Transcriptional Regulation of Arabidopsis MIR168a and
ARGONAUTE1 Homeostasis in Abscisic Acid and Abiotic
Stress Responses1[W]
Wei Li, Xiao Cui, Zhaolu Meng, Xiahe Huang, Qi Xie, Heng Wu, Hailing Jin,
Dabing Zhang, and Wanqi Liang*
School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China (W.L., X.C.,
Z.M., H.W., D.Z., W.L.); State Key Laboratory of Plant Genomics, National Center for Plant Gene Research,
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (X.H.,
Q.X.); and Department of Plant Pathology and Microbiology, University of California, Riverside, Riverside,
California 92521 (H.J.)
The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the
underlying mechanisms are poorly understood. The miR168-mediated feedback regulatory loop regulates ARGONAUTE1
(AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here, we reveal a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses
in Arabidopsis (Arabidopsis thaliana). Plants overexpressing MIR168a and the AGO1 loss-of-function mutant ago1-27 display
ABA hypersensitivity and drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no
obvious decrease for the target of miR168, AGO1, was shown under the same conditions. However, promoter activity analysis
indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional
elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we
showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four abscisic acid-responsive
element (ABRE) binding factors, which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also
found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of
miR168 and posttranscriptional control of AGO1 homeostasis may play an important and conserved role in stress response and
signal transduction in plants.
As sessile organisms, plants have evolved sophisticated mechanisms to circumvent the adverse environment. The phytohormone abscisic acid (ABA) is a
key inducer of plant responses to abiotic stress. Much
effort has been made to decipher the mechanism underlying ABA signal transduction, in which posttranscriptional regulation has been shown to be one of the
most important modes of regulation (Chinnusamy
et al., 2008). Mutants of several components of the
microRNA (miRNA) biogenesis machinery, such as
hyponastic leaves1, serrate, dicer-like1, hua enhancer1
(hen1), and cap-binding protein (cbp20 and cbp80), are
hypersensitive to ABA (Lu and Fedoroff, 2000;
Hugouvieux et al., 2001; Kim et al., 2008; Zhang et al.,
2008). These mutants, except hen1, were shown to have
1
This work was supported by the National Natural Science
Foundation of China (grant no. 30600347).
* Corresponding author; e-mail [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.plantphysiol.org) is:
Wanqi Liang ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.188789
lower miRNA levels but higher primary miRNA precursor (pri-miRNA) levels compared with wild-type
plants (Laubinger et al., 2008). In cbp20 and cbp80
mutants, ABA induction of miR159 was delayed and
the miR159 target transcripts, which encode two positive regulators of ABA response, MYB DOMAIN
PROTEIN33 (MYB33) and MYB101, accumulated to a
higher level (Reyes and Chua, 2007; Kim et al., 2008).
ARGONAUTE (AGO) proteins, which contain the
well-characterized Piwi-Argonuaute-Zwille (PAZ)
and P element-Induced WImpy testis (PIWI) domains,
are considered to be integral players in all known
small RNA-targeted regulatory pathways (Vaucheret,
2008). Among the AGO proteins in Arabidopsis (Arabidopsis thaliana), AGO1 is a core component of the
RNA-induced silencing complex, which associates
with miRNAs and inhibits target genes by mRNA
cleavage and/or translational repression (Vaucheret
et al., 2004; Vaucheret, 2008; Voinnet, 2009). Mutations
in AGO1 cause increased accumulation of miRNA
targets (Vaucheret et al., 2004; Ronemus et al., 2006;
Kurihara et al., 2009). A hypomorphic allele, ago1-27
exhibits hypersusceptibility to Cucumber mosaic virus
(Morel et al., 2002), and AGO1 was proved to be a
major determinant for virus-induced gene silencing
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Li et al.
(Zhang et al., 2006). ago1-27 displays resistance to the
infection of a fungal pathogen, Verticillium dahliae, supporting the view that AGO1 is involved in the regulation
of pathogen defense responses (Ellendorff et al., 2009).
Although several AGO proteins have been shown to
regulate biotic stresses (Katiyar-Agarwal et al., 2007; Li
et al., 2010; Zhang et al., 2011), the role of AGO proteins
in regulating abiotic stress responses is still not well
understood. A recent report showed that a decrease in
AGO1 confers hypersensitivity to ABA, whereas an increase in the level of AGO1 leads to ABA hyposensitivity (Earley et al., 2010). However, the role of AGO1 in
abiotic stress response pathways is still unclear.
It is well known that miRNAs play crucial roles in
controlling a variety of developmental processes, such
as organ identity establishment, organ separation, hormone signaling, and flowering time control (Chen,
2009). Recent evidence also uncovered the role of small
RNA molecules in regulating plant stress responses
(Sunkar, 2010; Khraiwesh et al., 2012). Microarray
analysis and deep-sequencing data demonstrated that
the accumulation of a number of miRNAs is affected by
abiotic stresses (Sunkar and Zhu, 2004; Reyes and Chua,
2007; Zhou et al., 2007, 2008; Liu et al., 2008; Jia et al.,
2009), implying a potential role of miRNAs in abiotic
stress responses. In Arabidopsis, the expression of
MIR395, MIR398, and MIR399 is induced by sulfate,
copper, and phosphate deprivation, respectively, and
their subsequent down-regulation of target genes is
essential for nutrient reallocation (Fujii et al., 2005;
Jagadeeswaran et al., 2009; Kawashima et al., 2009). Upregulation of miR159 and miR160 by ABA and the subsequent degradation of their target transcripts have
been reported to be important for Arabidopsis seed
germination and seedling development (Liu et al., 2007;
Reyes and Chua, 2007). Down-regulation of miR169 by
ABA and drought leads to the suppression of its cleavage
of NUCLEAR FACTOR Y A5 (NFYA5), the positive regulator of drought resistance (Li et al., 2008). In addition,
natural cis-antisense small interfering RNAs (siRNAs)
and transacting siRNAs also have been reported as important players in adaptation to a variety of abiotic stresses (Borsani et al., 2005; Moldovan et al., 2010). However,
the roles and regulatory mechanisms of most of the plant
small RNAs in abiotic stresses remain largely unknown.
MIR168 is one of the most commonly detected
stress-inducible MIR genes. Homologs of MIR168 exist
in various plant species, including dicots such as
poplar (Populus trichocarpa), tobacco (Nicotiana tabacum), and Arabidopsis and monocots such as maize
(Zea mays) and rice (Oryza sativa). These homologs
have been found to also respond to salt, drought, and
cold stresses or ABA treatment (Liu et al., 2008; Ding
et al., 2009; Jia et al., 2009, 2010; Zhou et al., 2010). A
newly identified MIR168a mutant, mir168a-2, exhibits
developmental defects when exposed to temperature,
light, and water fluctuations (Vaucheret, 2009). These
findings led to the speculation that miR168 and its
exclusive target AGO1 may play a critical role in the
stress regulatory network.
