCR. 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 (48C). 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.
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.
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
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 59 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).
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.
U-Box E3 Ub Ligases, At PUB22 and At PUB23
Figure 1. Sequence Analysis of Arabidopsis PUB22 and PUB23.
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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.
We next went on to estimate the capacity of wild-type and 35S:
PUB22 and 35S:PUB23 plants to respond to water deficit. Fourweek-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 PUB23overexpressors, 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 wildtype 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 wildtype plants in germination rate and in stomatal closure with
Figure 1. (continued).
(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.
U-Box E3 Ub Ligases, At PUB22 and At PUB23
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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 308C 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 48C), 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 mM). 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 6 SD (n = 3).
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
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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.
and RPN12a in yeast cells. As a specificity control, we tested the
association between RPN12a and PUB221-160 and PUB22155-435
mutant proteins. As shown in Figure 6A, the PUB22155-435
derivative interacted with RPN12a, while the PUB221-160 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.
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 antiMBP 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, PUB22155-435, 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 antiMyc 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:6XMycPUB22 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 308C 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 HARPN12a 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
U-Box E3 Ub Ligases, At PUB22 and At PUB23
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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 59 and 39 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.
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).
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 PUB22overexpressing 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 mM MG132, an inhibitor of the 26S
proteasome complex, to stabilize ubiquitinated proteins. Total
protein was prepared from treated tissues and used for
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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 6 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 6 SD (n = 44).
U-Box E3 Ub Ligases, At PUB22 and At PUB23
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).
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
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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 PUBoverexpressing plants, and furthermore, that the Ub-tagged
RPN12a is dissociated from the 19S RP.
DISCUSSION
The U-box motif was originally identified as a modified RING
domain, and many U-box–containing proteins were subsequently revealed to function as E3 Ub ligases (Azevedo et al.,
2001; Hatakeyama and Nakayama, 2003; Andersen et al., 2004;
Mudgil et al., 2004). Despite limited insight into the plant U-box
E3 Ub ligases compared with those in yeast and mammalian
systems, plant U-box proteins have recently attracted much
interest, as it is becoming apparent that they are critically
involved in a wide range of physiological processes. In this
study, we examined the effects of inactivating and overexpressing PUB22 and PUB23, which encode homologous cytosolic
U-box E3 Ub ligases (Figures 1, 2A, and 3), on the response to
water deficit in Arabidopsis. We found that the pub22 pub23
double mutant line had markedly enhanced tolerance to drought,
whereas both PUB-overexpressors displayed hypersensitivity to
dehydration (Figure 5). Thus, the activity of PUB22 and PUB23
genes seems to be inversely correlated with water stress tolerance, suggesting that PUB22 and PUB23 participate in negative
control of the drought stress response. It was previously shown
that overexpression of the extreme temperature–induced U-box
CHIP rendered Arabidopsis plants more sensitive to both low
and high temperatures (Yan et al., 2003). Mutation of the rice
U-box SPOTTED LEAF11 gene resulted in a spontaneous cell
death phenotype and increased resistance to bacterial and
fungal pathogens (Zeng et al., 2004).
Figure 5. (continued).
(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 6 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.
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The Plant Cell
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 (PUB221-160 and PUB22155-435) 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 308C.
(B) In vitro pull-down assay. MBP-PUB22, MBP-PUB23, and deletion mutants (MBP-PUB221-160 and PUB22155-435) 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 mg of proteins were prepared from wild-type and 35S:6XMycPUB22 T3 transgenic seedlings and then incubated with anti-Myc antibody. Immunoprecipitated proteins were detected by immunoblotting using antiRPN12a or anti-Myc antibody.
These results, along with our data, imply a role for a subset of
U-box ubiquitination systems as a negative regulator in the
control of biotic and abiotic defense mechanisms. PUB22 and
PUB23 were rapidly induced by abiotic stresses, but their
expression was unaffected by exogenously applied abscisic
acid (Figure 2B). In addition, neither pub22 pub23 mutant nor
35S:PUB transgenic plants exhibited different phenotypes relative to wild-type plants in response to abscisic acid with regard to
germination rate and stomatal closure (see Supplemental Figures 2 and 3 online). Thus, although it is still possible that abscisic
acid affects the E3 Ub ligase activity of PUB22 and PUB23, these
results invoke a working hypothesis in which PUB22 and PUB23
regulate an abscisic acid–independent drought signaling pathway by ubiquitinating as yet unidentified proteins.
