The Arabidopsis RING E3 Ubiquitin Ligase AtAIRP2
Plays Combinatory Roles with AtAIRP1 in Abscisic
Acid-Mediated Drought Stress Responses1[C][W][OA]
Seok Keun Cho2, Moon Young Ryu2, Dong Hye Seo, Bin Goo Kang, and Woo Taek Kim*
The ubiquitin (Ub)-26S proteasome pathway is implicated in various cellular processes in higher plants. AtAIRP1, a C3H2C3type RING (for Really Interesting New Gene) E3 Ub ligase, is a positive regulator in the Arabidopsis (Arabidopsis thaliana)
abscisic acid (ABA)-dependent drought response. Here, the AtAIRP2 (for Arabidopsis ABA-insensitive RING protein 2) gene
was identified and characterized. AtAIRP2 encodes a cytosolic C3HC4-type RING E3 Ub ligase whose expression was
markedly induced by ABA and dehydration stress. Thus, AtAIRP2 belongs to a different RING subclass than AtAIRP1 with a
limited sequence identity. AtAIRP2-overexpressing transgenic (35S:AtAIRP2-sGFP) and atairp2 loss-of-function mutant plants
exhibited hypersensitive and hyposensitive phenotypes, respectively, to ABA in terms of seed germination, root growth, and
stomatal movement. 35S:AtAIRP2-sGFP plants were highly tolerant to severe drought stress, and atairp2 alleles were more
susceptible to water stress than were wild-type plants. Higher levels of drought-induced hydrogen peroxide production were
detected in 35S:AtAIRP2-sGFP as compared with atairp2 plants. ABA-inducible drought-related genes were up-regulated in
35S:AtAIRP2-sGFP and down-regulated in atairp2 progeny. The positive effects of AtAIRP2 on ABA-induced stress genes were
dependent on SNF1-related protein kinases, key components of the ABA signaling pathway. Therefore, AtAIRP2 is involved in
positive regulation of ABA-dependent drought stress responses. To address the functional relationship between AtAIRP1 and
AtAIRP2, FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2-2 and atairp1 plants, respectively.
Constitutive expression of FLAG-AtAIRP1 and AtAIRP2-sGFP in atairp2-2 and atairp1 plants, respectively, reciprocally rescued
the loss-of-function ABA-insensitive phenotypes during germination. Additionally, atairp1/35S:AtAIRP2-sGFP and atairp2-2/
35S:FLAG-AtAIRP1 complementation lines were more tolerant to dehydration stress relative to atairp1 and atairp2-2 single
knockout plants. Overall, these results suggest that AtAIRP2 plays combinatory roles with AtAIRP1 in Arabidopsis ABAmediated drought stress responses.
Dehydration and continuous water deficit drastically hinder plant growth and development. To survive under such severe environmental conditions,
sessile plants have developed adaptive strategies that
involve integrated molecular, cellular, and metabolic
programs (Fujita et al., 2006; Yoo et al., 2009; Ahuja
1
This work was supported by the National Research Foundation
(project no. 2010–0000782, funded by the Ministry of Education,
Science, and Technology, Republic of Korea) and the National Center
for GM Crops (project no. PJ008152 of the Next Generation BioGreen
21 Program, funded by the Rural Development Administration,
Republic of Korea; to W.T.K.) and by the Korea Institute of Science
and Technology Information (to B.G.K.).
2
These authors contributed equally to the article.
* Corresponding author; e-mail wtkim@yonsei.ac.kr.
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.plantphysiol.org) is:
Woo Taek Kim (wtkim@yonsei.ac.kr).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.185595
et al., 2010; Hirayama and Shinozaki, 2010; Hummel
et al., 2010). Inhibition of plant growth and development by water stress conditions is directly correlated
with worldwide reductions in crop yields. Thus, elucidation of stress defense mechanisms and the development of resistant transgenic crops against drought
have attracted much interest for many years.
The plant stress hormone abscisic acid (ABA) regulates drought stress responses as a main modulator. In
particular, ABA induces stomata closing to mitigate
undesirable transpirational water loss and activates
various gene groups to initiate rapid and efficient
defense programs (Xiong et al., 2002; YamaguchiShinozaki and Shinozaki, 2006; Tuteja, 2007; Cho
et al., 2009; Kim et al., 2010b; Raghavendra et al.,
2010). Among the diverse gene sets induced by ABA
are the E3 ubiquitin (Ub) ligases. This suggests the
existence of a functional network between ABAmediated stress responses and Ub-dependent protein
degradation.
Ub is a conserved 76-amino acid polypeptide that
functions as a posttranslational protein tag. Ub-mediated
protein modification is ubiquitously found in eukaryotic cells (Dye and Schulman, 2007; Hunter, 2007;
Vierstra, 2009). In higher plants, the ubiquitination
2240 Plant PhysiologyÒ, December 2011, Vol. 157, pp. 2240–2257, www.plantphysiol.org Ó 2011 American Society of Plant Biologists. All Rights Reserved.
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Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120–749,
Korea (S.K.C., M.Y.R., D.H.S., W.T.K.); and ReSEAT Program, Korea Institute of Science and Technology
Information, Seoul 130–741, Korea (B.G.K.)
Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
Plant Physiol. Vol. 157, 2011
and RING E3 Ub ligases in response to dehydration
stress in Arabidopsis. Previously, we identified 100
RING E3 Ub ligase genes, which appeared to be upregulated in response to abiotic stresses based on in
silico data (http://www.genevestigator.com). Mutant
seeds with T-DNA insertion knockouts of these selected genes were obtained from the Arabidopsis
Biological Resource Center and were screened for
ABA sensitivity during the germination stage (Ryu
et al., 2010). Several mutants that displayed ABAinsensitive phenotypes in comparison with wild-type
plants were isolated. One of these mutants was named
atairp1 (for Arabidopsis ABA-insensitive RING protein
1). Phenotypic analysis suggested that AtAIRP1, a
C3H2C3-type RING E3 Ub ligase, is a positive regulator
in the Arabidopsis ABA-dependent drought response.
In this study, another loss-of-function mutant that
was insensitive to ABA during the germination stage
was characterized. This mutant was referred to as
atairp2. The AtAIRP2 gene encodes a C3HC4-type
RING E3 Ub ligase, and its expression was markedly
induced in response to ABA and a broad spectrum of
abiotic stresses, including drought, cold, and high salt
levels. AtAIRP2 overexpressors and atairp2 loss-offunction mutant plants exhibited inverse phenotypes
in terms of ABA-responsive seed germination, root
growth, and stomatal movement. Furthermore, 35S:
AtAIRP2-sGFP transgenic plants were highly tolerant
of severe drought stress; in contrast, atairp2 alleles
were more susceptible to mild water stress than were
wild-type plants. These results suggest that AtAIRP2,
an Arabidopsis C3HC4-type RING E3 Ub ligase, is
involved in positively regulating ABA-dependent
drought stress responses. To address the functional
relationship between AtAIRP1 and AtAIRP2, the
FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2 and atairp1 mutant plants,
respectively. These complementation transgenic
(atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAGAtAIRP1) plants were subsequently used for the analysis of ABA-related phenotypes. The results showed
that constitutive expression of FLAG-AtAIRP1 and
AtAIRP2-sGFP in atairp2 and atairp1, respectively, reciprocally rescued the loss-of-function ABA-insensitive phenotypes. Collectively, the results presented
in this report suggest that the RING E3 Ub ligase
AtAIRP2 plays combinatory roles with AtAIRP1 in
ABA-mediated drought stress responses in Arabidopsis.
RESULTS
Identification of AtAIRP2 Encoding a C3HC4-Type RING
E3 Ub Ligase in Arabidopsis
Germination tests of 100 different T-DNA insertion
loss-of-function Arabidopsis mutants, in which RING
E3 Ub ligase genes were silenced, revealed that the #72
mutant seedlings were significantly less sensitive to
ABA as compared with the wild-type seedlings. In
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system is associated with many cellular processes as
diverse as environmental stress responses, circadian
rhythms, cell cycles, and hormone signaling (Moon
et al., 2004; Smalle and Vierstra, 2004; Dreher and
Callis, 2007; Vierstra, 2009; Lee and Kim, 2011). Ub
tagging of target proteins is performed by three consecutive actions of E1 Ub-activating enzymes, E2 Ubconjugating enzymes, and E3 Ub ligases (Glickman
and Adir, 2004; Smalle and Vierstra, 2004). Polyubiquitinated substrate proteins are degraded by the 26S
proteasome complex, while monoubiquitination or
multiubiquitination confers nonproteolytic functions,
such as DNA repair, protein trafficking, protein activity, and protein-protein interactions (Mukhopadhyay
and Riezman, 2007; Jacobson et al., 2009).
Approximately 6% of the Arabidopsis (Arabidopsis
thaliana) proteome is involved in the Ub 26S proteasome pathway; in particular, there are more than 1,400
different E3 Ub ligase genes in the Arabidopsis genome (Smalle and Vierstra, 2004; Vierstra, 2009). Proteins encoded by Ub ligase genes contain distinct
functional motifs, such as RING (for Really Interesting
New Gene), U Box, HECT (for Homology to E6-AP
Carboxyl Terminus), SCF (for Skp1-Cullin-F Box), or
APC (for Anaphase-Promoting Complex). Arabidopsis contains at least 477 RING domain-containing E3
Ub ligase genes (Kraft et al., 2005; Stone et al., 2005;
Vierstra, 2009). These RING E3 Ub ligase members
play various physiological roles, including hormonal
perception and signaling, seed germination and seedling development, and nitrogen and sugar responses
(Xie et al., 2002; Zhang et al., 2005; Stone et al., 2006;
Peng et al., 2007; Bu et al., 2009; Huang et al., 2010;
Liu and Stone, 2010). Particularly, RING E3 proteins
are shown to function as key mediators in defense
mechanisms against salt and osmotic stresses by increasing ABA biosynthesis (Ko et al., 2006) and ABAdependent drought signaling (Zhang et al., 2007,
2008). Furthermore, DRIP-RING E3 Ub ligase functions as a negative regulator in the drought stress
response by ubiquitinating the drought-induced DREB2A transcription factor (Qin et al., 2008), whereas the
ER-localized RING E3 Rma1H1 is a positive regulator
that induces Ub/26S proteasome-dependent degradation of a water channel protein, aquaporin PIP2;1 (Lee
et al., 2009). Recently, it was reported that RHA2a and
RHA2b RING E3s play positive roles in ABA signaling
and drought responses (Li et al., 2011). These studies
indicate that different isoforms of RING E3 Ub ligases
are crucially involved either positively or negatively
in Arabidopsis drought stress responses. In addition,
RING E3 Ub ligases function in drought stress responses in rice (Oryza sativa), a monocot model crop
(Liu et al., 2008; Park et al., 2010; Bae et al., 2011; Ning
et al., 2011).