In this paper, we reveal the important role of MIR168a
and its target AGO1 in plant responses to ABA and
several abiotic stresses. Plants either overexpressing
MIR168a or carrying a mutation in AGO1 (ago1-27) display ABA hypersensitivity and drought tolerance, while
the mir168a-2 mutant shows ABA hyposensitivity and
drought hypersensitivity. Consistent with this, MIR168a
is transcriptionally up-regulated by ABA and various
stresses, resulting in posttranscriptional control of AGO1
homeostasis. Furthermore, we demonstrate that MIR168a
is activated by abscisic acid-responsive element (ABRE)binding transcription factors ABF1, ABF2, ABF3, and
ABF4. A typical ABRE motif within the MIR168a promoter, which can be bound by the four ABRE-binding
transcription factors, is conserved in the MIR168 homologs in other plant species. These results imply a
common and conserved mechanism of MIR168 transcriptional control in plant stress responses.
RESULTS
Transcriptional Regulation of MIR168a and AGO1
under ABA and Abiotic Stresses
Recent investigations using microarray followed by
reverse transcription (RT)-PCR analyses revealed increased accumulation of miR168 transcripts under
high salinity, drought, and cold conditions in Arabidopsis (Liu et al., 2008). To investigate how the level of
miR168 is induced, we analyzed the abundance of
precursor and mature miR168 under stress conditions.
Given that MIR168a is ubiquitously expressed and
coexpressed with AGO1 while MIR168b expression is
restricted to the shoot apical meristem (Jiang et al.,
2006; Gazzani et al., 2009; Vaucheret, 2009), we focused
on MIR168a in this study.
Northern-blot analysis was performed to measure
the abundance of pre-miR168a transcripts and mature
miR168 in 2-week-old wild-type Arabidopsis seedlings treated by drought, 300 mM NaCl, and cold for
0, 6, and 12 h. The levels of mature miR168 and premiR168a were increased under drought, salt, and cold
treatment (Fig. 1A). ABA is an important signal in
abiotic stress responses. Thus, we investigated the
effect of 100 mM ABA on the accumulation of miR168
and pre-miR168a in 2-week-old wild-type Arabidopsis
seedlings at 0, 2, 4, 6, and 12 h. The response to ABA
and stress treatments was confirmed by the increased
expression of RESPONSIVE TO DESICCATION29A
(RD29A; Supplemental Fig. S1), a gene known to be
induced under such conditions (Narusaka et al., 2003).
Northern-blot analysis showed the elevation of miR168
and pre-miR168a levels 2 h after treatment with 100 mM
ABA (Fig. 1A). Furthermore, drought-induced accumulation of miR168 was substantially reduced in the ABAdeficient mutant aba1-5 (Léon-Kloosterziel et al., 1996;
Fig. 1C), suggesting that the enhanced accumulation
of miR168 by abiotic stress is at least partially ABA
dependent.
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Transcriptional Regulation of MIR168a
Figure 1. Expression analysis of mature miR168, pre-miR168a, AGO1, and GUS in wild-type, aba1-5, and pAGO1:GUS
transgenic plants under ABA and abiotic stress treatments. A and B, Northern-blot analysis of mature miR168 and pre-miR168a
(A) and qRT-PCR analysis of AGO1 mRNA (B) in 2-week-old wild-type Arabidopsis seedlings (Col) with drought, 300 mM NaCl,
cold, and 100 mM ABA treatments. The significant induction of AGO1 mRNA by a 12-h drought treatment is indicated with the
asterisk (n = 3, P , 0.05, Student’s t test). nt, Nucleotides. C, Northern-blot analysis of miR168 and qRT-PCR analysis of AGO1
mRNA in 2-week-old wild-type and aba1-5 mutant plants (n = 10) under standard and drought conditions. Numbers below the
blots in A and C show the relative abundance compared with the control U6; the level of each target in the nontreated wild type
(Col) was set to 1. These experiments were repeated three times, and similar results were observed. D, qRT-PCR analysis of GUS
and AGO1 mRNA in three independent 2-week-old pAGO1:GUS transgenic lines with drought treatment for 0, 6, and 12 h. E,
qRT-PCR analysis of GUS and AGO1 mRNA in three independent 2-week-old pAGO1:GUS transgenic lines with the application
of 100 mM ABA over time. The significant difference between GUS and AGO1 is indicated with asterisks (n = 3, P , 0.05,
Student’s t test). Error bars denote SD. Quantifications were normalized to the expression of TUBULIN4. The level of AGO1 and
GUS in the wild type (Col) or nontreated sample was set to 1.
miR168 is critical for modulating the level of AGO1
by miR168-targeted cleavage of the AGO1 mRNA and
translational repression of AGO1 (Vaucheret et al.,
2006; Vaucheret, 2009; Várallyay et al., 2010). To determine whether ABA and stress treatments affect AGO1
accumulation, we performed quantitative (q) RT-PCR
analyses. Unexpectedly, AGO1’s mRNA level was
weakly increased by ABA or other stresses and showed
about 2-fold increase at 12 h of drought treatment (Fig.
1B). In order to clarify the role of miR168 in ABA and
stress responses, we compared the expression levels of
endogenous AGO1 and exogenous GUS in pAGO1:GUS
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Li et al.
transgenic plants with ABA or drought treatment. The
data showed that the increase of GUS transcript free
from miR168 regulation was significantly higher as
compared with the increase of endogenous AGO1 transcript after ABA and drought treatments (Fig. 1, D and
E). These results suggest that AGO1 is induced by ABA
and drought at the transcriptional level and that miR168mediated repression occurs at the posttranscriptional
level. Both regulatory mechanisms modulate the expression of AGO1 in response to ABA and drought stress.
The ago1-27 Mutant and MIR168a Overexpression Plants
Display ABA and Salt Hypersensitivity
Although the accumulation of miR168 is affected by
ABA treatment and several abiotic stresses, the function of miR168 and its exclusive target AGO1 in plant
stress responses is still unclear. To assess the role of
miR168 and AGO1 in stress responses, we examined
MIR168a overexpression lines (Jiang et al., 2006) and
the AGO1 hypomorphic mutant allele ago1-27 (Morel
et al., 2002) under ABA and abiotic stress treatments.
The levels of mature miR168 in two 35S:MIR168a transgenic lines (lines 4 and 6) were more than 10-fold
higher than that in the wild type, while the AGO1
transcript levels decreased to 35% and 28%, respec-
tively, of the wild-type levels (Supplemental Fig. S2).
The overexpression lines 35S:MIR168a#4 and 35S:
MIR168a#6 displayed some developmental defects,
including shorter roots, smaller plant stature, and
delayed flowering.