With the aid of a series of in vitro and in vivo experiments, we
identified RPN12a as a potential substrate for the Ub ligase
activity of PUB22 and PUB23. Yeast two-hybrid screening and in
vitro pull-down and in vivo coimmunoprecipitation assays indicated a physical interaction between PUB22/23 and RPN12a
(Figure 6). Bacterially expressed RPN12a was effectively ubiquitinated in a PUB-dependent fashion (Figures 7A and 7B). More
importantly, in PUB22-overexpressing plants, RPN12a was
highly ubiquitinated, whereas ubiquitination of RPN12a was
only seen at a background level in the pub22 pub23 double
U-Box E3 Ub Ligases, At PUB22 and At PUB23
1909
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-PUB22V24I and MBP-PUB23V29I) 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 mL). 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 mM 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 mL). The bound proteins
were eluted and analyzed using anti-HA, anti-RPN12a, or anti-Ub antibody as described above.
mutant (Figures 7C and 7D). Taken together, these results
strengthen the model that PUB22 and PUB23 ubiquitinate
RPN12a, thereby altering its cellular stability or activity.
RPN12a is a non-ATPase subunit of 19S RP in the 26S proteasome; hence, generation of Ub-tagged RPN12a may result in
changes in the structure or function of 19S RP. Consistent with
this view, we found that in water-stressed wild-type and PUBoverexpressing plants, a significant amount of the RPN12a
subunit was dissociated from the 19S RP and appeared to be
associated with smaller molecular mass protein complexes in
the cytosol, where PUB22 and PUB23 are predominantly localized (Figure 8). These results are reminiscent of a previous report
that RPN6, another non-ATPase subunit, was present not only in
the 19S RP but also in a low-molecular-mass protein complex of
;500 kD named PR500 in heat-shocked or canavanine-treated
Arabidopsis seedlings and cauliflower (Brassica oleracea) florets
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The Plant Cell
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 mg) 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 SDSPAGE, 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.
U-Box E3 Ub Ligases, At PUB22 and At PUB23
1911
Table 1. Primer List
Gene Name
RT-PCR and genotyping PCR
PUB22
22 FP1 and 22 RP1
22 FP2 and 22 RP2
22 FP3 and 22 RP3
PUB23
23 FP1 and 23 RP1
23 FP2 and 23 RP2
23 FP3 and 23 RP3
RD29a
RD22
18s rRNA
UBC10
Ub
Site-directed mutagenesis
PUB22 V24I
PUB23 V29I
Construction
PUB22
PUB221-160
PUB22155-435
PUB23
RPN12a
Forward Primers (FP)
Reverse Primers (RP)
59-GTTTAGATTCGTTCGTCGTTTC-39
59-GAATCTCAACGACATGTGTCAGT-39
59-GCTTGAACAACGTACATGTTTG-39
59-ACTGACACATGTCGTTGAGATTC-39
59-AAGCAGGATACGAATCATACAAA-39
59-CTACACATAATGATAAACATTGAAAGA-39
59-GATCAATATATTCGTCAGGGTTC-39
59-GCAACGACTCAGAAAATGGG-39
59-AACAATACAAACAAGTCCAATTCTC-39
59-CAGGTGAATCAGGAGTTGTT-39
59-GCAGCGAAGGAGACTCAGCT-39
59-GCATTTGCCAAGGATGTTTT-39
59-ATGGCGTCGAAGCGGATC-39
59-ATGCAGATCTTTGTTAAGACTCTC-39
59-CCCATTTTCTGAGTCGTTGC-39
59-CATCAGCAGGGATATGCAAG-39
59-AGCCTGGTAACTTGTGATTTTAT-39
59-CCGGAAATTTATCCTCTTCT-39
59-GTTCCAAGCTGAGGTGTTCTT-39
59-GTACAAAGGGCAGGGACGTA-39