Because ABA is a well-characterized plant stress
hormone (Xiong et al., 2002; Yamaguchi-Shinozaki and
Shinozaki, 2006; Tuteja, 2007; Cutler et al., 2010;
Hubbard et al., 2010; Kim et al., 2010b), we wanted
to elucidate the functional relationship between ABA
Cho et al.
AtAIRP2 Expression Is Induced in Response to ABA and
Other Abiotic Stresses
AtAIRP2 was initially considered an ABA- and
abiotic stress-induced gene based on the microarray
2242
data (http://www.genevestigator.com). To address
the in planta induction patterns of AtAIRP2, lightgrown 10-d-old seedlings were subjected to ABA
treatment or various environmental stresses. Total
RNA was isolated from the treated tissues and used
for RT-PCR. The results in Figure 2A demonstrate that
steady-state levels of AtAIRP2 mRNAs were heightened in response to ABA (100 mM for 1.5–3 h), drought
(1–2 h), high salinity (300 mM NaCl for 1.5–3 h), and
cold temperature (4°C for 12–24 h; Fig. 2A). The
induction kinetics of AtAIRP2 was comparable to
those of the marker genes (RAB18 for ABA and
RD29A for abiotic stress).
To further analyze the AtAIRP2 expression profile, a
transcriptional fusion of the 1.3-kb AtAIRP2 upstream
region with the GUS reporter gene was constructed and
introduced into Arabidopsis. GUS activity was monitored in T3 transgenic plants. AtAIRP2 promoter activity was detected in embryos and testa in very young
seedlings 72 h after imbibition and 1 to 2 d after
germination, respectively (Fig. 2B). In 4-d-old lightgrown seedlings, AtAIRP2 expression was low and
restricted to limited areas, including leaf hydathodes,
shoot apical meristems, and vascular tissues of shoots
and roots (Fig. 2B). In 10-d-old plants, a low basal level
of the promoter activity was markedly induced by both
ABA and abiotic stresses throughout the plant tissues.
ABA- and stress-induced gene expression was clearly
identified in guard cells (Fig. 2C). This raises the possibility that AtAIRP2 may play a role in ABA-mediated
stomatal movement. A significant amount of GUS
staining was also observed in floral organs from fully
matured plants, such as anthers, upper stigma regions,
and siliques (Fig. 2D). Taken together, gene expression
studies suggest that AtAIRP2 is indeed an ABA- and
abiotic stress-inducible gene in Arabidopsis.
AtAIRP2 Has in Vitro E3 Ub Ligase Activity and Is
Predominantly Localized to Cytosolic Fractions
The deduced AtAIRP2 protein possesses a single
C3HC4-type RING motif in its C-terminal region,
suggesting that AtAIRP2 functions as an E3 Ub ligase.
AtAIRP2 was expressed in Escherichia coli as a fusion
protein with maltose-binding protein (MBP). The purified recombinant protein was subjected to an in vitro
E3 Ub ligase assay. Incubation of MBP-AtAIRP2 with
Ub, ATP, UBA1 (Arabidopsis E1), and UBC8 (Arabidopsis E2) at 30°C for 1 h gave rise to high-molecularmass smearing ladders detected by either anti-MBP or
anti-Ub antibody (Fig. 3A). In contrast, MBP-AtAIRP2
failed to display E3 activity in the absence of Ub, E1, or
E2. Furthermore, a single-amino acid substitution derivative (MBP-AtAIRP2H163A), in which the conserved
His-163 residue was modified to Ala-163, did not
exhibit ligase activity (Fig. 3B). Thus, bacterially expressed AtAIRP2 possessed in vitro E3 Ub ligase
activity.
To explore the subcellular localization of AtAIRP2,
35S:AtAIRP2-sGFP and control 35S:sGFP constructs
Plant Physiol. Vol. 157, 2011
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terms of cotyledon greening, germination percentages
of wild-type seedlings were clearly reduced in response to ABA. In our experimental conditions, less
than 20% of the wild-type cotyledons were able to
green and expand in the 7-d incubation period in the
presence of 0.5 mM ABA (Supplemental Fig. S1A). However, approximately 44% of the #72 mutant seedlings
exhibited normal greening and expanded cotyledons
7 d after germination. Mutant #72 was subsequently
referred to as atairp2-1 (SAIL_686_G08). Search of the
Arabidopsis Biological Resource Center database
(http://abrc.osu.edu) identified the second allele of
the mutant (atairp2-2; Salk_005082). The atairp2-2 mutant
seedlings also displayed an ABA-insensitive phenotype
highly similar to the atairp2-1 mutant at the germination
stage (Supplemental Fig. S1B).
The AtAIRP2 gene (At5g01520; GenBank accession
no. NM_120230) is located on chromosome 5 and is
composed of 2,180 bp with five exons and four introns.
The T-DNA insertions were mapped to the first exon
(atairp2-1) and the first intron (atairp2-2) in AtAIRP2
(Fig. 1A). Homozygous mutant plants were selected
based on genotyping PCR using primer sets FW1/RV3
and LB_6313R/RV3 (Fig. 1B). Reverse transcription
(RT)-PCR revealed that, while partial AtAIRP2 transcripts were detected in the atairp2-1 and atairp2-2
mutant seedlings, full-length mRNAs were undetectable in both alleles, indicating that expression of
functional AtAIRP2 mRNAs was repressed in the
atairp2 mutant plants (Fig. 1C).
The predicted AtAIRP2 protein consists of 242
amino acids (molecular mass of 28 kD) with a single
RING domain in its C-terminal region (Fig. 1D). Consistent with the notion that RING E3 Ub ligases are
encoded by a multigene family (Kraft et al., 2005; Stone
et al., 2005; Vierstra, 2009), the amino acid sequence
identity of AtAIRP2 to other Arabidopsis RING proteins was relatively low (63% identical to At5g58787
and 60% identical to At3g47160; Fig. 1E). Additionally,
AtAIRP2 is 64% to 77% identical to rice, poplar
(Populus trichocarpa), grape (Vitis vinifera), and sorghum
(Sorghum bicolor) RING proteins whose cellular functions are unknown (Supplemental Fig. S2). The CysX2-Cys-X11-Cys-X1-His-X2-Cys-X2-Cys-X10-Cys-X2-Cys
motif is conserved in the C-terminal RING domain of
AtAIRP2, indicating that AtAIRP2 is a C3HC4-type
RING E3 Ub ligase (Fig. 1F). Recently, AtAIRP1
(NM_118474) was identified as a stress- and ABAinducible E3 Ub ligase (Ryu et al., 2010). AtAIRP1
contains a C3H2C3-RING motif with a predicted molecular mass of 16.9 kD (153 amino acids), which is
significantly smaller than that of AtAIRP2. AtAIRP2 is
only 13% identical to AtAIRP1; therefore, they belong
to different subclasses of the RING multigene family.
Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
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Figure 1. Identification of atairp2 loss-of-function mutants, sequence analysis of the AtAIRP2 gene, and construction of AtAIRP2
overexpressors. A, Schematic representation of the atairp2-1 (SAIL_686_G08) and atairp2-2 (Salk_005082) alleles with T-DNA
insertions. Gray bars indicate coding regions, black bars indicate the 5# and 3# untranslated regions, and solid lines represent
introns of the AtAIRP2 gene (GenBank accession no. NM_120230). T-DNA insertions are indicated by triangles. T-DNA-specific
(LB1 and LB2) and gene-specific (FW1, FW2, FW3, RV1, RV2, and RV3) primers used in genotyping PCR and RT-PCR are
indicated with arrows. B, Genotyping PCR of the two atairp2 T-DNA insertion mutant alleles (atairp2-1 and atairp2). Genespecific and T-DNA-specific primer sets used for genomic PCRs are indicated on the right. WT, Wild type. C, Expression levels of
AtAIRP2 transcripts in wild-type and atairp2 mutant plants. Gene-specific primer sets for RT-PCR are indicated on the right.
Constitutively expressed UBC10 (for E2 ubiquitin-conjugating enzyme) mRNA was used as a loading control. Primer sequences
are listed in Supplemental Table S1. D, Schematic structure of the full-length AtAIRP2 cDNA clone and its deduced protein. The
gray bar indicates the coding region, and solid lines represent the 5# and 3# untranslated regions. The C-terminal C3HC4-type
RING domain is indicated by the black bar. E, Phylogenetic analysis of the seven AtAIRP2 homologs from Arabidopsis
(At5g58787 and At3g47160), rice (GenBank accession no. NP_001060539), poplar (XP_002309135), grape (XP_002280008),
and sorghum (XP_002447334). F, Amino acid sequence alignment of the RING motifs of AtAIRP2 and other C3HC4-type RING
proteins. Potential Zn2+-interacting amino acid residues (C-X2-C-X11-C-X1-H-X2-C-X2-C-X10-C-X2-C) are indicated. Amino acid
residues identical in all seven RING domains are shown in black, and those conserved in at least four of the seven sequences are
shaded. G, Real-time qRT-PCR analysis of the wild type and AtAIRP2 overexpressors. Expression levels of AtAIRP2 transcripts in
wild-type and T3 35S:AtAIRP2-sGFP transgenic (independent lines 10 and 19) plants were determined by real-time qRT-PCR
using gene-specific primer sets. UBC10 mRNA levels were used as a loading control. H, Immunoblot analysis of wild-type and
AtAIRP2-sGFP (lines 10 and 19) plants. Expression levels of the AtAIRP2-sGFP fusion protein were determined using an anti-GFP
antibody. Rubisco large subunit (RbcL) was used as a loading control.
Plant Physiol. Vol. 157, 2011
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Cho et al.
were transformed into onion (Allium cepa) epidermal
cells using particle bombardment. Localization of
expressed fusion proteins was visualized by fluorescence microscopy under dark and bright fields.
As shown in Figure 3B, the fluorescence signal of
sGFP was uniformly distributed throughout the
onion cells. The localization signal of the AtAIRP2sGFP fusion protein was similar to that of the sGFP
control, suggesting that AtAIRP2 is present in the
cytosolic fractions. The cytosolic localization of
AtAIRP2-sGFP was more evident in plasmolyzed
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Figure 2. Expression profiles of
AtAIRP2 in response to ABA and
different abiotic stress conditions.
A, Light-grown, 10-d-old Arabidopsis seedlings were treated with
100 mM ABA (1.5–3 h), drought
(1–2 h), high salinity (300 mM
NaCl for 1.5–3 h), or cold (4°C for
12–24 h). Total RNA was isolated
from the treated tissues and used for
RT-PCR. The RAB18 and RD29A
genes were positive controls for
ABA and abiotic stress responses,
respectively. UBC10 was used as a
loading control. B to D, AtAIRP2
promoter activity. AtAIRP2-promoter:
GUS transgenic T3 plants were incubated with 5-bromo-4-chloro-3indolyl-b-glucuronic acid for 12 h.
AtAIRP2 promoter activity was visualized by GUS-specific staining.