We first examined the effects of ABA treatment on
seed germination and seedling growth in the MIR168a
overexpression lines and the ago1-27 mutant. Under
standard growth conditions, the germination of 35S:
MIR168a lines was delayed by about 2 h compared
with wild-type seeds, and a 4-h delay in germination
was observed for ago1-27 seeds (Fig. 2A). We next
determined their ABA response during and after seed
germination. Treatment with 0.3 mM ABA caused a
slight inhibition of seed germination and seedling
growth of the wild type, while the growth of 35S:
MIR168a and ago1-27 seedlings was severely arrested
after radicles emerged (Fig. 2B). Consistent with this,
the root growth of 35S:MIR168a and ago1-27 seedlings
was hypersensitive to ABA treatment (Fig. 2C). After
growing on Murashige and Skoog (MS) medium
containing 30 mM ABA for 5 d, the root length of
ago1-27 and the two 35S:MIR168a lines decreased 85%,
70%, and 75%, respectively, compared with a 40%
decrease in wild-type plants. These results indicate
that disturbance of AGO1 function leads to enhanced
Figure 2. ago1-27 and MIR168a overexpression plants display ABA and salt hypersensitivity. A, 35S:MIR168a and ago1-27
seeds showed delayed germination compared with wild-type seeds (Col) on ABA-free medium. Each data point represents the
mean of triplicate experiments (n = 100 each), and error bars indicate SE. B, Seven-day-old 35S:MIR168a and ago1-27 seedlings
on MS medium containing 0.3 mM ABA. Representative images are shown. C, ABA inhibition of primary root elongation of 35S:
MIR168a (lines 4 and 6) and ago1-27 seedlings. Seeds were germinated on MS medium for 4 d, transferred to medium containing
30 mM ABA, and root length was measured after a 5-d treatment. Representative images are shown. D, Seed germination on
medium containing 0, 50, 100, or 150 mM NaCl. Experiments were performed in triplicate (n = 100 each), and error bars indicate SE.
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Transcriptional Regulation of MIR168a
sensitivity to exogenous ABA treatment and are consistent with the results of Earley et al. (2010).
In addition, we investigated the response of 35S:
MIR168a and ago1-27 lines to salt treatment. The
germination efficiencies of 35S:MIR168a and ago1-27
seeds were much lower on medium containing 50 or
100 mM NaCl compared with wild-type seeds (Fig.
2D). Under the treatment with 100 mM NaCl, the germination efficiencies were 70% for the wild type, 42%
and 37% for 35S:MIR168a#4 and -#6, respectively, and
only 18% for ago1-27. These results suggest that 35S:
MIR168a and ago1-27 plants are hypersensitive to salt
stress. After germination, the difference in growth
between these lines and the wild type became less
apparent.
MIR168a Overexpression Plants and the ago1-27 Mutant
Display an Enhanced Resistance to Drought
Guard cells form the stoma on the leaf epidermis and
respond to environmental factors by changing stomatal
aperture. Drought stress is able to promote the synthesis of ABA in plants, leading to a quick closure of stoma
to prevent water loss by transpiration (Blatt, 2000).
Previous transcriptomic and proteomic analyses detected
AGO1 transcripts and proteins in Arabidopsis guard
cells (Leonhardt et al., 2004; Zhao et al., 2008). Consistent with this, we observed MIR168a expression in
guard cells in transgenic lines carrying pMIR168a:GUS
(Supplemental Fig. S3) in this study.
Given the drought-inducible expression of MIR168a
as well as the expression of MIR168a and AGO1 in
guard cells, we hypothesized that miR168a and AGO1
may play a role in plant drought tolerance. To address
this point, we performed a drought tolerance experiment and found that both 35S:MIR168a and ago1-27
plants displayed higher survival rates under water
deficit conditions. Three-week-old plants were treated
with drought for 15 d before being rewatered. Two
days after the rewatering, only 46 out of 90 wild-type
plants survived, whereas all of the 35S:MIR168a and
ago1-27 plants recovered (Fig. 3A). We hypothesized
that the enhanced drought tolerance of 35S:MIR168a
and ago1-27 plants might have resulted, at least partially, from their lower transpiration rate. To test this
prediction, we examined the transpiration rate and
stomatal aperture of 35S:MIR168a and ago1-27 plants.
The water loss rates of 35S:MIR168a and ago1-27 lines
were 71% to 77% of that of wild-type plants (Fig. 3B).
We also measured the stomatal opening of 35S:
MIR168a and ago1-27 plants under drought conditions.
The stomatal apertures of 35S:MIR168a and ago1-27
plants were 78% to 84% of the wild-type value. The
stomatal closure was significantly enhanced in 35S:
MIR168a and ago1-27 lines under drought conditions
(Fig. 3C). These data suggest that overexpression of
MIR168a or mutation in AGO1 promotes stomatal
closure, resulting in drought tolerance.
The mir168a-2 Mutant Displays ABA Hyposensitivity
and Drought Hypersensitivity
To determine whether a loss-of-function mutant of
MIR168a exhibits the opposite phenotype to the MIR168a
overexpression plant in ABA and stress responses, we
analyzed the mir168a-2 mutant, which had been shown
to have reduced levels of miR168 and increased AGO1
transcripts (Vaucheret, 2009). Under standard conditions, mir168a-2 plants had more lateral roots than wildtype plants, but otherwise they grew normally. Under
ABA treatment, wild-type plants showed inhibitions of
germination and root growth, but these inhibitions were
not as serious in the mir168a-2 mutants (Fig. 4, A and B).
Under 0.3 mM ABA treatment, the germination efficiency
of the wild type decreased more than 90%, while that of
mir168a-2 decreased only about 60%. Consistently, when
Figure 3. ago1-27 and MIR168a overexpression
plants display enhanced drought tolerance. A,
Three-week-old plants treated with drought for 15
d before being rewatered. The photographs were
taken 2 d after rewatering. Number codes indicate the number of surviving plants out of the total
number (three repeats, with 90 plants in total). B,
Transpiration rate of 4-week-old plants. Rosette
leaves (third and fourth leaves) at the same developmental stages were detached and weighed
at various times. Each data point represents the
mean of triplicate experiments (n = 10 each), and
error bars show SE. C, Stomatal aperture of leaves
from 3-week-old plants with drought treatment
for 6 d. Asterisks indicate significant difference
compared with wild type (n = 80, P , 0.05,
Student’s t test). Error bars denote SE.
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the seedlings were treated with 10 mM ABA, the root
growth rate of the wild type was reduced nearly 50%,
while that of mir168a-2 displayed much less change.
These results suggest that the mir168a-2 mutant has
reduced ABA sensitivity. When measured by the fresh
weight loss of detached rosette leaves, the water loss rate
of mir168a-2 was 30% higher than that of the wild-type
plants (Fig. 4C). At the 6th d of water withholding, the
stomatal aperture index of mir168a-2 leaves was 0.24,
which was 18% greater than that of the wild type (Fig.
4D). In contrast to that of 35S:MIR168a and ago1-27
plants, the stomatal closure of the mir168a-2 mutant was
significantly reduced under water limitation conditions.
Additionally, when wild-type and mir168a-2 plants were
subjected to 11 d of water-withholding conditions, as
shown in Figure 4E, 2 d after the rewatering all of the
wild-type plants recovered, whereas only 45 among 87
mir168a-2 mutant lines survived, indicating that the
mir168a-2 mutation led to a reduction in plant tolerance
to drought stress.
ABRE Motifs within the MIR168a Promoter Are
Responsible for ABA-Responsive MIR168a Expression
To further elucidate the regulatory mechanism of
MIR168a in the ABA response, we screened the pro-
moter region of MIR168a using the PLACE prediction
software (http://www.dna.affrc.go.jp/PLACE/signalscan.
html) and identified five putative ABRE motifs (M1–
M5), all of which contain a core sequence of ACGT.