59-TTAGCCCATGGCATACTTCTG-39
59-TCAACCACCACGGAGCCTGA-39
59-CATGAAAGATCCGATCATAGTTTCCACCG-39
59-CATGAAAGATCCAATCATAGTCTCTACCG-39
59-CGGTGGAAACTATGATCGGATCTTTCATG-39
59-CGGTAGAGACTATGATTGGATCTTTCATG-39
59-CTGCAGATGGATCAAGAGATAGA-39
59-GAATTCATGGATCAAGAGATAGA-39
59-CTGCAGTCTCCTTCTTCTTCACT-39
59-GAATTCATGTCCGGAGGAATAATG-39
59-CTGCAGCAATGGATCCGCAGCTAACT-39
59-CTGCAGTTAGCCTTTTCTTTAGTC-39
59-GTCGACTCAAAGTGAAGAAGAAGGAGA-39
59-CTGCAGTTAGCCTTTTCTTTAGTC-39
59-GAATTCTCAGCAGGGATATGCAAGAATC-39
59-CTGCAGTTACACGATACGCTCCAG-39
(Peng et al., 2001). It was proposed that PR500 is necessary for
higher plants to deal with the frequently encountered environmental stresses. With this in mind, we speculate that ubiquitination
of RPN12a alters the subunit composition and/or characteristics
of the 19S RP upon water stress. This event could be captured
by cells as a signal for triggering a subset of physiological processes to cope with severe dehydration stress. This regulatory
interaction between PUB22/23 and RPN12a would permit
the plant to fine-tune its cellular responses to the environmental cue.
In yeast and mammalian systems, there are several types of
Ub modification. Polyubiquitination has been generally regarded
as a signal for proteasomal degradation, while monoubiquitination or multiubiquitination is associated with nonproteolytic signaling (Hershko and Ciechanover, 1992; Pickart and Eddins,
2004). Emerging evidence, however, indicates that polyubiquitination is also involved in nonproteolytic functions. The Lys-63–
linked poly-Ub chain in particular appears to play important roles
in signaling for inflammatory responses, endocytosis, and DNA
damage response (Kawadler and Yang, 2006; Mukhopadhyay
and Riezman, 2007). Most recently, roles of Lys-63–linked polyubiquitination in the control of apical dominance and the DNA
damage response have been reported in Arabidopsis (Yin et al.,
2007; Wen et al., 2008), suggesting that modification of proteins
by Lys-63–linked polyubiquitination also occurs in higher plants.
Thus, further experiments are required to define whether or
not PUB22 and PUB23 are responsible for Lys-63–linked polyubiquitination of RPN12a.
It is worth noting that PUB-overexpressors have significantly
longer roots than the wild type (Figure 5A; see Supplemental
Figure 1 online). Thus, it is plausible that PUB22 and PUB23 are
also involved in cell and tissue growth during root development.
In this regard, we could not rule out the possibility that ubiquitination of RPN12a by PUB22 and PUB23, which affects 19S
regulatory particle function, may be more closely linked with the
enhanced root growth than drought stress tolerance. More
detailed studies about the functional relationship between
RPN12a and drought stress adaptation are necessary.
Compared with the extensive studies of the ubiquitination
pathway in yeast and mammals, we are only beginning to understand the functions of Ub in higher plants. In conclusion, our
results argue that the combinatory actions of the U-box E3 Ub
ligases PUB22 and PUB23 function as negative regulators in the
water stress signaling pathway by ubiquitinating the cytosolic
RPN12a, one of the subunits of 26S proteasome complex, in
Arabidopsis.