B, Histochemical localization of
GUS activity in young seedlings
(72 h after imbibition, 1 d after
germination, 2 d after germination,
and 4-d-old seedlings). Arrows indicate GUS signals. Bars = 0.25 cm.
C, GUS-specific staining patterns in
10-d-old seedlings in response to
ABA, drought, salt, and cold treatments. GUS signals were markedly
induced in guard cells in rosette
leaves and roots. Bar lengths are
indicated to the right. D, GUS activity in mature plants. GUS signals
were detected in anthers (flower
buds), upper region of stigma (2–3 d
after flowering [DAF]), and siliques
(6–16 d after flowering). Bars =
0.25 cm. [See online article for
color version of this figure.]
onion epidermal cells. AtAIRP1 and Arabidopsis
AREB1 (for ABA response element-binding protein
1) were used as specificity controls. AtAIRP1 is a
cytosolic E3 Ub ligase (Ryu et al., 2010), while
AREB1 is a nuclear transcription factor (Yoshida
et al., 2010). Consistent with previous findings,
AtAIRP1 was found in the cytosol of both unplasmolyzed and plasmolyzed cells, whereas AREB1
was exclusively detected in the nuclei (Fig. 3B).
Collectively, it is concluded that AtAIRP2 is a cytosolic RING E3 Ub ligase.
Plant Physiol. Vol. 157, 2011
Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
Germination Percentages of Arabidopsis Seedlings Are
Intimately Linked with the Expression Levels of
AtAIRP2 in the Presence of ABA and NaCl Stress
Before exploring detailed phenotypes of the atairp2-1
and atairp2-2 mutant alleles, transgenic Arabidopsis
plants that overexpressed the AtAIRP2 gene were
constructed using the cauliflower mosaic virus 35S
promoter. Several T3 transgenic plants were obtained
based on resistance to the herbicide BASTA (glufosinate ammonium), and two independent transgenic
lines (lines 10 and 19) were selected for further experiments (Supplemental Fig. S3). Real-time quantitative (q)RT-PCR and immunoblotting with an anti-GFP
antibody indicated that both lines 10 and 19 effectively
expressed AtAIRP1 mRNA and AtAIRP1-sGFP protein without stress treatments (Fig. 1, G and H).
The atairp2 mutants were initially selected due to
their ABA-insensitive phenotypes (Supplemental Fig. S1).
For phenotypic analysis, seed germination percentages were examined in the presence or absence of
ABA. Sterilized seeds from wild-type, atairp2 mutant, and 35S:AtAIRP2-sGFP plants were plated on
Murashige and Skoog (MS) growth medium containing different concentrations (0, 0.2, 0.4, or 0.8 mM) of
Plant Physiol. Vol. 157, 2011
ABA. Germination rates were monitored in terms of
radicle emergence and cotyledon greening 3 and 7 d
after stratification, respectively. As ABA concentrations increased, radicle emergence rates of wild-type
seeds concomitantly decreased from 97.2% (0.2 mM
ABA) to 71.8% (0.4 mM ABA) and 37.6% (0.8 mM ABA)
3 d after germination (Fig. 4A). However, both
atairp2-1 and atairp2-2 knockout mutant seeds showed
hyposensitivity to ABA as compared with wild-type
seeds. Approximately 85% of the mutant seeds were
able to germinate on medium containing 0.4 mM
ABA, and more than 55% of the mutants still germinated normally with 0.8 mM ABA. In contrast,
AtAIRP2-overexpressing plants displayed a hypersensitive phenotype toward ABA. Only 26.2%
(line 10) and 12.9% (line 19) of the 35S:AtAIRP2-sGFP
seeds could germinate in the presence of 0.8 mM ABA
(Fig. 4A).
Subsequently, cotyledon greening percentages were
monitored 7 d after germination. The results indicate
that mutant and overexpressing seedlings were hyposensitive and hypersensitive to ABA, respectively,
relative to wild-type plants. Approximately 53% of
the mutant and 10% of the 35S:AtAIRP2-sGFP seed2245
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Figure 3. AtAIRP2 is a cytosolic RING
E3 Ub ligase. A, In vitro E3 Ub ligase
assay. Left panel, bacterially expressed
MBP-AtAIRP2 was incubated with ATP
in the presence or absence of Ub,
Arabidopsis E1 (His-UBA1), and Arabidopsis E2 (His-UBC8) at 30°C for 2 h.
Reaction mixtures were separated by
SDS-PAGE and subjected to immunoblot analysis using either anti-MBP antibody or anti-Ub antibody. Right
panel, MBP-AtAIRP2 and single-amino
acid substitution mutant MBP-AtAIRP1H163A were incubated at 30°C for 2 h
in the presence of ATP, Ub, E1, and E2.
Ubiquitinated proteins were detected
by either anti-MBP or anti-Ub antibody. WT, Wild type. B, Cytosolic localization of AtAIRP2. 35S:sGFP, 35S:
AtAIRP2-sGFP, 35S:AtAIRP1-sGFP, and
35S:AREB1-sGFP gene constructs were
transformed into onion epidermal cells
using particle bombardment. Localization of the expressed proteins was visualized by fluorescence microscopy
(dark and bright fields) in both unplasmolyzed and plasmolyzed onion cells.
Arabidopsis AREB1 and RING E3 Ub
ligase AtAIRP1 were used as specificity controls for nuclear and cytosolic
proteins, respectively. DAPI, 4#,
6-Diamino-phenylindole. Bars = 100
mm. [See online article for color version of this figure.]
Cho et al.
lings developed true green cotyledons on medium
supplemented with 0.4 mM ABA, while 20% of wildtype plants developed normal cotyledons (Fig. 4A). In
the presence of 0.8 mM ABA, no wild-type or 35S:
AtAIRP2-sGFP plants could display normal cotyledons. In contrast, 13% of both mutant alleles were still
able to develop green cotyledons.
Germination tests were repeated in the presence of
NaCl (0, 75, 100, and 125 mM). Again, mutant and
overexpressing seedlings exhibited hyposensitivity
and hypersensitivity to NaCl, respectively, relative to
wild-type seedlings in both radicle emergence and
cotyledon greening (Supplemental Fig. S4). Phenotypic differences became more evident as salinity
increased. A significant number of atairp2 mutants
(32%–44%) displayed normal cotyledons with 100 mM
NaCl 7 d after germination, whereas the growth of
most 35S:AtAIRP2-sGFP seedlings was arrested under
the same conditions (Fig. 4B). Wild-type seedlings
exhibited intermediate phenotypes between mutant
and overexpressing plants under high-salinity conditions (Fig. 4B). Taken together, these results provide
evidence that the atairp2 mutants and AtAIRP2 overexpressors have inverse phenotypes in response to
ABA and NaCl during the germination stage, suggest2246
ing that AtAIRP2 is positively involved in Arabidopsis
ABA-modulated germination processes.
AtAIRP2 Is Positively Involved in ABA-Mediated Root
Growth Inhibition and Stomatal Closure
Since reverse effects of ABA on seed germination
were clearly identifiable in atairp2 mutants and
AtAIRP2 overexpressors (Fig. 4), we next investigated
the effects of ABA on postgermination growth. Inhibition of seedling root growth is a typical action of
ABA (Quiroz-Figueroa et al., 2010; Ryu et al., 2010). As
shown in Figure 5A, atairp2 and 35S:AtAIRP2-sGFP
plants displayed hyposensitivity and hypersensitivity,
respectively, to ABA in terms of young root growth.
When wild-type, atairp2 allele, and 35S:AtAIRP2-sGFP
(lines 10 and 19) seedlings were grown for 10 d with
0.2 mM ABA, the mutant root growth appeared to be
unaffected. In contrast, elongation of 35S:AtAIRP2sGFP roots was significantly reduced by approximately 43% under the same ABA concentration (Fig.
5A). With 0.4 mM ABA, growth of 35S:AtAIRP2-sGFP
roots was inhibited by 74.7%, while that of loss-offunction mutant roots was reduced by only 42.4%. 35S:
AtAIRP2-sGFP root growth was severely impaired and
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Figure 4. Germination rates of wildtype, atairp2, and 35S:AtAIRP2-sGFP
plants in response to ABA and NaCl. A,
ABA sensitivity of the wild type (WT),
two atairp2 mutant alleles (atairp2-1
and atairp2-2), and AtAIRP2 overexpressors (transgenic lines 10 and 19)
during the germination stage. Sterilized seeds were imbibed in water for
2 d at 4°C and incubated on MS medium in the presence of different concentrations of ABA (0, 0.2, 0.4, and
0.8 mM) at 22°C under a 16-h-light/8-hdark photoperiod. Germination percentages were determined in terms of
radical emergence 3 d after germination and cotyledon greening 7 d after
germination. SD values were determined from four biological replicates
(n . 36). Bars = 0.5 cm. B, NaCl
sensitivity of the wild type, two atairp2
mutant alleles (atairp2-1 and atairp2-2),
and AtAIRP2 overexpressors (transgenic
lines 10 and 19) during the germination stage. Germination rates were determined in the presence of different
concentrations of NaCl (0, 75, 100,
and 125 mM) as described above. Data
represent means 6 SD (n . 36) from
three independent experiments. [See
online article for color version of this
figure.]
Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
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Figure 5. Root growth and stomatal aperture of wild-type, atairp2, and 35S:AtAIRP2-sGFP plants in response to ABA treatment.
A, Root-growth phenotypes of the wild type (WT), two atairp2 mutant alleles (atairp2-1 and atairp2-2), and AtAIRP2
overexpressors (transgenic lines 10 and 19) in response to different concentrations (0, 0.2, 0.4, and 0.8 mM) of ABA. Sterilized
seeds were imbibed in water for 2 d and grown vertically on MS medium supplemented with the indicated concentrations of ABA
for 10 d. Root growth patterns were monitored and analyzed using Scion Image software. Data represent means 6 SD (n = 20).
Bars = 0.5 cm. B, Stomatal aperture of the wild type, two atairp2 mutant alleles (atairp2-1 and atairp2-2), and AtAIRP2
overexpressors (transgenic lines 10 and 19) in response to different concentrations (0, 0.1, 1.0, and 10 mM) of ABA. Mature leaves
from wild-type, atairp2 allele, and AtAIRP2-overexpressing plants were treated with a stomatal opening solution for 2 h and
incubated with the indicated concentrations of ABA for 2 h. Stomata on abaxial surfaces were photographed by light microscopy.
Bars = 10 mm. Stomatal aperture (the ratio of width to length) was quantified using at least 30 guard cells from each sample. Data
represent means 6 SD (n = 30). [See online article for color version of this figure.]
elongation ceased in the presence of 0.8 mM ABA.
However, mutant roots were still alive and growing
under the same conditions. Wild-type roots showed
intermediate phenotypes in response to all of the
different ABA concentrations examined (Fig. 5A).