M1 to M5 are located in the MIR168a promoter at
2981 to 2978, 2875 to 2871, 2301 to 2297, 2275 to
2271, and 2126 to 2122, respectively. The ABRE motif
is a major class of ABA-responsive cis-elements and is
found in most of the genes regulated by ABA (Kim,
2006). To test which of the ABRE motifs in the MIR168a
promoter are mainly responsible for the ABA response,
a series of truncated fragments of the MIR168a promoter
were fused to GUS (Fig. 5A), and the resulting constructs were transformed into wild-type plants. Transgenic plants containing an 800-bp upstream region from
the transcription start site of the MIR168a promoter
(pMIR168aD1:GUS) had similar GUS activities to plants
with the 1,000-bp MIR168a promoter (pMIR168aD0:
GUS) with or without ABA treatment, suggesting that
the M1 and M2 motifs are not essential for MIR168a
expression in response to ABA (Fig. 5B). When M3 and
M4 were deleted, the pMIR168aD2:GUS plants showed
reduced GUS activity, but they still could be significantly induced by the ABA treatment. When M5 was
removed (pMIR168aD3:GUS), a decrease of the MIR168a
promoter activity was observed with or without ABA
Figure 4. mir168a-2 plants display ABA hyposensitivity and drought hypersensitivity. A, Germination of 4-d-old wild-type (WT) and mir168a-2
seedlings on MS medium containing different
concentrations (0, 0.1, 0.3, 0.5 mM) of ABA. Each
data point represents the mean of triplicate experiments (n = 100 each), and error bars show SE.
B, ABA inhibition of the primary root elongation
of mir168a-2 seedlings. Seeds were germinated
on MS medium for 3 d, transferred to medium
containing 10 mM ABA, and root length was
measured after a 5-d treatment. Representative
images are shown. C, Transpiration rate of
4-week-old mir168a-2 leaves. Rosette leaves
(third and fourth leaves) at the same developmental stages were detached and weighed at various
times. Each data point represents the mean of
triplicate experiments (n = 10 each), and error
bars show SE. D, Stomatal aperture of 3-week-old
leaves with drought treatment for 6 d (n = 80, P ,
0.05, Student’s t test). Error bars denote SE. E,
Three-week-old plants treated with drought for
11 d before being rewatered. The photographs
were taken 2 d after rewatering. Number codes
indicate the number of surviving plants out of
the total number (three repeats, with 87 plants in
total).
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Transcriptional Regulation of MIR168a
Figure 5. ABF1, ABF2, ABF3, and ABF4 bind the MIR168a promoter in vitro and in vivo. A, Different constructs of the MIR168a
promoter. The blue boxes indicate ABRE; the white boxes indicate mutated ABRE. The five ABREs were named M1 to M5,
respectively. pMIR168aD0:GUS contains a 1,000-bp promoter region beginning from the transcription start site. pMIR168aD1:
GUS contains an 800-bp promoter beginning from the transcription start site with M1 and M2 deleted. pMIR168aD2:GUS
contains a 1,000-bp promoter with mutated M3 and M4. pMIR168aD3:GUS contains mutated M5. pMIR168aD4:GUS contains
mutated M3, M4, and M5. B, GUS activity in 10 independent transgenic Arabidopsis plants (n = 15) harboring various constructs
shown in A with or without 6 h of 100 mM ABA treatment. Data show means 6 SD for four independent experiments (* P , 0.05,
Student’s t test). C, The bait DNA sequence used in yeast one-hybrid analysis contained three tandem repeats of a 25-bp fragment
(5#-CAAGAGAACACGTGTCGGAAAATGA-3#) that includes the ABRE motif M5 within the MIR168a promoter. Six plasmids
containing insert DNA fragments of ABF1, ABF2, ABF3, ABF4, ABI5, and TDR were retransformed into yeast strain YM4271
carrying the reporter genes HIS and lacZ under the control of the 75-bp fragment containing three M5 motifs. The transformants
were examined for growth in the presence of 3-AT and b-galactosidase (b-gal) activity. D, Schematic diagram of the MIR168a
promoter. The bent arrow indicates the translational start site. The blue boxes indicate ABRE motifs. F1, F2, and F3 represent
DNA fragments amplified in the quantitative ChIP-PCR assay. E, ChIP enrichment test showing the binding of ABF1-Myc, ABF2Myc, ABF3-HA, and ABF4-HA to the MIR168a promoter after application of 100 mM ABA for 6 h. F, ChIP enrichment test showing
the binding of ABF1-Myc, ABF2-Myc, ABF3-HA, and ABF3-HA to the MIR168a promoter under drought conditions for 6 h. Error
bars denote SD. Asterisks in E and F indicate significant difference between enrichment fold of F3 and that of F1 (n = 5, P , 0.05,
Student’s t test).
treatment, suggesting that M5 is crucial for the basal
expression and response of MIR168a to ABA. Furthermore, when the M3, M4, and M5 motifs were eliminated
simultaneously (pMIR168aD4:GUS), the MIR168a promoter activity was reduced and almost no ABA response was detected. These results indicate that the
three ABREs near the transcription start site of MIR168a
mainly contribute to MIR168a expression and the ABA
response.
ABF1, ABF2, ABF3, and ABF4 Directly Interact with the
ABRE Motif in the MIR168a Promoter
The ABF family members, also referred to as AREB
(for ABA-responsive element-binding protein), have
been shown to bind ABRE in yeast one-hybrid screen
analysis (Choi et al., 2000). ABF/AREB proteins belong to the bZIP class of transcription factors and are
required for the ABA-mediated stress response by
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binding to the G-ABRE motif. In Arabidopsis, the ABF/
AREB family consists of 13 members, eight of which
have been reported to be involved in ABA signal
transduction (Bensmihen et al., 2002; Jakoby et al.,
2002). These eight members can be further divided
into two classes. One class includes ABF1, ABF2/
AREB1, ABF3/AtDPBF5, and ABF4/AREB2, which are
ubiquitously expressed and induced by ABA and various abiotic stresses (Jakoby et al., 2002; Kang et al.,
2002; Kim et al., 2004). The other class includes ABA
INSENSITIVE5 (ABI5), AREB3, DC3 PROMOTERBINDING FACTOR2, and ENHANCED EM LEVEL,
which are mainly expressed in embryos and involved
in seed maturation, germination, and early seedling
development (Bensmihen et al., 2002; Jakoby et al.,
2002). Given that members of the ABF class share a
similar expression pattern with MIR168a (Kang et al.,
2002; Kim et al., 2004; Gazzani et al., 2009; Vaucheret,
2009), we speculated that ABFs may be associated with
MIR168a expression.