METHODS
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
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The Plant Cell
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 mg 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 mL containing 1 mL of the first-strand cDNA reaction products, 1 mM
primers, 10 mM Tris (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin,
200 mM deoxynucleotides, and 2.5 units of high-fidelity Ex-Taq polymerase (Takara). Twenty thermal cycles were performed, each consisting of
45 s at 958C, 1 min at 608C, and 90 s at 728C 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 mL with ubiquitination reaction buffer (50 mM Tris-HCl, pH
7.5, 2.5 mM MgCl2, 0.5 mM DTT, 4 mM ATP, 5 mg of Ub [Sigma-Aldrich],
100 ng of Arabidopsis E1 [UBA1], and 100 ng of Arabidopsis E2 [UBC8])
and incubated at 308C 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 nickelnitrilotriacetic 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 59 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 pAS2-1PUB22 as a bait, followed by transformation with a 3-d-old etiolated
seedling cDNA library in pACT vector (http://www.Arabidopsis.org).
Approximately 3 3 106 yeast transformants were screened on the
selective medium SD/2His/2Trp/2Leu 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/2His/2Trp/2Leu medium including 10 mM 3-amino1,2,4,-triazole and allowed to grow for 4 d at 308C.
In Vitro Pull-Down Assay
For the in vitro pull-down assay, the bacterially expressed HA-RPN12a and
MBP-PUB22, MBP-PUB23, MBP-PUB221-160, and MBP-PUB22155-435
were coincubated in 30 mL 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 mM 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
13 complete protease inhibitor cocktail). For coimmunoprecipitation
assay, 500 mg of total proteins was incubated for 2 h at 48C with 1 mg of
monoclonal anti-Myc antibody (Applied Biological Materials), and then 40
mL of protein A–Sepharose (GE Healthcare), previously equilibrated with
IP buffer, was added and incubated for 1 h at 48C. 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 308C 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 mL 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 mM 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 13 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 antiHA antibody and 40 mL of protein A–Sepharose and then washed three
times extensively with IP buffer. The bound proteins were eluted and
U-Box E3 Ub Ligases, At PUB22 and At PUB23
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 13
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-mm
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 WildType, 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.
ACKNOWLEDGMENTS
We thank the members of the W.T.K. laboratory for their help and
discussion. This work was supported by grants from the Plant Diversity
Research Center (21st Century Frontier Research Program funded by
the Ministry of Science and Technology of Korea) and the Korea
Research Foundation (Basic Research Program Project Grant 2005C00129) to W.T.K. and by grants from the National Science Foundation
(Grant MCB-0614203) and the U.S. Department of Agriculture (Grant
2004-35100-14909) to J.M.K. S.K.C. and M.Y.R. were recipients of
Brain Korea 21 graduate student scholarships.
Received May 13, 2008; revised June 17, 2008; accepted July 14, 2008;
published July 29, 2008.
1913
REFERENCES
Amador, V., Monte, E., Garcı́a-Martı́nez, J.L., and Prat, S.
(2001). Gibberellins signal nuclear import of PHOR1, a photoperiodresponsive protein with homology to Drosophila armadillo. Cell 106:
343–354.
Andersen, P., Kragelund, B.B., Olsen, A.N., Larsen, F.H., Chua,
N.-H., Poulsen, F.M., and Skriver, K. (2004). Structure and biochemical function of a prototypical Arabidopsis U-box domain. J. Biol.
Chem. 279: 40053–40061.
Azevedo, C., Santos-Rosa, M.J., and Shirasu, K. (2001). The U-box
protein family in plants. Trends Plant Sci. 6: 354–358.
Baumeister, W., Walz, J., Zuhl, F., and Seemuller, E. (1998). The
proteasome: Paradigm of a self-compartmentalizing protease. Cell
92: 367–380.
Boyer, J.S. (1982). Plant productivity and environment. Science 218:
443–448.
Bray, E.A. (1997). Plant responses to water deficit. Trends Plant Sci. 2: 48–54.
Byun, M.Y., Hong, J.-P., and Kim, W.T. (2008). Identification and
characterization of three telomere repeat-binding factors in rice.
Biochem. Biophys. Res. Commun. 372: 85–90.
Cho, S.K., Chung, H.S., Ryu, M.Y., Park, M.J., Lee, M.M., Bahk, Y.-Y.,
Kim, J., Pai, H.S., and Kim, W.T. (2006). Heterologous expression and
molecular and cellular characterization of CaPUB1 encoding a hot pepper
U-box E3 ubiquitin ligase homolog. Plant Physiol. 142: 1664–1682.