ABA-dependent stomatal closure was next examined in wild-type, atairp2, and 35S:AtAIRP2-sGFP
plants. Light-grown 4-week-old rosette leaves were
pretreated with stomatal opening solution to induce
full opening of the guard cells (Kwak et al., 2003). The
leaves were subsequently incubated with different
concentrations of ABA (0, 0.1, 1.0, and 10 mM) for 2 h
Plant Physiol. Vol. 157, 2011
and stomatal behavior was monitored. While stomatal
apertures in all leaves examined were indistinguishable without ABA, there were clear differences in
response to ABA. In the presence of 0.1 mM ABA,
average stomatal apertures (the ratio of width to
length) of wild-type, atairp2-1, and 35S:AtAIRP2sGFP (line 10) plants were 0.15 6 0.02, 0.20 6 0.02,
and 0.10 6 0.02, respectively (Fig. 5B). Differences in
average stomatal apertures of these plants became
progressively more evident as ABA concentrations
increased. With 1 mM ABA, the stomatal apertures of
the atairp2-1 mutant and AtAIRP2 overexpressor line
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Cho et al.
Expression Levels of AtAIRP2 Are Closely Associated
with Drought Tolerance in Arabidopsis
Several recent studies reported that ectopic expression of drought-induced RING E3 Ub ligases results
in a tolerant phenotype to water stress in an ABAdependent or ABA-independent manner. For example,
transgenic Arabidopsis plants that constitutively express the hot pepper (Capsicum annuum) RING E3 Ub
ligase Rma1H1 are highly tolerant to severe dehydration
stress via an ABA-independent pathway (Lee et al.,
2009). On the other hand, overexpression of SDIR1,
AtAIRP1, or RHA2a, all of which encode Arabidopsis
RING E3 ligases, conferred resistance to water deficit in
an ABA-dependent fashion (Zhang et al., 2007; Ryu
et al., 2010; Li et al., 2011). AtAIRP2 was induced by
ABA as well as drought stress (Fig. 2). Furthermore,
AtAIRP2 was positively involved in ABA-mediated
responses, including seed germination (Fig. 4), seedling
root growth (Fig. 5A), and stomatal movement (Fig. 5B).
Thus, it is postulated that AtAIRP2 participates in the
ABA-dependent drought response.
Light-grown, 2-week-old, healthy wild-type and
atairp2 mutant (atairp2-1 and atairp2-2) plants were
further grown for 12 d under normal conditions without irrigation. These water-stressed plants were then
irrigated, and their survival ratios were determined
after 3 d of irrigation. As shown in Figure 6A, 81.0%
(47 of 58) of the wild-type plants grew normally after
reirrigation. On the other hand, significantly lower
percentages of mutant plants (36.2% of atairp2-1 and
39.2% of atairp2-2 mutants) resumed their growth.
Therefore, atairp2 knockout mutant alleles were more
susceptible than were the wild-type plants to mild
drought conditions. Subsequently, 2-week-old wildtype and 35S:AtAIRP2-sGFP (lines 10 and 19) plants
were grown for 15 d without irrigation. This drought
condition resulted in complete drying of the potted
soil and induced severe dehydration stress. Survival
rates were then estimated 3 d after reirrigation.
2248
AtAIRP2-overexpressing plants displayed a markedly
resistant phenotype, and their survival percentages
reached 71.5% (50 of 70 for line 10) and 74.0% (55 of 77
for line 19; Fig. 6B). The survival rate of wild-type
plants was only 20.7% (12 of 58). Thus, AtAIRP2
overexpressors were more tolerant of severe water
deficits; in contrast, atairp2 mutants were more sensitive to the stress than were the wild-type plants.
Consistent with the drought-tolerant phenotype,
2-week-old detached rosette leaves from 35S:AtAIRP2sGFP plants lost water more slowly than did wild-type
leaves. After a 5-h incubation under dim light at room
temperature, 35S:AtAIRP2-sGFP leaves retained approximately 60% of their fresh weights and wild-type leaves
retained approximately 55% of their fresh weights
(Fig. 6C). However, atairp2 alleles retained only approximately 40% to 45% of their fresh weights after the 5-h
incubation. It is worth noting that the reduction in fresh
weight of the mutant leaves was more rapid and evident
earlier during the initial stages of the incubation period
(within 15 to 30 min) than later, suggesting that the
mutants were susceptible to the initial stage of dehydration (Fig. 6C, inset).
Reactive oxygen species, such as hydrogen peroxide
(H2O2), are critical participants in the ABA-mediated
drought stress responses in guard cells (Wang and
Song, 2008; Cho et al., 2009; Jammes et al., 2009; Song
and Matsuoka, 2009). To evaluate the degree of H2O2
production in response to drought stress, normal and
water-stressed leaves from wild-type, atairp2 allele,
and 35S:AtAIRP2-sGFP lines were incubated with 3,3#diaminobenzidine (DAB). DAB interacts with H2O2 in
the presence of endogenous peroxidases and produces
a dark-brown color (Thordal-Christensen et al., 1997).
Figure 6D demonstrates that higher levels of droughtinduced H2O2 were produced in 35S:AtAIRP2-sGFP
(lines 10 and 19) rosette leaves relative to that of atairp2
mutant leaves. H2O2 levels in wild-type leaves were
intermediate between those in overexpressor and
mutant plants before and after drought treatments
(Fig. 6D), indicating that AtAIRP2 is positively involved in drought-induced H2O2 production. Overall,
35S:AtAIRP2-sGFP and atairp2 plants exhibited inverse
phenotypes toward drought, indicating that expression
levels of AtAIRP2 are closely associated with drought
tolerance in Arabidopsis. These results are consistent
with the hypothesis that AtAIRP2 is a positive component of an ABA-dependent response to drought.
The Positive Role of AtAIRP2 in ABA Induction of
Drought Stress-Related Genes Is Dependent on SnRK
Protein Kinase Activities
abi1-1 is an ABA-insensitive dominant mutant
(Hubbard et al., 2010; Kim et al., 2010b; Raghavendra
et al., 2010). As shown in Figure 7A, AtAIRP2 and
RAB18, a marker gene for ABA induction, were not
induced by exogenously applied ABA in abi1-1 mutant
plants, confirming that AtAIRP2 is an ABA-induced
gene.
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10 were 0.11 6 0.02 and 0.03 6 0.01, respectively. With
10 mM ABA, the stomatal aperture of mutant leaves
was 0.07 6 0.01, which was approximately 7-fold
greater than that of the overexpressors (0.01 6 0.01;
Fig. 5B). In addition, stomatal movement of the atairp2-2
allele and 35S:AtAIRP2-sGFP line 19 displayed similar
opposite profiles in response to ABA. Stomatal behavior patterns in wild-type leaves were intermediate
between the mutants and overexpressors under all
ABA concentrations examined (Fig. 5B). These results
indicate that ABA-mediated stomatal closure in atairp2
mutant leaves was markedly hindered as compared
with wild-type and AtAIRP2 overexpressors. Because
morphological differences between wild-type, atairp2,
and 35S:AtAIRP2-sGFP plants were undetectable in the
absence of exogenously applied ABA, the data presented
in Figures 4 and 5 strongly suggest that AtAIRP2 is
positively involved in ABA responses during germination and postgermination growth.
Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
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Figure 6. AtAIRP2 expression levels were closely associated with drought tolerance. A, atairp2 loss-of-function mutants were
more sensitive to drought than were wild-type (WT) plants. Light-grown, 2-week-old wild-type and atairp2 mutant allele
(atairp2-1 and atairp2-2) plants were further grown for 12 d under normal conditions but without irrigation. The water-stressed
plants were irrigated, and their survival ratios were determined after 3 d of irrigation. B, Overexpression of AtAIRP2 conferred
tolerance to drought stress. Light-grown, 2-week-old wild-type and 35S:AtAIRP2-sGFP (lines 10 and 19) plants were grown for 15
d without irrigation. Survival percentages were determined 3 d after irrigation. C, Water loss rates of detached rosette leaves.
Mature rosette leaves from 2-week-old wild-type, atairp2 allele, and 35S:AtAIRP2-sGFP lines were detached, and their fresh
weights were measured at the indicated time points. Water loss rates were calculated as the percentage of fresh weight of the
excised leaves. Data represent means 6 SD (n = 7) from eight independent experiments. D, H2O2 production in response to
drought stress. Control and water-stressed rosette leaves from wild-type, atairp2-1, atairp2-2, and 35S:AtAIRP2-sGFP plants were
stained with 100 mg mL21 DAB overnight. Levels of drought-induced H2O2 production were visualized as a dark brown color.
[See online article for color version of this figure.]
The SNF1-related protein kinase (SnRK) protein
kinase family is a major component of the ABA signaling pathway and acts upstream of the AREB/ABF
transcription factors (Hubbard et al., 2010; Kim et al.,
2010b; Raghavendra et al., 2010). SnRKs were shown to
be key positive regulators in ABA-dependent reactive
oxygen species production and in water and osmotic
Plant Physiol. Vol. 157, 2011
stress responses (Mustilli et al., 2002; Fujita et al., 2009;
Fujii et al., 2011). Consistent with their roles, triple
knockout mutation of three SnRK genes (SnRK2.2,
SnRK2.3, and SnRK2.6) greatly impaired ABA- and
dehydration-induced gene expression (Fujii and Zhu,
2009; Fujita et al., 2009). Because AtAIRP2 was not only
induced by ABA but was also positively involved in
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Figure 7. The positive role of AtAIRP2 in ABA induction of drought stress-related gene expression required SnRK protein kinase
activity. A, ABA induction profiles of AtAIRP2 in wild-type (WT), abi1-1, snrk2.2, snrk2.3, and snrk2.6 single knockout mutant,
and snrk2.2 snrk2.3 snrk2.6 triple mutant plants. Light-grown, 10-d-old wild-type and various snkr2 mutant seedlings were
treated with 100 mM ABA. Total RNA was extracted from the treated tissues and analyzed by real-time qRT-PCR. RAB18 was a
positive control for ABA induction, and UBC10 was used as a loading control. B, ABA induction profiles of drought-related genes
in wild-type, atairp2-2, and AtAIRP2-overexpressing plants. Light-grown, 3-week-old plants were incubated with 100 mM ABA
for 6 h. Induction patterns of various ABA- and drought-responsive genes (ABI1, ABI2, ABF3, ABF4, RD26, RD20, KIN2, and
RAB18) were analyzed by real-time qRT-PCR. Data represent the fold induction of each gene by ABA (100 mM) relative to the
control treatment (0 mM ABA). Mean values from three independent technical replicates were normalized to the levels of an
internal control, glyceraldehyde-3-phosphate dehydrogenase C subunit mRNA. [See online article for color version of this
figure.]