To test our prediction, we first used the yeast onehybrid assay to test the binding activity of ABFs to the
M5 motif within the MIR168a promoter (Fig. 5C). Yeast
cells carrying plasmids containing the cDNAs of ABF1,
ABF2, ABF3, and ABF4 were able to grow on a medium
lacking His in the presence of 60 mM 3-aminotriazole
(3-AT), but cells carrying plasmids containing the
cDNAs of ABI5 (Bensmihen et al., 2002) and the
negative control, Tapetum Degeneration Retardation
(TDR; Li et al., 2006), did not. All of the 3-AT-resistant
yeast strains had induced lacZ activity and formed
blue colonies on filter papers containing 5-bromo-4chloro-3-indolyl-b-D-galactoside. These data indicate
that cDNAs of ABF1, ABF2, ABF3, and ABF4 encode
proteins that specifically bind to the M5 motif of the
MIR168a promoter and activate the transcription of the
reporter genes in yeast.
To further investigate whether ABF1, ABF2, ABF3, and
ABF4 are able to physically interact with the MIR168a
promoter in planta, chromatin immunoprecipitation
(ChIP)-PCR was performed (Zhang et al., 2010). Transgenic T2 lines of 35S:ABF1-Myc, 35S:ABF2-Myc, 35S:
ABF3-HA, and 35S:ABF4-HA treated with 100 mM ABA
or drought for 6 h were used for ChIP assays. Three
primer pairs were designed to scan the MIR168a promoter (F1–F3; Fig. 5D). ChIP-PCR enrichment revealed
that ABF1, ABF2, ABF3, and ABF4 were mainly associated with the F3 region, which contains M3, M4, and
M5 (Fig. 5, E and F), suggesting that ABF1, ABF2, ABF3,
and ABF4 directly bind to the MIR168a promoter in
vivo in response to ABA or drought treatment.
The Expression of MIR168a Is Positively Regulated
by ABF1, ABF2, ABF3, and ABF4
To further determine whether ABF1, ABF2, ABF3,
and ABF4 can regulate the expression of MIR168a in
vivo, the levels of mature miR168 and pre-miR168a
were analyzed in 2-week-old lines overexpressing
ABF1, ABF2, ABF3, and ABF4 (Supplemental Fig. S4).
Without ABA treatment, the mature miR168 level
changed less in the lines overexpressing ABF1 and
ABF2 and slightly increased in ABF3- and ABF4-overexpressing lines (Fig. 6A). Likewise, the levels of premiR168a were also slightly elevated in ABF2-, ABF3-,
and ABF4-overexpressing lines. Surprisingly, AGO1
transcript was increased also in the lines overexpressing ABF1, ABF2, ABF3, and ABF4 (Fig. 6B). Previous
studies show that ABA treatment enhances the activity
of overexpressed ABF proteins to activate the downstream genes (Finkelstein et al., 2005; Fujita et al., 2005).
In accordance, under 6- and 12-h treatment with 100 mM
ABA, a much stronger induction of pre-miR168a and
mature miR168 was detected in all of the ABF overexpression lines (Fig. 6A). Furthermore, the AGO1
mRNA levels were significantly lower after a 12-h
treatment, although they were still higher than those
in 12-h treated wild-type plants (Fig. 6B). Collectively,
ABF1, ABF2, ABF3, and ABF4 may directly activate the
expression of MIR168a, but how they activate the
expression of AGO1 remains to be elucidated. In response to ABA, these four ABFs induce miR168, which
partially contributes to the reduction of the AGO1
transcript. We did not detect significant changes in the
expression of MIR168a in the individual abf mutant,
which may be explained by the functional redundancy
of ABF family members (Yoshida et al., 2010).
The ABRE Motif Is Conserved in Plant
MIR168 Promoters
The increase of miR168 accumulation during ABA
treatment was also observed in poplar and tobacco (Jia
et al., 2009, 2010). Homologs of miR168 were found in
15 plant species (Supplemental Table S1; miRBase,
version16.0 [http://www.mirbase.org/index.shtml]).
We also detected the ABA-induced expression of
miR168 in rice and maize after a 6-h treatment with
100 mM ABA (Supplemental Fig. S5). To understand
whether these diverse plant species share a similar
regulatory mechanism for miR168 accumulation to
Arabidopsis, we scanned MIR168 upstream sequences
in 10 dicot and monocot species (Arabidopsis, Brassica
napus, Glycine max, Medicago truncatula, rice, poplar,
Ricinus communis, Sorghum bicolor, Vitis vinifera, and
maize; Supplemental Table S2) using “profit” from
EMBOSS (Rice et al., 2000). The promoter regions of
these MIR168 genes were predicted by defining
the transcription start site using the transcription
start site identification program for plants (http://linux1.
softberry.com/berry.phtml?topic=tssp&group=programs&
subgroup=promoter). We found that most of the transcription start sites were located within the 150-bp
upstream region of the predicted hairpins of MIR168
genes (Supplemental Table S3). For each upstream
sequence, a list of candidate ABRE sites was generated
by profit with percentage scores representing their
similarities to the known ABRE motifs (Gómez-Porras
et al., 2007). Using an arbitrary 90% threshold of
similarity score, we found 24 putative ABRE motifs in
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Transcriptional Regulation of MIR168a
Figure 6. Expression analysis of miR168 and AGO1 in the wild type and ABF1, ABF2, ABF3, and ABF4 overexpression lines with
or without ABA treatment. A, Mature miR168 and pre-miR168a accumulated in the individual ABF1, ABF2, ABF3, and ABF4
overexpression lines (ABF1OE, ABF2OE, ABF3OE, and ABF4OE) with 0, 6, and 12 h of ABA treatment by northern-blot analysis.
This experiment was repeated three times independently, and similar results were observed. Numbers below the blots show the
relative abundance compared with the control U6; the level of each target in the nontreated wild type (Col) was set to 1. B, AGO1
mRNA was also induced in the individual ABF1, ABF2, ABF3, and ABF4 overexpression lines with 0, 6, and 12 h of ABA
treatment by qRT-PCR analysis. However, the scale of the inductions was decreased by ABA. The expression level of AGO1
mRNA in the wild-type plants under nonstressed conditions was defined as 1.0. Error bars denote SD (n = 3).
10 MIR168 upstream sequences from eight species (Fig.
7; Supplemental Table S3). Some of the ABRE motifs
share high similarity to M3, M4, and M5 in Arabidopsis
MIR168. The putative ABRE motifs displayed a high
occurrence in the 500-bp upstream region starting from
the beginning of the miR168 hairpin precursors
(Fig. 7A). WebLogo analysis (http://weblogo.berkeley.
edu) and a frequency matrix of the 24 putative ABRE
motifs of the eight species showed that the core
sequence ACGTG is conserved within the promoter
region of MIR168a homologs (Fig. 7, B and C). As the
negative control, we checked ABRE occurrences within
every known promoter in the genome of Arabidopsis
on the POBO Web site (http://ekhidna.biocenter.
helsinki.fi/poxo/pobo/pobo). POBO analysis showed
that the distribution of ABRE in MIR168 upstream
sequences was significantly higher than the background set (Supplemental Fig. S6). These observations
indicate that ABA-regulated MIR168 expression is conserved in diverse plant species.