Dreher, K., and Callis, J. (2007). Ubiquitin, hormones and biotic stress
in plants. Ann. Bot. (Lond.) 99: 787–822.
Gonzalez-Lamothe, R., Tsitsigiannis, D.I., Ludwig, A.A., Panicot, M.,
Shirasu, K., and Jones, J.D. (2006). The U-box protein CMPG1 is required
for efficient activation of defense mechanisms triggered by multiple
resistance genes in tobacco and tomato. Plant Cell 18: 1067–1083.
Hatakeyama, S., and Nakayama, K.I. (2003). U-box proteins as a new
family of ubiquitin ligases. Biochem. Biophys. Res. Commun. 302:
635–645.
Hershko, A., and Ciechanover, A. (1992). The ubiquitin system for
protein degradation. Annu. Rev. Biochem. 61: 761–807.
Joo, S., Seo, Y.S., Kim, S.M., Hong, D.K., Park, K.Y., and Kim, W.T.
(2006). Brassinosteroid induction of AtACS4 encoding an auxinresponsive 1-aminocyclopropane-1-carboxylate synthase 4 in Arabidopsis seedlings. Physiol. Plant. 126: 592–604.
Kawadler, H., and Yang, X. (2006). Lys63-linked polyubiquitin chains.
Cancer Biol. Ther. 5: 1273–1274.
Kim, J., Kim, J.E., Lee, J.-H., Lee, M.H., Jung, H.W., Bahk, Y.Y.,
Hwang, B.K., Hwang, I., and Kim, W.T. (2004). The Vr-PLC3 gene
encodes a putative plasma membrane-localized phosphoinositidespecific phospholipase C whose expression is induced by abiotic
stress in mung bean. FEBS Lett. 556: 127–136.
Kim, Y.W., Park, D.S., Park, S.C., Kim, S.H., Cheong, G.W., and
Hwang, I. (2001). Arabidopsis dynamin-like2 that binds specifically to
phosphatidylinositol 4-phosphate assembles into a high-molecular
weight complex in vivo and in vitro. Plant Physiol. 127: 1243–1255.
Kirsch, C., Logemann, E., Lippok, B., Schmelzer, E., and Hahlbrock,
K. (2001). A highly specific pathogen-responsive promoter element
from the immediate-early activated CMPG1 gene in Petroselinum
crispum. Plant J. 26: 217–227.
Lee, J.-H., Deng, X.W., and Kim, W.T. (2006). Possible role of light in
the maintenance of EIN3/EIL1 stability in Arabidopsis seedlings.
Biochem. Biophys. Res. Commun. 350: 484–491.
Liu, P., Sherman-Broyles, S., Nasrallah, M.E., and Nasrallah, J.B.
(2007). A cryptic modifier causing transient self-incompatibility in
Arabidopsis thaliana. Curr. Biol. 17: 734–740.
Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., YamaguchiShinozaki, K., and Shinozaki, K. (1998). Two transcription factors,
1914
The Plant Cell
DREB1 and DREB2, with an EREBP/AP2 DNA binding domain
separate two cellular signal transduction pathways in drought- and
low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10: 1391–1406.
Luo, J., Shen, G., Yan, J., He, C., and Zhang, H. (2006). AtCHIP
functions as an E3 ubiquitin ligase of protein phosphatase 2A subunits
and alters plant response to abscisic acid treatment. Plant J. 46:
649–657.
Moon, J., Parry, G., and Estelle, M. (2004). The ubiquitin-proteasome
pathway and plant development. Plant Cell 16: 3181–3195.
Mudgil, Y., Shiu, S.H., Stone, S.L., Salt, J.N., and Goring, D.R. (2004).
A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box ubiquitin ligase family. Plant Physiol.
134: 59–66.
Mukhopadhyay, D., and Riezman, H. (2007). Proteasome-independent
functions of ubiquitin in endocytosis and signaling. Science 315:
201–205.