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Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
Functional Relationship of AtAIRP1 and AtAIRP2
In our previous study, AtAIRP1, a C3H2C3-type
RING E3 Ub ligase, was shown to play a positive
role in the ABA-dependent drought response in Arabidopsis. AtAIRP1-overexpressing and atairp1 lossof-function mutant plants had opposite phenotypes,
including germination rates, root elongation, stomatal
closure, and tolerance to drought stress, following
ABA-mediated responses (Ryu et al., 2010). AtAIRP1
belongs to a different RING subfamily than does
AtAIRP2 (C3H2C3 type versus C3HC4 type), and its
deduced molecular mass is quite smaller than that of
AtAIRP2 (16.9 versus 28.0 kD; Fig. 1). However, both
E3 ligases were predominantly localized to cytosolic
Plant Physiol. Vol. 157, 2011
fractions (Fig. 3). In addition, phenotypic properties of
35S:AtAIRP1-sGFP and atairp1 plants are reminiscent of
those of 35S:AtAIRP2-sGFP and atairp2 plants, respectively, in terms of ABA-mediated responses (Figs. 4–6).
With these findings in mind, we hypothesized that
AtAIRP1 and AtAIRP2 play a combinatory role in
ABA-dependent drought stress responses. Alternatively, it is possible that AtAIRP1 and AtAIRP2 work
independently. To test these possibilities, complementation tests were conducted. FLAG-AtAIRP1 and
AtAIRP2-sGFP fusion genes were ectopically expressed
in atairp2-2 and atairp1 mutant plants, respectively.
Independent complementation lines were selected
and confirmed by genomic Southern blotting (Supplemental Fig. S3). RT-PCR and immunoblot analyses
revealed that AtAIRP2 and AtAIRP1 transgenes were
clearly expressed in atairp1/35S:AtAIRP2-sGFP (lines
5 and 7) and atairp2-2/35S:FLAG-AtAIRP1 (lines 3 and
24) complementation T3 transgenic plants, respectively (Fig. 8, A and B). These T3 complementation
lines were used to analyze ABA- and stress-related
phenotypes to determine whether mutant phenotypes were reciprocally rescued. The results in Figure
8C show that both atairp1/35S:AtAIRP2-sGFP and
atairp2-2/35S:FLAG-AtAIRP1 lines were more sensitive
to ABA at all concentrations examined (0.2–0.8 mM) than
were atairp1 and atairp2-2 single mutants, respectively,
during the germination stage. For example, in the
presence of 0.4 mM ABA, germination (cotyledon
greening) percentages for wild-type, atairp1, atairp2-2,
atairp1/35S:AtAIRP2-sGFP (lines 5 and 7), and atairp22/35S:FLAG-AtAIRP1 (lines 3 and 24) plants were
31.3%, 76.4%, 67.6%, 17.4% to 33.0%, and 11.7%
to 31.5%, respectively. Thus, the degree of ABA sensitivity for both complementation progeny was approximately the same as the average for wild-type and
overexpressing plants (compare Figs. 4 and 8). This
indicates that the insensitive phenotypes of atairp1 and
atairp2-2 young seedlings in response to ABA were
efficiently rescued by ectopic expression of AtAIRP2
and AtAIRP1, respectively.
Mature atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:
FLAG-AtAIRP1 complementation lines were also
markedly more tolerant to dehydration stress as compared with the atairp1 and atairp2-2 single knockout
mutant plants. After 13 d of water stress, survival rates
of wild-type, atairp1, and atairp2-2 progeny were determined to be 61.6%, 16.7%, and 26.7%, respectively,
whereas those of atairp1/35S:AtAIRP2-sGFP (lines 5
and 7) and atairp2-2/35S:FLAG-AtAIRP1 (lines 3 and
24) plants were 63.3% to 86.7% and 60.0% to 75.0%,
respectively (Fig. 8D). In addition, detached leaves
from complementation lines lost water more slowly
than those from single knockout mutant plants (Fig. 8E).
Overall, these results strongly suggest that constitutive expression of AtAIRP1 and AtAIRP2 in atairp2-2
and atairp1 mutant plants, respectively, reciprocally
rescued the loss-of-function ABA-insensitive phenotypes during both the germination and postgermination stages.
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ABA-dependent responses, our next question was
whether the mode of action of AtAIRP2 is downstream
of SnRKs. To answer this question, ABA induction of
AtAIRP2 was first examined in wild-type, snrk2.2,
snrk2.3, and snrk2.6 single knockout mutant, and
snrk2.2 snrk2.3 snrk2.6 triple mutant plants. Real-time
qRT-PCR analysis indicated that ABA induction of
AtAIRP2 in the single knockout mutants was very
comparable to that in wild-type plants (Fig. 7A). In
contrast, AtAIRP2 transcript levels remained unchanged before and after ABA treatment in the
snrk2.2 snrk2.3 snrk2.6 triple loss-of-function mutant
plants. Thus, the ABA induction of AtAIRP2 in the
single snrk mutant lines may be due to the redundant
functions of SnRK kinase family members. Similar
induction profiles were also obtained for RAB18 (Fig.
7A). These results indicate that SnRK protein kinase
activity is necessary for ABA-induced activation of
AtAIRP2 as well as RAB18.
Expression patterns of various ABA-responsive
genes were compared in wild-type, atairp2 mutant,
and AtAIRP2-overexpressing plants using real-time
qRT-PCR. As demonstrated in Figure 7B, following
ABA induction, the gene expression of ABI1, ABI2,
ABF3, and ABF4 was down-regulated and up-regulated
in atairp2-2 mutant and AtAIRP2-overexpressing plants,
respectively, relative to wild-type plants. ABI1 and
ABI2 are ABA-responsive protein phosphatase 2C genes
(Ma et al., 2009; Santiago et al., 2009), while ABF3 and
ABF4 encode ABA-activated basic Leu zipper transcription factors (Finkelstein et al., 2002; Gómez-Porras
et al., 2007; Lee et al., 2010). Furthermore, mRNA levels of various ABA- and stress-induced downstream
marker genes (RD20, RD26, KIN2, and RAB18) were
also lower in knockout mutant alleles and, in contrast,
markedly higher in 35S:AtAIRP2-sGFP plants as compared with those in wild-type plants (Fig. 7B). Thus,
AtAIRP2 positively regulates ABA induction of protein phosphatases, ABF/AREB transcription factors,
and downstream marker gene expression. Collectively, the results presented in Figure 7 indicate that
SnRK protein kinase activity is necessary for a positive role of AtAIRP2 in ABA induction of drought
stress-related genes.
Cho et al.
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Figure 8. Construction and characterization of atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation
transgenic plants. A and B, RT-PCR and immunoblot analyses. AtAIRP2-sGFP and FLAG-AtAIRP1 fusion genes were ectopically
expressed in atairp1 and atairp2-2 mutant plants, respectively. Transcript (A) and protein (B) levels of AtAIRP2-sGFP and FLAG-AtAIRP1
were examined in atairp1/35S:AtAIRP2-sGFP (lines 5 and 7) and atairp2-2/35S:FLAG-AtAIRP1 (lines 3 and 24) complementation T3
transgenic plants. Rubisco large subunit (RbcL) was used as a loading control. C, Phenotypic properties of atairp1/35S:AtAIRP2-sGFP
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Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
DISCUSSION
Figure 8. (Continued.)
and atairp2-2/35S:FLAG-AtAIRP1 complementation T3 transgenic plants during the germination stage. After imbibition in water
for 2 d at 4°C, wild-type (WT), atairp1 and atairp2-2 mutant, and atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1
complementation T3 seeds were treated with different concentrations of ABA (0, 0.2, 0.4, and 0.8 mM) at 22°C under a 16-h-light/
8-h-dark photoperiod. Germination percentages were determined in terms of cotyledon greening 7 d after germination. SD values
were determined from four biological replicates (n = 40). Bars = 0.5 cm. D, Water stress tolerance of atairp1/35S:AtAIRP2-sGFP
and atairp2-2/35S:FLAG-AtAIRP1 complementation T3 transgenic plants. Light-grown, 2-week-old wild-type, atairp1 and
atairp2-2 mutant, and atairp1/35S:AtAIRP2-sGFP (lines 5 and 7) and atairp2-2/35S:FLAG-AtAIRP1 (lines 3 and 24) complementation T3 transgenic plants were further grown for 13 d without irrigation. Water-stressed plants were irrigated, and their
survival ratios were determined after 3 d of irrigation. E, Water loss rates of detached rosette leaves. Mature rosette leaves from
2-week-old wild-type, atairp1 and atairp2-2 mutant, and atairp1/35S:AtAIRP2-sGFP (lines 5 and 7) and atairp2-2/35S:FLAGAtAIRP1 (lines 3 and 24) complementation T3 transgenic plants were detached, and their fresh weights were measured at the
indicated time points. Water loss rates were calculated as the percentage of fresh weight of the excised leaves. Data represent
means 6 SD (n = 7) from three independent experiments. [See online article for color version of this figure.]
Plant Physiol. Vol. 157, 2011
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In this report, we identified atairp2 allele Arabidopsis mutants that were less sensitive to ABA treatment
than were wild-type plants at the germination stage.
The AtAIRP2 gene encodes a C3HC4-type RING E3 Ub
ligase (Fig. 1). AtAIRP2 transcript levels were markedly heightened in response to ABA treatment and
dehydration stress (Fig. 2). Consistent with other
RING domain-containing proteins, bacterially expressed AtAIRP2 displayed in vitro E3 Ub ligase
activity and was localized to cytosolic fractions of
onion epidermal cells (Fig. 3). 35S:AtAIRP2-sGFP and
atairp2 loss-of-function mutant plants were tested for a
broad spectrum of ABA responsiveness, including
seed germination, root growth, and stomatal movement. It was found that AtAIRP2 overexpressors and
atairp2 alleles exhibited hypersensitive and hyposensitive phenotypes, respectively, toward ABA treatment
during seed germination (Fig. 4A). Because high salinity (75–125 mM NaCl) exerted a similar opposite
effect on seed germination in AtAIRP2 overexpressors
and atairp2 mutants, AtAIRP2 likely controls early
seedling development by inhibiting seed germination
under unfavorable growth conditions, including high
ABA concentrations and salt stress (Fig. 4B). Such an
inhibitory function during seed germination was recently reported for the Arabidopsis PUB44/SAUL1
U-box E3 Ub ligase (Salt et al., 2011). PUB44/SAUL1
prevents seed germination under stress conditions,
including those involving ABA, Glc, NaCl, and mannitol. In addition, 35S:AtAIRP2-sGFP and atairp2 progeny displayed opposite phenotypes in response to
ABA treatment in all categories examined (Fig. 5).
The initial aim of this study was to illuminate the
functional relationship between ABA and RING E3 Ub
ligases in drought stress responses. 35S:AtAIRP2-sGFP
transgenic plants were highly tolerant to severe
drought stress; in contrast, atairp2 alleles were more
susceptible to mild water stress than were wild-type
plants (Fig. 6C). Higher levels of drought-induced
H2O2 production were detected in AtAIRP2 overexpressors as compared with atairp2 alleles (Fig. 6D).