DISCUSSION
Interference with AGO1 Function Alters ABA and
Several Abiotic Stress Responses
A number of reports on the identification and functional analysis of stress-responsible miRNAs and siRNAs
suggest that small RNAs may play an essential role in
stress responses (Zhou et al., 2007; Sunkar, 2010). In addition, mutations in some genes involved in small RNA
biogenesis cause altered ABA and stress responses,
confirming the regulatory role of the small RNA pathway in plant stress responses (Hugouvieux et al., 2001;
Vazquez et al., 2004; Kim et al., 2008; Zhang et al., 2008).
Our data showed that not only proteins responsible
for miRNA biogenesis but also the slicer required for
miRNA action is employed in the posttranscriptional
regulation of plant abiotic stress. The reduced AGO1
level in both the hypomorphic allele ago1-27 and the
MIR168a overexpression lines leads to significantly
enhanced sensitivity to ABA and multiple abiotic stresses, such as increased seed dormancy, inhibition of
seed germination and root elongation, and wholeplant drought tolerance. In contrast, the mir168a-2
mutant, which contains an increased level of AGO1
(Vaucheret, 2009), displayed ABA hyposensitivity and
drought hypersensitivity. These results together indicate that AGO1 is a negative modulator of ABA signaling and stress responses in Arabidopsis.
Hypomorphic alleles of the AGO1 mutation do not
significantly affect the stability of most miRNAs but
reduce the cleavage efficiency of AGO1 (Vaucheret
et al., 2004; Kurihara et al., 2009). Previous microarray
data revealed an overaccumulation of a large portion
of miRNA target transcripts in ago1-9, ago1-11, and
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Li et al.
2008). Down-regulation of UBC24 by miR399 is required
for primary root inhibition and phosphate transporter
induction in response to inorganic phosphate starvation
(Fujii et al., 2005). Up-regulation of these targets affects
the response of the plant to adverse environments.
Indeed, we observed that the transcripts of CSD1/2,
NFYA5, and APS4 were up-regulated in ago1-27 and
35S:MIR168a lines (Supplemental Fig. S7).
miR168 Modulates AGO1 Homeostasis during ABA
and Several Abiotic Stress Treatments
Figure 7. Plants have conserved ABRE motifs within the MIR168
promoters. A, The localizations of ABRE motifs in 2-kb upstream
sequences of 10 plant MIR168 hairpin precursors in eight species (ath,
Arabidopsis thaliana; bna, Brassica napus; gma, Glycine max; osa,
Oryza sativa; ptc, Populus trichocarpa; rco, Ricinus communis; vvi,
Vitis vinifera; zma, Zea mays). Yellow boxes denote the ABRE motif in
the sense strand (ABRE+); red boxes denote the ABRE motif in the
antisense strand (ABRE2); blue arrows denote the transcription start site
(TSS); two-way arrows denote the TATA box; blue lines denote the
hairpin precursor of the MIR168 gene. B, Base composition of ABRE
motifs that were found in the promoters of MIR168 from the eight
common species. C, Frequency matrix of the ABRE motifs in the
promoters of MIR168 from the eight species.
ago1-25 (Ronemus et al., 2006; Kurihara et al., 2009).
Among the list are a number of targets of stressresponsive miRNAs, including the well-characterized
COPPER/ZINC SUPEROXIDE DISMUTASE1/2
(CSD1/2), NFYA5, ATP SULFURYLASE4 (APS4),
AUXIN RESPONSE FACTOR8 (ARF8), and UBIQUITINCONJUGATING ENZYME24 (UBC24). Targets of
miR398, CSD1 and CSD2 encode reactive oxygen species scavengers that prevent the deleterious effects of
oxidants. Arabidopsis plants overexpressing a miR398resistant version of CSD2 exhibit enhanced resistance to
oxidative stresses (Sunkar et al., 2006). NFYA5, a target
of miR169, which is the positive regulator of drought
resistance, is up-regulated by ABA and drought (Li
et al., 2008). APS4 is involved in miR395-mediated
sulfate assimilation (Hatzfeld et al., 2000). The circuit
between miR167a and ARF8 mediates nitrogen-induced
lateral root initiation and emergence (Gifford et al.,
In wild-type Arabidopsis, AGO1 homeostasis is
maintained by an autoregulatory feedback loop that
involves the coexpression of MIR168 and AGO1 as
well as their interaction at the posttranscriptional level
(Vaucheret et al., 2006; Vaucheret, 2009; Várallyay
et al., 2010). AGO1 mRNA abundance is also controlled by siRNAs derived from its own transcripts
(Mallory and Vaucheret, 2009). In addition, AGO1
activity is further regulated negatively by AGO10 and
positively by a cyclophilin protein, SQUINT (Mallory
et al., 2009; Smith et al., 2009).
Here, we show that a coordinate transcriptional
regulation of MIR168 and AGO1 also plays an important role in modulating plant stress responses. Previous studies have demonstrated that miR168 is induced
by various stresses and ABA treatment (Liu et al., 2008;
Jia et al., 2009, 2010). Our results further demonstrate
that the increase of miR168 level is at least partially
caused by the transcriptional activation of MIR168a, a
ubiquitously expressed miR168 gene. On the contrary,
no significant change of AGO1 mRNA level was
detected after a 12-h treatment with ABA, salt, and
cold. Simultaneously, the AGO1 protein level displays
only subtle changes in response to ABA and cold and
is slightly induced by drought (Supplemental Fig. S8).
Less repression of AGO1 abundance might result from
distinct regulatory mechanisms at different regulatory
levels. Consistent with this hypothesis, we observed
that the promoter activity of the transgenic plants
harboring the pAGO1:GUS construct, which is free
from miR168 inhibition, is induced by ABA treatment.
The opposite effects of ABA on AGO1 at the transcriptional and posttranscriptional levels might reflect an
arms race between the forces transmitting and attenuating ABA signals.
AGO1 is one of the core factors crucial for miRNAand siRNA-mediated gene silencing. The fluctuation
in AGO1 level affects normal development and responses to environmental stimuli in plants (Morel
et al., 2002; Ellendorff et al., 2009; Várallyay et al.,
2010). Thus, it is not surprising that plants evolve
complicated mechanisms to fine-tune AGO1 levels
under stress conditions. Recently, miR168 was reported to be hijacked by plant viruses to alleviate plant
antiviral function through inhibiting the translational
capacity of AGO1 mRNA (Várallyay et al., 2010),
indicating that miR168 acts as a rheostat exploited by
different regulatory loops to control AGO1 abun-
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Transcriptional Regulation of MIR168a
dance. A novel negative regulator of AGO1, F-BOX
WITH WD-40 2 (FBW2), was also reported to be
responsible for fine-tuning ABA signaling by controlling AGO1 protein level (Earley et al., 2010). Similar to
mir168a-2, mutations in FBW2 have nearly no effect on
plant morphology but affect the sensitivity of plants to
ABA (Earley et al., 2010). These observations reveal
that the AGO1 level is modulated by multiple mechanisms in response to external stimuli. The observation that AGO1 is transcriptionally up-regulated by
ABA suggests that there might be other yet unknown
mechanisms involved in the transcriptional regulation
of AGO1 in response to ABA.
miR168 Is a Component of the ABF Transcriptional
Cassette and Mediates ABA-Dependent Stress Signaling
A wealth of evidence reveals that miRNAs play a
crucial role in controlling gene expression at the posttranscriptional level by targeting mRNA cleavage or
translational repression. However, little is known
about the transcriptional regulation of miRNA genes.
Previously, myeloid transcription factors, PU.1 and
CAAT enhancer-binding protein a, were shown to directly regulate the transcription of pri-miR223, and this
regulatory mechanism seems to be conserved in mouse
and human (Fukao et al., 2007). In Arabidopsis, the
MIR398 expression regulator SQUAMOSA PROMOTERBINDING PROTEIN-LIKE7 (SPL7) is essential for the
copper deficiency response and is required for the
degradation of mRNAs that encode copper/zinc superoxide dismutases (Yamasaki et al., 2009). A negative feedback loop, which involves the direct induction
of MIR172b transcription by the miR156 targets SPL9
and SPL10, contributes to the stabilization of the
transition from juvenile to adult phases (Wu et al.,
2009). Recently, the expression of MIR164 was shown to
be directly induced by TEOSINTE BRANCHED1 AND
CYCLOIDEA AND PCF TRANSCRIPTION FACTOR3
to repress the expression of CUP-SHAPED COTYLEDON genes (Koyama et al., 2010). The above observations indicate that many of these posttranscriptional
regulators are transcriptionally controlled by developmental or environmental cues.
Rapid accumulation of the MIR168a precursor after
ABA and stress treatments indicates that MIR168a is
regulated by stress signaling at the transcriptional
level. ABA is involved in the activation of MIR168,
since the up-regulation of miR168 by drought treatment was attenuated in the ABA-deficient mutant
aba1-5. A previous survey for cis-acting elements in
stress-responsive miRNAs revealed the presence of
multiple ABREs in the MIR168a promoter (Liu et al.,
2008), suggesting a possible link between MIR168a
transcription and ABRE-binding proteins. A small clade
of basic Leu zipper transcription factors, called AREB/
ABF, has been shown to convey ABA-dependent stress
signaling by binding to ABRE motifs within promoters
of downstream genes and inducing gene expression
(Kim, 2006). The AREB/ABF regulon is the most impor-
tant transcriptional cassette regulating ABA-dependent
gene expression (Jakoby et al., 2002; Yoshida et al., 2010).
AREB/ABF proteins function at an early step of ABA
signal transduction and regulate a large spectrum of
stress-responsive genes (Kim, 2006).
In this study, we present biochemical and genetic
evidence for the direct activation of MIR168a expression by four members of the AREB/ABF family. Surprisingly, an elevated AGO1 expression level was also
observed in these ABF overexpression lines. This
might have resulted from an unknown feedback regulatory loop of AGO1 by ABA or the ABF family. The
fluctuation of MIR168 and AGO1 expression impacts
global miRNA action (Vaucheret et al., 2004; Kurihara
et al., 2009). Therefore, it is efficient to employ an initial
regulator of one pathway to modulate the central
modifier of another pathway, thus coordinating the
cross talk of two pathways. In addition, the findings
that miR168 is up-regulated by ABA and abiotic
stresses as well as that the conserved ABRE motifs
were found in the MIR168 upstream sequences in
multiple species lead us to the conclusion that higher
plants may share a common regulatory mechanism to
control miR168 expression in response to ABA and
abiotic stimuli. However, we cannot exclude the possibility that there may be some other transcriptional
factors besides ABFs binding to the same or other ciselements within the promoter of MIR168. Promoter
deletion analysis will help to uncover other players
involved in stress-regulated miR168 accumulation.
MATERIALS AND METHODS
Plant Materials and Stress Treatments
Arabidopsis (Arabidopsis thaliana) ecotypes Columbia (Col-0) and Landsberg erecta were used as the wild type. ago1-27 and aba1-5 mutants in the Col-0
background were kindly provided by Dr. Hervé Vaucheret and Hao Yu,
respectively. CS25625 (mir168a-2) in the Landsberg erecta background was
obtained from the Arabidopsis Biological Resource Center. Plants were grown
in a controlled chamber at 22°C, 70% relative humidity, under continuous
illumination. Two-week-old seedlings were subjected to various treatments.
For ABA treatment, plants were sprayed with 100 mM ABA and harvested at
various time points. For salt treatment, seedlings were transferred to solutions
containing 300 mM NaCl. For drought treatment, seedlings were dehydrated
on filter paper. For cold stress treatment, seedlings were kept at 4°C.
Plasmid Construction
The upstream fragment of MIR168a, spanning nucleotides 22,065 to
21 with respect to the MIR168a transcription start site (Jiang et al., 2006),
was amplified from Arabidopsis genomic DNA and then fused in frame with
the GUS coding region in pCAMBIA1301. To generate internal deletion
constructs, the ABREs located at nucleotides 2302 to 2272 and nucleotides
2126 to 2119 were replaced with restriction endonuclease-binding sites (NcoI
and BglII) by PCR. The fusion between the promoter of AGO1 and GUS was
constructed according to Vaucheret et al. (2006).
The cauliflower mosaic virus 35S promoter-MIR168a construct was described previously (Jiang et al., 2006). The coding regions of ABF1 and ABF2
were retrieved from SalI/BamHI-digested pEASY-Blunt vector and ligated to
SalI/BamHI-digested pCAMBIA1300-221-Myc vector to generate 35S:ABF1Myc and 35S:ABF2-Myc. 35S:ABF3-HA and 35S:ABF4-HA were generated by
inserting the fusion of the ABF coding region and the hemagglutinin (HA) tag
to the pCAMBIA1300-221 vector. These gene fusions were controlled by the
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Li et al.
cauliflower mosaic virus 35S promoter and used for the transformation of
Arabidopsis.
Histochemical GUS Staining and Quantification of
GUS Activity
Rosette leaves of 3-week-old homozygous transgenic plants were harvested. Histochemical localization of GUS activity was determined as described
previously (Kang et al., 2002). For GUS quantification, at least 10 independent
transgenic lines for each construct were used, and 15 2-week-old T2 seedlings
from each line were combined to measure GUS activity. At least four repeats
were carried out for each line. SD between lines was calculated. GUS activity
was quantified using the fluorometric 4-methylumbelliferyl-b-D-glucuronide
assay method (Jefferson, 1987).
RNA Analysis
Total RNA was extracted from control or treated plants with Trizol reagent
(Invitrogen). miR168 and pre-miR168a were detected by the probe complemented with miR168 and pre-miR168a loop sequences, which were modified
with 3# and 5# biotin. Forty micrograms of total RNA was loaded in each lane,
and U6 was used as a loading control (Li et al., 2007). After stringent washes,
signals on the membranes were detected according to the manufacturer’s
instructions for the Chemiluminescent Nucleic Acid Detection Module
(Thermo). qRT-PCR analyses were carried out with total RNA using BioRad’s real-time PCR amplification systems. Each gene was assayed on three
biological replicates and normalized using the TUBULIN4 cDNA level
(Magnan et al., 2008).
Germination and Seedling Growth Assay
Seeds of wild-type, ago1-27, 35S:MIR168a, and mir168a-2 plants were
collected at the same time and stored in the same conditions. They were
plated on ABA-free medium for 4 d at 4°C before switching to 22°C to
germinate, then the germination (fully emerged radicle) efficiency was scored
at various times. For germination under various NaCl doses, seeds were
plated on MS medium containing 0, 50, 100, and 150 mM NaCl for 4 d before
counting. For germination under various ABA doses, seeds were plated on MS
medium containing 0, 0.1, 0.3, and 0.5 mM ABA for 1 week. For the root growth
assay, plants were germinated on MS plates and transferred after 4 d to fresh
plates containing ABA at 10 mM for the assay of mir168a-2 and 30 mM for the
assay of ago1-27 and 35S:MIR168a transgenic lines. Photographs of the
elongated roots were taken 5 d later.
Drought Treatment and Measurement of Transpiration
Rate and Stomatal Aperture
For drought treatment, 3-week-old soil-grown plants were not watered for
11 to 15 d. Photographs were taken 2 d after rewatering. The transpiration rate
of detached leaves was measured by weighing freshly harvested leaves placed
on open dishes on the laboratory bench, with the abaxial side up. Leaves of
similar developmental stages from 4-week-old soil-grown plants were used
(Kang et al., 2002; Kim et al., 2004).
Stomatal aperture analysis was conducted according to a previous description (Li et al., 2008). Stomatal apertures were measured 6 d after
dewatering. For each treatment, 80 measurements were collected, and each
experiment was repeated at least three times.
Yeast One-Hybrid Analysis
The coding regions of the transcription factors ABF1, ABF2, ABF3, ABF4,
and ABI5 were amplified from Arabidopsis Col-0 cDNA using gene-specific
primers. The PCR products were then cloned into the pGADT7 prey vector
(Clontech), creating a translational fusion between the GAL4 activation
domain and the transcription factor. Three tandem repeats of the 2135 to
2110 MIR168a promoter region were synthesized and cloned into pHISi-1
vector (Clontech) carrying the HIS3 reporter gene and pLacZi vector
(Clontech) containing the LacZ reporter gene. Induction of HIS3 and LacZ
indicates positive interaction. The yeast one-hybrid assay was performed
according to the Clontech Yeast Protocols Handbook.
Quantitative ChIP-PCR Analysis
The procedure for ChIP of ABF-DNA complexes in the 35S:ABF1-Myc, 35S:
ABF2-Myc, 35S:ABF3-HA, and 35S:ABF4-HA transgenic Arabidopsis leaves
was modified from previous descriptions (Bowler et al., 2004). Chromatin was
isolated from 1.5 g of formaldehyde cross-linked tissue from leaves of 4-weekold transgenic plants sprayed with 100 mM ABA for 6 h and seedlings of
2-week-old transgenic plants treated with drought for 6 h. Preabsorption
with protein A-Sepharose beads and immunoprecipitation with c-Myc or
HA monoclonal antibody (Sigma-Aldrich) were performed as described by
Bowler et al. (2004). A small aliquot of untreated sonicated chromatin was
reverse cross-linked and used as the total input DNA control. Quantitative
ChIP-PCR was performed with primers specific to different regions upstream
of MIR168a. ChIP populations from immune or control immunoprecipitations
were used as templates and normalized by the amount of the DNA fragments
in corresponding input DNA populations. The difference between the normalized mean cycle threshold of the immunized population and the preimmunized population was calculated to obtain the relative enrichment of
each upstream fragment (Zhang et al., 2010). The primers used in this article
are listed in Supplemental Table S5.
Sequence Analysis of MIR168 Promoters in Plants
Sequences of miR168 from Arabidopsis, Brassica napus, Glycine max,
Medicago truncatula, rice (Oryza sativa), poplar (Populus trichocarpa), Ricinus
communis, Sorghum bicolor, Vitis vinifera, and maize (Zea mays) and their
corresponding genome coordinates were obtained from miRBase (version
16.0; http://www.mirbase.org/index.shtml). Upstream sequences 2 kb relative to the beginning of the predicted hairpins were extracted from available
genome sequences according to their coordinates. These 2-kb upstream
sequences were then scanned to identify ABRE sites on both plus and minus
strands using profit from EMBOSS (Rice et al., 2000). A previously discovered
matrix of ABRE was adopted as the input matrix for profit (Gómez-Porras
et al., 2007). In addition, the plant promoter identification program (http://
linux1.softberry.com/berry.phtml?topic=tssp&group=programs&subgroup=
promoter) was used to check the transcription start site of these sequences.
Western-Blot Analysis
The AGO1 antibody was purchased from Agrisera and was used at 1:3,000
dilution in the immunoblot analysis. Anti-b-tubulin was purchased from
Santa Cruz Biotechnology and was used at 1:1,000 dilution in the experiments.
Accession numbers for the genes in this article are as follows: MIR168a
(AT4G19395), AGO1 (AT1G48410), TUBULIN4 (AT5G44340), RD29A (AT5G5
2310), ABA1 (AT5G67030), ABF1 (AT1G49720), ABF2 (AT1G45249), ABF3 (AT
4G34000), ABF4 (AT3G19290), ABI5 (AT2G36270), NFYA5 (AT1G54160), APS4
(AT5G43780), CSD1 (AT1G08830), and CSD2 (AT2G28190).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. qRT-PCR analysis of RD29A mRNA under ABA
and abiotic stress treatments.
Supplemental Figure S2. Expression analysis of mature miR168, premiR168a, and AGO1 mRNA in the wild-type and MIR168a overexpression lines.
Supplemental Figure S3. Histochemical GUS staining in the guard cells of
pMIR168a:GUS transgenic plants.
Supplemental Figure S4. qRT-PCR analysis of ABF1, ABF2, ABF3, and
ABF4 transcript levels in overexpression lines.
Supplemental Figure S5. Northern-blot analysis of mature miR168 in
2-week-old seedlings of rice and maize with or without ABA treatment.
Supplemental Figure S6. POBO output interface.
Supplemental Figure S7. qRT-PCR analysis of miRNA targets in ago1-27
and 35S:MIR168a lines.
Supplemental Figure S8. Western-blot analysis of AGO1 in response to
ABA, cold, and drought.
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Transcriptional Regulation of MIR168a
Supplemental Table S1. Putative miR168 in 15 species.
Supplemental Table S2. Upstream sequences (2 kb) of MIR168 in 10
species.
Supplemental Table S3. The putative transcription start sites of MIR168
genes.
Supplemental Table S4. Candidate ABRE sites in the promoters of MIR168
genes.
Supplemental Table S5. Primers and probers used in this article.
ACKNOWLEDGMENTS
We thank Dr. Hervé Vaucheret for kindly providing the mutant ago1-27,
Dr. Hao Yu for providing the mutant aba1-5, Dr. Jianping Hu for editing the
manuscript, and The Arabidopsis Information Resource for supplying
mir168a-2 seeds. We also thank Dr. Yuke He for providing the protocol for
small RNA blot.
Received October 12, 2011; accepted January 12, 2012; published January 13,
2012.
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