Peng, Z., Staub, J.M., Serino, G., Kwok, S.F., Kurepa, J., Bruce, B.,
Vierstra, R.D., Wei, N., and Deng, X.-W. (2001). The cellular level of
PR500, a protein complex related to the 19S regulatory particle of the
proteasome, is regulated in response to stresses in plants. Mol. Biol.
Cell 12: 383–392.
Pickart, C.M., and Eddins, M.J. (2004). Ubiquitin: Structures, functions,
mechanisms. Biochim. Biophys. Acta 1695: 55–72.
Saitou, N., and Nei, M. (1987). The neighbor-joining method: A
new method for reconstructing phylogenetic trees. Mol. Biol. Evol.
4: 406–425.
Smalle, J., Kurepa, J., Yang, P., Babiychuk, E., Kushnir, S., Durski, A.,
and Vierstra, R.D. (2002). Cytokinin growth responses in Arabidopsis
involve the 26S proteasome subunit RPN12. Plant Cell 14: 17–32.
Smalle, J., and Vierstra, R.D. (2004). The ubiquitin 26S proteasome
proteolytic pathway. Annu. Rev. Plant Biol. 55: 555–590.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4:
Molecular evolutionary genetics analysis (MEGA) software version 4.0.
Mol. Biol. Evol. 24: 1596–1599.
Wen, R., Torres-Acosta, A., Pastushok, L., Lai, X., Pelzer, L., Wang,
H., and Xiao, W. (2008). Arabidopsis UEV1D promotes lysine-63linked polyubiquitination and is involved in DNA damage response.
Plant Cell 20: 213–227.
Yan, J., Wang, J., Li, Q., Hwang, J.R., Patterson, C., and Zhang, H.
(2003). AtCHIP, a U-box-containing E3 ubiquitin ligase, plays a critical
role in temperature stress tolerance in Arabidopsis. Plant Physiol. 132:
861–869.
Yang, C.W., Gonzalez-Lamothe, R., Ewan, R.A., Rowland, O.,
Yoshioka, H., Shenton, M., Ye, H., O’Donnell, E., Jones, J.D., and
Sadanandom, A. (2006). The E3 ubiquitin ligase activity of Arabidopsis
PLANT U-BOX17 and its functional tobacco homolog ACRE276 are
required for cell death and defense. Plant Cell 18: 1084–1098.
Yin, X.J., et al. (2007). Ubiquitin lysine 63 chain-forming ligases regulate
apical dominance in Arabidopsis. Plant Cell 19: 1898–1911.
Zeba, N., Ashrafuzzaman, M., and Hong, C.B. (2006). Molecular
characterization of the Capsicum annuum RING zinc finger protein
1 (CaRZFP1) gene induced by abiotic stresses. J. Plant Biol. 49:
484–490.
Zeng, L.R., Qu, S., Bordeos, A., Yang, C., Baraoidan, M., Yan, H.,
Xie, Q., Nahm, B.H., Leung, H., and Wang, G.L. (2004). Spotted
leaf11, a negative regulator of plant cell death and defense, encodes a
U-box/armadillo repeat protein endowed with E3 ubiquitin ligase
activity. Plant Cell 16: 2795–2808.
Zhu, J.K. (2002). Salt and drought stress signal transduction in plants.
Annu. Rev. Plant Biol. 53: 247–273.
Zuckerkandl, E., and Pauling, L. (1965). Evolutionary divergence and
convergence in proteins. In Evolving Genes and Proteins, V. Bryson
and H.J. Vogel, eds (New York: Academic Press). pp. 97–166.
Arabidopsis PUB22 and PUB23 Are Homologous U-Box E3 Ubiquitin Ligases That Play
Combinatory Roles in Response to Drought Stress
Seok Keun Cho, Moon Young Ryu, Charlotte Song, June M. Kwak and Woo Taek Kim
Plant Cell 2008;20;1899-1914; originally published online July 29, 2008;
DOI 10.1105/tpc.108.060699
This information is current as of June 15, 2017
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References
This article cites 37 articles, 19 of which can be accessed free at:
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