Furthermore, ABA-induced drought-related gene expression was up-regulated in 35S:AtAIRP2-sGFP and
down-regulated in atairp2 progeny (Fig. 7). The positive effects of AtAIRP2 on the ABA induction of stress
genes were dependent on the protein kinase activity of
SnRKs, a key component in the ABA signaling pathway (Fig. 7). Therefore, it is concluded that AtAIRP2 is
involved in positive regulation of the ABA-dependent
drought stress response in Arabidopsis.
RING E3 Ub ligase isoforms are implicated not only
in normal growth and developmental processes but
also in induced defense mechanisms against biotic and
abiotic environmental stresses (Moon et al., 2004;
Smalle and Vierstra, 2004; Dreher and Callis, 2007;
Vierstra, 2009; Lee and Kim, 2011). However, the
current understanding of the functional relationships
between RING E3s and ABA-mediated drought stress
responses is rudimentary. The Arabidopsis RING E3
Ub ligases XERICO and SDIR1 positively regulate
drought responses by heightening ABA synthesis and
acting upstream of ABA-responsive basic Leu zipper
transcription factors, respectively (Ko et al., 2006;
Zhang et al., 2007). In addition, the RING E3s AtAIRP1
and RHA2b are positive regulators of ABA signaling
and drought responses (Ryu et al., 2010; Li et al., 2011).
Through these studies and our data here, it is becoming increasingly apparent that there is a functional
network(s) between RING E3 Ub ligases and the stress
hormone ABA that helps plants fine-tune their cellular
responses to dehydration stress, one of the most serious environmental stresses crop plants face. Overall,
the results presented in this report implicate the RING
E3 AtAIRP2 as a positive regulator of ABA-mediated
drought stress responses in Arabidopsis.
AtAIRP1 was previously reported to be a C3H2C3type RING E3 Ub ligase that works as a positive
mediator in the Arabidopsis ABA-dependent drought
response (Ryu et al., 2010). The 35S:AtAIRP1-sGFP and
atairp1 lines showed opposite germination and postgermination growth phenotypes in response to ABA
treatment. Therefore, the phenotypic properties of the
35S:AtAIRP1-sGFP and atairp1 progeny were reminiscent of those of the 35S:AtAIRP2-sGFP and atairp2
lines, respectively. In this context, we theorized two
possible modes of action for AtAIRP1 and AtAIRP2.
The first possibility was that AtAIRP1 and AtAIRP2
play coordinate roles in ABA-dependent drought
Cho et al.
2254
masses are quite different (16.9 versus 28.0 kD), with
limited deduced amino acid sequence identity (13%;
Fig. 1). One notable common feature of AtAIRP1 and
AtAIRP2 was that they were localized to cytosolic
fractions (Fig. 3). Nevertheless, AtAIRP1 and AtAIRP2
were able to reciprocally complement loss-of-function
ABA-insensitive mutant phenotypes (Fig. 8). One
could thus postulate that AtAIRP1 and AtAIRP2 share
a common substrate protein(s) that acts negatively during drought stress responses. Ubiquitinated negative
regulators are targeted for 26S proteasome-dependent
proteolysis, conferring tolerance to dehydration stress.
This possibility, however, seems to be somewhat unlikely, because the structural properties of AtAIRP1 and
AtAIRP2 may not be close enough for sharing common
substrates. Alternatively, AtAIRP1 and AtAIRP2 could
ubiquitinate different target proteins that are functionally interconnected. In this scenario, the output of
two distinct ubiquitination pathways by AtAIRP1
and AtAIRP2 could influence each other by an as yet
unknown metabolic mechanism, which in turn would
increase ABA sensitivity and tolerance to water deficit. The results in Figure 8, C to E, indicate that the
degrees of ABA sensitivity and drought tolerance for
both complementation lines (atairp1/35S:AtAIRP2sGFP and atairp2-2/35S:FLAG-AtAIRP1) were not as
high as in overexpressors (35S:AtAIRP2-sGFP) but
rather were approximately the same as the average for wild-type and overexpressing plants. These
results may suggest that AtAIRP1 and AtAIRP2
ubiquitinate different target proteins rather than
share a common substrate protein. Therefore, it is
essential to identify the target proteins of AtAIRP1
and AtAIRP2 to decipher the dynamic mechanism
and combinatory roles of these two cytosolic RING
E3 Ub ligases.
Urbanization and global warming have had causal
effects on the worldwide reduction of freshwater availability for crop plants. Continuously increasing human
and industrial water consumption could pose a future
threat to agricultural crop plants as well as humans
(Hightower and Pierce, 2008; Yoo et al., 2009). Thus, it is
of immense importance to develop drought-tolerant
transgenic crops. In conclusion, the data presented in
this report provide evidence that AtAIRP2 plays integrated roles with AtAIRP1 in ABA-mediated drought
stress responses in Arabidopsis.
MATERIALS AND METHODS
Plant Materials
Arabidopsis (Arabidopsis thaliana ecotype Columbia-0) seeds were soaked
in 30% bleach solution (1.5% sodium hypochlorite and 0.1% Triton X-100) for
10 min and washed 10 times with sterilized water. Young seedlings were
grown in 13 MS medium (Duchefa Biochemie) supplemented with 1% to
3% Suc and 0.8% phytoagar (pH 5.7) or in soil (Sunshine Mix 5; Sun Gro) in
a growth chamber at 22°C with a 16-h-light/8-h-dark cycle. The atairp2-1
(SAIL_686_G08) and atairp2-2 (Salk_005082) T-DNA insertion mutant alleles
were obtained from the Arabidopsis Biological Resource Center (http://
www.arabidopsis.org).
Plant Physiol. Vol. 157, 2011
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stress responses. The second possibility was that they
work in nonoverlapping or parallel pathways. Our
results demonstrated that overexpression of AtAIRP1
and AtAIRP2 reciprocally rescued the loss-of-function
ABA-insensitive phenotypes of atairp2-2 and atairp1,
respectively, in terms of seed germination and water
stress tolerance (Fig. 8). Thus, it is highly likely that
the Arabidopsis RING E3 Ub ligases AtAIRP1 and
AtAIRP2 play combinatory roles in ABA-mediated
drought stress responses. In addition, the Arabidopsis
genome contains two AtAIRP2 homologs. At5g58787
and At3g47160 are 63% and 60% identical to AtAIRP2,
respectively (Fig. 1E; Supplemental Fig. S2). Because
the identities of these two homologs to AtAIRP2 are
significantly higher than that of AtAIRP1, it is possible
that At5g58787 and At3g47160 may also play combinatory roles with AtAIRP2 in ABA and drought stress
responses. This possibility is currently under investigation.
The adaptive mechanisms that plants have developed in response to abiotic stresses are combinatorial and interconnected defensive webs that work
coordinately for concomitant metabolic reprogramming (Ahuja et al., 2010; Hummel et al., 2010; Tardieu
et al., 2011). In this sense, it is plausible that the E3
Ub ligase multigene family also functions in combination to cope with water deficit conditions. For example, two homologous Arabidopsis U-box E3 Ub
ligases, AtPUB22 and AtPUB23, coordinately control
a drought signaling pathway by sharing cytosolic
RPN12a, a non-ATPase subunit of the 26S proteasome
complex, as a substrate (Cho et al., 2008). Ubiquitination of RPN12a may result in a conformational
change of the 26S proteasome complex, which in turn
serves as a negative signal for the drought response.
Similarly, the dehydration-responsive element-binding
protein DREB2A is a common substrate for the homologous nuclear RING E3s DRIP1 and DRIP2. The
DRIPs down-regulated dehydration stress-responsive
gene expression by ubiquitinating and targeting
DREB2A for 26S proteasome proteolysis (Qin et al.,
2008). RHA2a and RHA2b may play redundant, yet
distinguishable, roles in the control of ABA signaling
and drought responses. RHA2a and RHA2b are
plasma membrane and nuclear dual-localized RING
E3s that act downstream of protein phosphatase 2C
and ABI2 and in parallel with ABI3/4/5 (Li et al.,
2011). Thus, it would not be uncommon to consider
that E3 Ub ligase isoforms function in a coordinated
manner to interact with their common target proteins
more effectively. This notion is in agreement with the
fact that plants contain over 1,400 E3 Ub ligases as
compared with the approximately 600 human E3s
(Vierstra, 2009; Liu and Walters, 2010). The combinatory work patterns of plant E3s may increase the
efficiency of reprogramming metabolic responses to
environmental stresses.
On the other hand, AtAIRP1 belongs to a different
RING subfamily than does AtAIRP2 (C3H2C3 type
versus C3HC4 type), and their deduced molecular
Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
Sequence Analysis
AtAIRP2 and its homologous proteins were identified with the WU-BLST
program (http://arabidopsis.org/wublast/index2.jsp). Selected homolog protein sequences were analyzed with MEGA5 software (Tamura et al., 2007).
Multiple sequence alignments were edited using the GeneDoc program (http://
www.nrbsc.org/gfx/genedoc/). Phylogenetic trees were generated with MEGA5
software (Ryu et al., 2010).
RT-PCR and Real-Time qRT-PCR Analyses
restriction enzyme and inserted into modified pENTR vectors (Invitrogen).
AtAIRP2-sGFP and FLAG-AtAIRP1 clones were subsequently integrated into
pEarlygate 100 destination vectors using LR Clonase II (Invitrogen) and
transformed into wild-type, atairp1, or atairp2-2 plants using an Agrobacteriummediated floral dip method (Joo et al., 2006). Transformed seeds were selected
on MS plates containing 25 mg mL21 BASTA. Expression of each transgene
was examined by genomic Southern-blot, RT-PCR, and immunoblot analyses
as described by Lee et al. (2009). Homozygous T3 lines were selected through
self-crossing and were subsequently used in phenotypic analyses.
Seed Germination Assay
Seed germination assays were performed with greater than 36 seeds and
repeated three times. Seeds, 3 d after imbibition, from wild-type, atairp1, atairp2-1,
atairp2-2, 35S:AtAIRP2-sGFP, atairp2-2/35S:FLAG-AtAIRP1, and atairp1/35S:
AtAIRP2-sGFP plants were grown on 13 MS medium supplemented with
different concentrations (0, 0.2, 0.4, or 0.8 mM) of ABA (Sigma-Aldrich) at 22°C
with a 16-h-light/8-h-dark photoperiod. The rates of radicle emergence and
cotyledon greening were measured after 3 and 7 d, respectively.
Histochemical GUS Assay
Root Growth and Stomatal Aperture Measurements
Arabidopsis genomic DNA was amplified using the AtAIPR2 pro-GUS FW
and pro-GUS RV primer set (Supplemental Table S1). PCR products were
inserted into a pCAMBIA1381 vector. The AtAIRP2 promoter-GUS construct was transformed into wild-type Arabidopsis using the Agrobacterium
tumefaciens-mediated floral dip method as described by Joo et al. (2006). For
the histochemical GUS assay, transgenic plant tissues were immersed in a
GUS staining solution containing 2 mM X-GlcA (cyclohexylammonium salt;
Duchefa Biochemie), 0.5 mM K3Fe(CN)6, and 0.5 mM K4Fe(CN)6 in 50 mM
sodium phosphate buffer (pH 7.2) and incubated for 12 h at 37°C (Joo et al.,
2004). To remove chlorophyll after GUS staining, GUS-stained tissues were
incubated in 70% ethanol for several hours.
To measure seedling root growth, seeds were vertically grown for 10 d on
13 MS medium containing 0.2 to 0.8 mM ABA, and root elongation was
monitored and analyzed using Scion Image software (www.scioncorp.com).
Mature rosette leaves from light-grown 4-week-old wild-type, atairp2-1,
atairp2-2, and 35S:AtAIRP2-sGFP plants were detached and incubated in a
stomatal opening solution (10 mM KCl, 100 mM CaCl2, and 10 mM MES, pH 6.1)
for 2 h at 22°C (Kwak et al., 2003). Treated leaves were transferred to a stomatal
opening solution containing ABA (0, 0.1, 1, or 10 mM) for 2 h. Epidermal strips
were observed using a light microscope (Olympus BX51). Stomatal aperture
was measured using Multigauge version 3.1 software (Fujifilm) as described
by Ryu et al. (2010).
In Vitro Self-Ubiquitination Assay
Drought Phenotype Analysis
The full-length coding region of AtAIRP2 cDNA was amplified with the
XbaI-FW and PstI-RV primer set (Supplemental Table S1). PCR products were
restricted by XbaI and PstI and inserted into pMAL C2 vectors (New England
Biolabs). The single amino acid substitution derivative (AtAIRP2H163A) of
wild-type AtAIRP2 was generated using the QuikChange site-directed mutagenesis kit (Stratagene) and the H163A-FW and H163A-RV primer set. MBPAtAIRP2 and MBP-AtAIRP2H163A fusion proteins (500 ng) were expressed in
Escherichia coli strain BL21 and purified using amylose resin (New England
Biolabs). In vitro self-ubiquitination assays were conducted as described
previously (Cho et al., 2006a). Immunoblot analyses were carried out with
anti-MBP antibody (New England Biolabs) or anti-Ub antibody (Santa Cruz
Biotechnology) according to Ryu et al. (2009). All primers used in this study
are provided in Supplemental Table S1.
Wild-type, atairp2-1, atairp2-2, 35S:AtAIRP2-sGFP, atairp2-2/35S:FLAGAtAIRP1, and atairp1/35S:AtAIRP2-sGFP plants were grown for 2 weeks
under normal growth conditions and then subjected to dehydration stress by
ceasing irrigation for 12 to 15 d (Cho et al., 2006b). Three days after rewatering,
surviving plants were counted as described previously (Kim et al., 2010a). Cut
rosette water loss experiments were performed according to the method
described by Ryu et al. (2010). For DAB staining, light-grown 2-week-old plants
were treated with drought stress for 10 d, and rosette leaves were incubated with
100 mg mL21 DAB solution as described previously (Ryu et al., 2010).
Subcellular Localization
The 35S:sGFP, 35S:AtAIRP2-sGFP, 35S:AtAREB1-sGFP, and 35S:AtAIRP1sGFP plasmids were inserted into pBI221 transient expression vectors. The
fusion constructs were introduced into onion (Allium cepa) epidermal cells by
means of the particle bombardment method described by Lee and Kim (2003).
Transiently expressed GFP signals were detected using a fluorescence microscope (BX51; Olympus). Images were acquired with a 1600 CCD camera (PCO)
and analyzed using Image Pro Plus software (Media Cybernetics). AREB1-sGFP
and AtAIRP1-sGFP were used as controls for nuclear and cytosolic localization,
respectively. All primers used are listed in Supplemental Table S1.
Construction of 35S:AtAIRP2-sGFP, atairp2-2/35S:
FLAG-AtAIRP1, and atairp1/35S:AtAIRP2-sGFP
Transgenic Plants
Full-length AtAIRP2 and AtAIRP1 cDNAs were PCR amplified using
AtAIRP2-specific primers (AtAIRP2 SacI-FW and AtAIRP2 SacI-RV) and
AtAIRP1-specific primers (AtAIRP1 EcoRI-FW and AtAIRP1 XbaI-RV), respectively (Supplemental Table S1). PCR products were digested with each
Plant Physiol. Vol. 157, 2011
Sequence data used in this report are found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession numbers:
AtAIRP2 (At5g01520), AtAIRP1 (At4g23450), AtAREB1 (At1g45249), two Arabidopsis homologs (At5g58787 and At3g47160), Oryza sativa (NP_001060539),
Populus trichocarpa (XP_002309135), Vitis vinifera (XP_002280008), and Sorghum
bicolor (XP_002447334).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Identification of atairp2 T-DNA insertion loss-offunction mutant alleles.
Supplemental Figure S2. Amino acid sequence comparison of seven
AtAIRP2 homologs.
Supplemental Figure S3. Genomic Southern blot analysis of wild-type and
T3 35S:AtAIRP2-sGFP, atairp2-2/35S:FLAG-AtAIRP1, and atairp1/35S:
AtAIRP2-GFP transgenic Arabidopsis plants.
Supplemental Figure S4. Germination rates of wild-type, atairp2, and 35S:
AtAIRP2-sGFP plants in response to NaCl.
Supplemental Table S1. PCR primer sequences used for this article.
Received August 16, 2011; accepted September 29, 2011; published October 3,
2011.
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Total RNA was isolated from abiotic stress- and ABA-treated 10-d-old
seedlings using an RNA extraction kit (Intron Biotechnology) according to the
manufacturer’s protocol. cDNA synthesis and RT-PCR were performed as
described previously (Kim et al., 2010a). qRT-PCR was carried out using an
IQ5 light cycler (Bio-Rad) with SYBR Premix Ex Taq II (Takara). qRT-PCR data
were analyzed with Genex_Macro_IQ5_conversion_Template and Genex
software (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase C subunit
mRNA level was used as an internal control for qRT-PCR data normalization.
Cho et al.
LITERATURE CITED
2256
Plant Physiol. Vol. 157, 2011
Downloaded from https://academic.oup.com/plphys/article/157/4/2240/6109270 by guest on 05 June 2022
Ahuja I, de Vos RCH, Bones AM, Hall RD (2010) Plant molecular stress
responses face climate change. Trends Plant Sci 15: 664–674
Bae H, Kim SK, Cho SK, Kang BG, Kim WT (2011) Overexpression of
OsRDCP1, a rice RING domain-containing E3 ubiquitin ligase, increased tolerance to drought stress in rice (Oryza sativa L.). Plant Sci
180: 775–782
Bu Q, Li H, Zhao Q, Jiang H, Zhai Q, Zhang J, Wu X, Sun J, Xie Q, Wang D,
et al (2009) The Arabidopsis RING finger E3 ligase RHA2a is a novel
positive regulator of abscisic acid signaling during seed germination
and early seedling development. Plant Physiol 150: 463–481
Cho DS, Shin DJ, Jeon BW, Kwak JM (2009) ROS-mediated ABA signaling.
J Plant Biol 52: 102–113
Cho SK, Chung HS, Ryu MY, Park MJ, Lee MM, Bahk Y-Y, Kim J, Pai HS,
Kim WT (2006a) Heterologous expression and molecular and cellular
characterization of CaPUB1 encoding a hot pepper U-box E3 ubiquitin
ligase homolog. Plant Physiol 142: 1664–1682
Cho SK, Kim JE, Park J-A, Eom TJ, Kim WT (2006b) Constitutive expression of abiotic stress-inducible hot pepper CaXTH3, which encodes a
xyloglucan endotransglucosylase/hydrolase homolog, improves drought
and salt tolerance in transgenic Arabidopsis plants. FEBS Lett 580:
3136–3144
Cho SK, Ryu MY, Song C, Kwak JM, Kim WT (2008) Arabidopsis PUB22
and PUB23 are homologous U-box E3 ubiquitin ligases that play
combinatory roles in response to drought stress. Plant Cell 20: 1899–1914
Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR (2010) Abscisic acid:
emergence of a core signaling network. Annu Rev Plant Biol 61: 651–679
Dreher K, Callis J (2007) Ubiquitin, hormones and biotic stress in plants.
Ann Bot (Lond) 99: 787–822
Dye BT, Schulman BA (2007) Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu Rev Biophys
Biomol Struct 36: 131–150
Finkelstein RR, Gampala SS, Rock CD (2002) Abscisic acid signaling in
seeds and seedlings. Plant Cell (Suppl) 14: S15–S45
Fujii H, Verslues PE, Zhu J-K (2011) Arabidopsis decuple mutant reveals the
importance of SnRK2 kinases in osmotic stress responses in vivo. Proc
Natl Acad Sci USA 108: 1717–1722
Fujii H, Zhu J-K (2009) Arabidopsis mutant deficient in 3 abscisic acidactivated protein kinases reveals critical roles in growth, reproduction,
and stress. Proc Natl Acad Sci USA 106: 8380–8385
Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, YamaguchiShinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic
stress responses: a current view from the points of convergence in the
stress signaling networks. Curr Opin Plant Biol 9: 436–442
Fujita Y, Nakashima K, Yoshida T, Katagiri T, Kidokoro S, Kanamori N,
Umezawa T, Fujita M, Maruyama K, Ishiyama K, et al (2009) Three
SnRK2 protein kinases are the main positive regulators of abscisic acid
signaling in response to water stress in Arabidopsis. Plant Cell Physiol
50: 2123–2132
Glickman MH, Adir N (2004) The proteasome and the delicate balance
between destruction and rescue. PLoS Biol 2: E13
Gómez-Porras JL, Riaño-Pachón DM, Dreyer I, Mayer JE, MuellerRoeber B (2007) Genome-wide analysis of ABA-responsive elements
ABRE and CE3 reveals divergent patterns in Arabidopsis and rice. BMC
Genomics 8: 260
Hightower M, Pierce SA (2008) The energy challenge. Nature 452: 285–286
Hirayama T, Shinozaki K (2010) Research on plant abiotic stress responses
in the post-genome era: past, present and future. Plant J 61: 1041–1052
Huang Y, Li CY, Pattison DL, Gray WM, Park S, Gibson SI (2010) SUGARINSENSITIVE3, a RING E3 ligase, is a new player in plant sugar
response. Plant Physiol 152: 1889–1900
Hubbard KE, Nishimura N, Hitomi K, Getzoff ED, Schroeder JI (2010)
Early abscisic acid signal transduction mechanisms: newly discovered
components and newly emerging questions. Genes Dev 24: 1695–1708
Hummel I, Pantin F, Sulpice R, Piques M, Rolland G, Dauzat M,
Christophe A, Pervent M, Bouteillé M, Stitt M, et al (2010) Arabidopsis
plants acclimate to water deficit at low cost through changes of carbon
usage: an integrated perspective using growth, metabolite, enzyme, and
gene expression analysis. Plant Physiol 154: 357–372
Hunter T (2007) The age of crosstalk: phosphorylation, ubiquitination, and
beyond. Mol Cell 28: 730–738
Jacobson AD, Zhang NY, Xu P, Han KJ, Noone S, Peng J, Liu CW (2009)
The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 S proteasome. J Biol Chem 284: 35485–35494
Jammes F, Song C, Shin D, Munemasa S, Takeda K, Gu D, Cho D, Lee S,
Giordo R, Sritubtim S, et al (2009) MAP kinases MPK9 and MPK12 are
preferentially expressed in guard cells and positively regulate ROSmediated ABA signaling. Proc Natl Acad Sci USA 106: 20520–20525
Joo S, Park KY, Kim WT (2004) Light differentially regulates the expression
of two members of the auxin-induced 1-aminocyclopropane-1-carboxylate
synthase gene family in mung bean (Vigna radiata L.) seedlings. Planta 218:
976–988
Joo S, Seo YS, Kim SM, Hong DK, Park KY, Kim WT (2006) Brassinosteroidinduction of AtACS4 encoding an auxin-responsive 1-aminocyclopropane1-carboxylate synthase 4 in Arabidopsis seedlings. Physiol Plant 126:
592–604
Kim EY, Seo YS, Lee H, Kim WT (2010a) Constitutive expression of
CaSRP1, a hot pepper small rubber particle protein homolog, resulted in
fast growth and improved drought tolerance in transgenic Arabidopsis
plants. Planta 232: 71–83
Kim T-H, Böhmer M, Hu H, Nishimura N, Schroeder JI (2010b) Guard cell
signal transduction network: advances in understanding abscisic acid,
CO2, and Ca2+ signaling. Annu Rev Plant Biol 61: 561–591
Ko JH, Yang SH, Han KH (2006) Upregulation of an Arabidopsis RING-H2
gene, XERICO, confers drought tolerance through increased abscisic
acid biosynthesis. Plant J 47: 343–355
Kraft E, Stone SL, Ma L, Su N, Gao Y, Lau OS, Deng XW, Callis J (2005)
Genome analysis and functional characterization of the E2 and RINGtype E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol 139:
1597–1611
Kwak JM, Mori IC, Pei Z-M, Leonhardt N, Torres MA, Dangl JL, Bloom
RE, Bodde S, Jones JDG, Schroeder JI (2003) NADPH oxidase AtrbohD
and AtrbohF genes function in ROS-dependent ABA signaling in
Arabidopsis. EMBO J 22: 2623–2633
Lee HK, Cho SK, Son O, Xu Z, Hwang IH, Kim WT (2009) Drought stressinduced Rma1H1, a RING membrane-anchor E3 ubiquitin ligase homolog, regulates aquaporin levels via ubiquitination in transgenic Arabidopsis
plants. Plant Cell 21: 622–641
Lee J-H, Kim WT (2003) Molecular and biochemical characterization of VREILs encoding mung bean ETHYLENE INSENSITIVE3-LIKE proteins.
Plant Physiol 132: 1475–1488
Lee JH, Kim WT (2011) Regulation of abiotic stress signal transduction by
E3 ubiquitin ligases in Arabidopsis. Mol Cells 31: 201–208
Lee SJ, Kang JY, Park HJ, Kim MD, Bae MS, Choi HI, Kim SY (2010)
DREB2C interacts with ABF2, a bZIP protein regulating abscisic acidresponsive gene expression, and its overexpression affects abscisic acid
sensitivity. Plant Physiol 153: 716–727
Li H, Jiang H, Bu Q, Zhao Q, Sun J, Xie Q, Li C (2011) The Arabidopsis
RING finger E3 ligase RHA2b acts additively with RHA2a in regulating
abscisic acid signaling and drought response. Plant Physiol 156: 550–563
Liu F, Walters KJ (2010) Multitasking with ubiquitin through multivalent
interactions. Trends Biochem Sci 35: 352–360
Liu H, Stone SL (2010) Abscisic acid increases Arabidopsis ABI5 transcription factor levels by promoting KEG E3 ligase self-ubiquitination and
proteasomal degradation. Plant Cell 22: 2630–2641
Liu H, Zhang H, Yang Y, Li G, Yang Y, Wang X, Basnayake BM, Li D, Song
F (2008) Functional analysis reveals pleiotropic effects of rice RING-H2
finger protein gene OsBIRF1 on regulation of growth and defense
responses against abiotic and biotic stresses. Plant Mol Biol 68: 17–30
Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E
(2009) Regulators of PP2C phosphatase activity function as abscisic acid
sensors. Science 324: 1064–1068
Moon J, Parry G, Estelle M (2004) The ubiquitin-proteasome pathway and
plant development. Plant Cell 16: 3181–3195
Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions
of ubiquitin in endocytosis and signaling. Science 315: 201–205
Mustilli A-C, Merlot S, Vavasseur A, Fenzi F, Giraudat J (2002) Arabidopsis
OST1 protein kinase mediates the regulation of stomatal aperture by
abscisic acid and acts upstream of reactive oxygen species production.
Plant Cell 14: 3089–3099
Ning Y, Jantasuriyarat C, Zhao Q, Zhang H, Chen S, Liu J, Liu L, Tang S,
Park CH, Wang X, et al (2011) The SINA E3 ligase OsDIS1 negatively
regulates drought response in rice. Plant Physiol 157: 242–255
Park GG, Park JJ, Yoon J, Yu SN, An G (2010) A RING finger E3 ligase gene,
Combinatory Roles of AtAIRP1 and AtAIRP2 RING E3s
Plant Physiol. Vol. 157, 2011
tionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol
24: 1596–1599
Tardieu F, Granier C, Muller B (2011) Water deficit and growth: coordinating processes without an orchestrator? Curr Opin Plant Biol 14:
283–289
Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular
localization of H2O2 in plants: H2O2 accumulation in papillae and
hypersensitive response during barley-powdery mildew interaction.
Plant J 11: 1187–1194
Tuteja N (2007) Abscisic acid and abiotic stress signaling. Plant Signal
Behav 2: 135–138
Vierstra RD (2009) The ubiquitin-26S proteasome system at the nexus of
plant biology. Nat Rev Mol Cell Biol 10: 385–397
Wang P, Song CP (2008) Guard-cell signalling for hydrogen peroxide and
abscisic acid. New Phytol 178: 703–718
Xie Q, Guo HS, Dallman G, Fang S, Weissman AM, Chua NH (2002)
SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate
auxin signals. Nature 419: 167–170
Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold,
drought, and salt stress. Plant Cell (Suppl) 14: S165–S183
Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory
networks in cellular responses and tolerance to dehydration and cold
stresses. Annu Rev Plant Biol 57: 781–803
Yoo CY, Pence HE, Hasegawa PM, Mickelbart MV (2009) Regulation of
transpiration to improve crop water use. Crit Rev Plant Sci 28: 410–431
Yoshida T, Fujita Y, Sayama H, Kidokoro S, Maruyama K, Mizoi J,
Shinozaki K, Yamaguchi-Shinozaki K (2010) AREB1, AREB2, and
ABF3 are master transcription factors that cooperatively regulate
ABRE-dependent ABA signaling involved in drought stress tolerance
and require ABA for full activation. Plant J 61: 672–685
Zhang X, Garreton V, Chua NH (2005) The AIP2 E3 ligase acts as a novel
negative regulator of ABA signaling by promoting ABI3 degradation.
Genes Dev 19: 1532–1543
Zhang Y, Yang C, Li Y, Zheng N, Chen H, Zhao Q, Gao T, Guo H, Xie Q
(2007) SDIR1 is a RING finger E3 ligase that positively regulates
stress-responsive abscisic acid signaling in Arabidopsis. Plant Cell 19:
1912–1929
Zhang YY, Li Y, Gao T, Zhu H, Wang DJ, Zhang HW, Ning YS, Liu LJ, Wu
YR, Chu CC, et al (2008) Arabidopsis SDIR1 enhances drought tolerance
in crop plants. Biosci Biotechnol Biochem 72: 2251–2254
2257
Downloaded from https://academic.oup.com/plphys/article/157/4/2240/6109270 by guest on 05 June 2022
Oryza sativa Delayed Seed Germination 1 (OsDSG1), controls seed
germination and stress responses in rice. Plant Mol Biol 74: 467–478
Peng M, Hannam C, Gu H, Bi YM, Rothstein SJ (2007) A mutation in NLA,
which encodes a RING-type ubiquitin ligase, disrupts the adaptability
of Arabidopsis to nitrogen limitation. Plant J 50: 320–337
Qin F, Sakuma Y, Tran LS, Maruyama K, Kidokoro S, Fujita Y, Fujita M,
Umezawa T, Sawano Y, Miyazono K, et al (2008) Arabidopsis DREB2Ainteracting proteins function as RING E3 ligases and negatively regulate plant drought stress-responsive gene expression. Plant Cell 20:
1693–1707
Quiroz-Figueroa F, Rodrı́guez-Acosta A, Salazar-Blas A, HernándezDomı́nguez E, Campos ME, Kitahata N, Asami T, Galaz-Avalos RM,
Cassab GI (2010) Accumulation of high levels of ABA regulates the
pleiotropic response of the nhr1 Arabidopsis mutant. J Plant Biol 53: 32–44
Raghavendra AS, Gonugunta VK, Christmann A, Grill E (2010) ABA
perception and signalling. Trends Plant Sci 15: 395–401
Ryu MY, Cho SK, Kim WT (2009) RNAi suppression of RPN12a decreases
the expression of type-A ARRs, negative regulators of cytokinin signaling pathway, in Arabidopsis. Mol Cells 28: 375–382
Ryu MY, Cho SK, Kim WT (2010) The Arabidopsis C3H2C3-type RING E3
ubiquitin ligase AtAIRP1 is a positive regulator of an abscisic aciddependent response to drought stress. Plant Physiol 154: 1983–1997
Salt JN, Yoshioka K, Moeder W, Goring DR (2011) Altered germination
and subcellular localization patterns for PUB44/SAUL1 in response to
stress and phytohormone treatments. PLoS ONE 6: e21321
Santiago J, Rodrigues A, Saez A, Rubio S, Antoni R, Dupeux F, Park S-Y,
Márquez JA, Cutler SR, Rodriguez PL (2009) Modulation of drought
resistance by the abscisic acid receptor PYL5 through inhibition of clade
A PP2Cs. Plant J 60: 575–588
Smalle J, Vierstra RD (2004) The ubiquitin 26S proteasome proteolytic
pathway. Annu Rev Plant Biol 55: 555–590
Song X-J, Matsuoka M (2009) Bar the windows: an optimized strategy to
survive drought and salt adversities. Genes Dev 23: 1709–1713
Stone SL, Hauksdóttir H, Troy A, Herschleb J, Kraft E, Callis J (2005)
Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol 137: 13–30
Stone SL, Williams LA, Farmer LM, Vierstra RD, Callis J (2006) KEEP ON
GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling. Plant Cell 18: 3415–3428
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolu-