Plant Physiology Preview. Published on January 13, 2012, as DOI:10.1104/pp.111.188789
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Running head:
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Transcriptional regulation of MIR168a
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*
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Wanqi Liang
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Address: School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shang-
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hai 200240, China
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Tel: 0086-21-34205073; Fax: 0086-21-34204869;
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E-mail: [email protected].
Corresponding author:
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Copyright 2012 by the American Society of Plant Biologists
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Transcriptional regulation of Arabidopsis MIR168a and ARGONAUTE1 homeostasis in
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ABA and abiotic stress responses
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Wei Li1, Xiao Cui1, Zhaolu Meng1, Xiahe Huang2, Qi Xie2, Heng Wu1, Hailing Jin3, Dabing
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Zhang1, Wanqi Liang1*
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School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai
200240, China
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of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,
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China
State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute
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verside, CA 92521, USA.
Department of Plant Pathology and Microbiology, University of California, Riverside, Ri-
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FOOTNOTES
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Financial support
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This work was supported by funds from the National Natural Science Foundation of China
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(30600347).
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*
Corresponding author: Wanqi Liang, e-mail: [email protected].
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ABSTRACT
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The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA)
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and abiotic stresses, but the underlying mechanisms are poorly understood. The
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miR168-mediated feedback regulatory loop regulates ARGONAUTE1 (AGO1) homeostasis,
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which is crucial for gene expression modulation and plant development. Here we revealed a
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transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during
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ABA treatment and abiotic stress responses in Arabidopsis thaliana. Plants overexpressing
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MIR168a and the AGO1 loss-of-function mutant (ago1-27) display ABA hypersensitivity and
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drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought
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hypersensitivity. Both the precursor and mature miR168 were induced under ABA and sever-
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al abiotic stress treatments, but no obvious decrease for the target of miR168, AGO1, was
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shown under the same conditions. However, promoter activity analysis indicated that AGO1
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transcription activity was increased under ABA and drought treatments, suggesting that tran-
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scriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level
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during the stress response. Furthermore, we showed both in vitro and in vivo that the tran-
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scription of MIR168a is directly regulated by four ABA-responsive element (ABRE) binding
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factors (ABF1-4), which bind to the ABRE cis-element within the MIR168a promoter. This
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ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species.
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Our findings suggest that transcriptional regulation of miR168 and post-transcriptional con-
20
trol of AGO1 homeostasis may play an important and conserved role in stress response and
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signal transduction in plants.
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Key words: Arabidopsis thaliana, transcriptional regulation, MIR168a, AGO1 homeostasis,
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ABA and abiotic stresses
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
INTRODUCTION
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As sessile organisms, plants have evolved sophisticated mechanisms to circumvent the ad-
3
verse environment. The phytohormone abscisic acid (ABA) is a key inducer of plant response
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to abiotic stresses. Much effort has been made to decipher the mechanism underlying ABA
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signal transduction, in which post-transcriptional regulation has been shown to be one of the
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most important modes of regulation (Chinnusamy et al., 2008). Mutants of several compo-
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nents of the miRNA biogenesis machinery, such as hyponastic leaves 1 (hyl1), serrate (se),
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dicer-like 1 (dcl1), hua enhancer1 (hen1) and cap-binding protein (cbp20, cbp80), are
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hypersensitive to ABA (Lu and Fedoroff, 2000; Hugouvieux et al., 2001; Kim et al., 2008;
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Zhang et al., 2008). These mutants, except hen1, were shown to have lower miRNA levels
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but higher primary miRNA precursor (pri-miRNA) levels compared to wild-type plants
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(Laubinger et al., 2008). In cbp20 and cbp80 mutants, ABA induction of miR159 was de-
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layed and the miR159 target transcripts, which encode two positive regulators of ABA re-
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sponse, MYB DOMAIN PROTEIN 33 (MYB33) and MYB DOMAIN PROTEIN 101
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(MYB101), accumulated to a higher level (Reyes and Chua, 2007; Kim et al., 2008).
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ARGONAUTE (AGO) proteins, which contain the well characterized PAZ and PIWI do-
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mains, are considered to be integral players in all known small RNA-targeted regulatory
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pathways (Vaucheret, 2008). Among the AGO proteins in Arabidopsis thaliana (Arabidopsis),
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AGO1 is a core component of the RNA-induced silencing complex (RISC), which associates
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with miRNAs and inhibits target genes by mRNA cleavage and/or translational repression
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(Vaucheret et al., 2004; Vaucheret, 2008; Voinnet, 2009). Mutations in AGO1 cause in-
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creased accumulation of miRNA targets (Vaucheret et al., 2004; Ronemus et al., 2006; Kuri-
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hara et al., 2009). A hypomorphic allele, ago1-27 exhibits hypersusceptibility to Cucumber
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mosaic virus (CMV) (Morel et al., 2002), and AGO1 was proved to be a major determinant
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for virus-induced gene silencing (VIGS) (Zhang et al., 2006). ago1-27 displays resistance to
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the infection of a fungal pathogen, Verticillium dahliae, supporting the view that AGO1 is
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involved in the regulation of pathogen defense responses (Ellendorff et al., 2009). Although
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several AGO proteins have been shown to regulate biotic stresses (Katiyar-Agarwal et al.,
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
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2007; Li et al., 2010; Zhang et al., 2011), the role of AGO proteins in regulating abiotic stress
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responses is still not well understood. A recent report showed that a decrease in AGO1 con-
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fers hypersensitivity to ABA, whereas an increase in the level of AGO1 leads to ABA hypo-
4
sensitivity (Earley et al., 2010). However, the role of AGO1 in abiotic stress response path-
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ways is still unclear.
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It is well known that miRNAs play crucial roles in controlling a variety of developmental
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processes such as organ identity establishment, organ separation, hormone signaling and flo-
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wering time control (Chen, 2009). Recent evidence also uncovers the role of small RNA mo-
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lecules in regulating plant stress response (Sunkar, 2010; Khraiwesh et al., 2011). Microarray
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analysis and deep-sequencing data demonstrated that the accumulation of a number of miR-
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NAs is affected by abiotic stresses (Sunkar and Zhu, 2004; Reyes and Chua, 2007; Zhou et
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al., 2007; Liu et al., 2008; Zhou et al., 2008; Jia et al., 2009), implying a potential role of
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miRNAs in abiotic stress response. In Arabidopsis, the expressions of MIR395, MIR398 and
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MIR399 are induced by sulfate, copper and phosphate deprivation, respectively, and their
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subsequent down-regulation of target genes is essential for nutrient reallocation (Fujii et al.,
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2005; Jagadeeswaran et al., 2009; Kawashima et al., 2009). Upregulation of miR159 and
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miR160 by ABA and the subsequent degradation of their target transcripts have been reported
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to be important for Arabidopsis seed germination and seedling development (Liu et al., 2007;
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Reyes and Chua, 2007). Downregulation of miR169 by ABA and drought leads to suppres-
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sion of its cleavage of NUCLEAR FACTOR Y A 5 (NFYA5), the positive regulator of drought
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resistance (Li et al., 2008). In addition, natural cis-antisense siRNAs (nat-siRNAs) and
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trans-acting siRNAs (ta-siRNAs) also have been reported as important players in adaption to
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a variety of abiotic stresses (Borsani et al., 2005; Wilson et al., 2010). However, the role and
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the regulatory mechanism of most of the plant small RNAs in abiotic stresses remain largely
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unknown.
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MIR168 is one of the most commonly detected stress-inducible MIR genes. Homologs of
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MIR168 exist in various plant species including dicots such as poplar (Populus trichocarpa),
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tobacco (Nicotiana tabacum), and Arabidopsis, and monocots such as maize (Zea mays) and
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
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rice (Oryza sativa). These homologs have been found to also respond to salt, drought, cold
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stresses or ABA treatment (Liu et al., 2008; Ding et al., 2009; Jia et al., 2009; Jia et al., 2010;
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Zhou et al., 2010). A newly identified MIR168a mutant, mir168a-2, exhibits developmental
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defects when exposed to temperature, light and water fluctuations (Vaucheret, 2009). These
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findings led to the speculation that miR168 and its exclusive target AGO1 may play a critical
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role in the stress regulatory network.
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In this paper, we reveal the important role of MIR168a and its target AGO1 in plant res-
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ponses to ABA and several abiotic stresses. Plants either over-expressing MIR168a or carry-
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ing mutation in AGO1 (ago1-27) display ABA hypersensitivity and drought tolerance, while
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the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Consistent
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with this, MIR168a is transcriptionally upregulated by ABA and various stresses, resulting in
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post-transcriptional control of AGO1 homeostasis. Furthermore, we demonstrate that
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MIR168a is activated by ABRE binding transcription factors ABF1, ABF2, ABF3 and ABF4.
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A typical ABRE motif within the MIR168a promoter, which can be bound by the four ABRE
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binding transcription factors, is conserved in the MIR168 homologs in other plant species.
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These results imply a common and conserved mechanism of MIR168 transcriptional control
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in plant stress response.
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RESULTS
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Transcriptional regulation of MIR168a and AGO1 under ABA and abiotic stresses
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Recent investigations using microarray followed by RT-PCR analyses revealed increased
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accumulation of miR168 transcripts under high salinity, drought and cold conditions in Ara-
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bidopsis (Liu et al., 2008). To investigate how the level of miR168 is induced, we analyzed
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the abundance of precursors and mature miR168 under stress conditions. Given that MIR168a
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is ubiquitously expressed and co-expressed with AGO1, while MIR168b expression is re-
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stricted to the shoot apical meristem (SAM) (Jiang et al., 2006; Gazzani et al., 2009; Vau-
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cheret, 2009), we focused on MIR168a in this study.
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Northern blot analysis was performed to measure the abundance of pre-miR168a tran-
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scripts and mature miR168 in 2-week-old wild-type Arabidopsis seedlings treated by drought,
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300 mM NaCl, and cold for 0, 6 and 12 hours. The levels of mature miR168 and
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pre-miR168a were increased under drought, salt and cold treatment (Figure 1A). ABA is an
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important signal in abiotic stress responses. Thus, we investigated the effect of 100 µM ABA
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on the accumulation of miR168 and pre-miR168a in 2-week-old wild-type Arabidopsis
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seedlings at 0, 2, 4, 6 and 12 hours. The response to ABA and stress treatments was con-
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firmed by the increased expression of RESPONSIVE TO DESSICATION 29A (RD29A) (Fig-
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ure S1), a gene known to be induced under such conditions (Yamaguchi-Shinozaki et al.,
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2003). Northern blot analysis showed elevation of miR168 and pre-miR168a levels 2 hours
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after treatment of 100 µM ABA (Figure 1A). Furthermore, drought-induced accumulation of
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miR168 was substantially reduced in an ABA-deficient (aba1-5) mutant (Léon-Kloosterziel
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KM, 1996) (Figure 1C), suggesting that enhanced accumulation of miR168 by abiotic stress
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is at least partially ABA dependent.
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miR168 is critical for modulating the level of AGO1 by miR168-targeted cleavage of the
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AGO1 mRNA and translational repression of AGO1 (Vaucheret et al., 2006; Vaucheret,
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2009; Varallyay et al., 2010). To determine whether ABA and stress treatments affect AGO1
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accumulation, we performed qRT-PCR analyses. Unexpectedly, AGO1’s mRNA level was
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weakly increased by ABA or other stresses, and showed about 2-fold increase at 12 h of
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drought treatment (Figure 1B). In order to clarify the role of miR168 in ABA and stress res-
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ponses, we compared the expression levels of endogenous AGO1 and exogenous GUS (Glu-
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curonidase) in pAGO1:GUS transgenic plants with ABA or drought treatment. The data
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showed that the increase of GUS transcript free from miR168 regulation was significantly
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higher as compared with the increase of endogenous AGO1 transcript after ABA and drought
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treatments (Figure 1D and 1E). These results suggest that AGO1 is induced by ABA and
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drought at the transcriptional level and miR168-mediated repression occurs at the
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post-transcriptional level. Both regulatory mechanisms modulate the expression of AGO1 in
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response to ABA and drought stress.
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ago1-27 mutant and MIR168a overexpression plants display ABA and salt hypersensi-
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tivity
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Although the accumulation of miR168 is affected by ABA treatment and several abiotic
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stresses, the function of miR168 and its exclusive target AGO1 in plant stress responses is
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still unclear. To assess the role of miR168 and AGO1 in stress response, we examined
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MIR168a overexpression lines (Jiang et al., 2006) and the AGO1 hypomorphic mutant allele
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ago1-27 (Vaucheret et al., 2004) under ABA and abiotic stress treatments. The levels of ma-
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ture miR168 in two 35S:MIR168a transgenic lines (line 4 and 6) were more than 10-fold
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higher than that in wild-type, while the AGO1 transcript levels decreased to 35% and 28%,
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respectively, of the wild type levels (Figure S2). The overexpression lines 35S:MIR168a#4
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and 35S:MIR168a#6 displayed some developmental defects, including shorter roots, smaller
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plant stature and delayed flowering.
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We first examined the effects of ABA treatment on seed germination and seedling growth
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in the MIR168a overexpression lines and ago1-27 mutant. Under standard growth conditions,
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the germination of 35S:MIR168a lines was delayed by about 2 hours compared to wild-type
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seeds, and a 4-hour delay in germination was observed for ago1-27 seeds (Figure 2A). We
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next determined their ABA response during and after seed germination. Treatment with 0.3
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µM ABA caused a slight inhibition of seed germination and seedling growth of the wild type,
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while the growth of 35S:MIR168a and ago1-27 seedlings was severely arrested after radicles
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emerged (Figure 2B). Consistent with this, the root growth of 35S:MIR168a and ago1-27
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seedlings were hypersensitive to ABA treatment (Figure 2C). After growing on the Mura-
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shige-Skoog (MS) medium containing 30 μM ABA for 5 days , the root length of ago1-27
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and the two 35S:MIR168a lines decreased 85%, 70% and 75%, respectively, compared with a
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40% decrease in wild-type plants. These results indicate that disturbance of AGO1 function
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leads to enhanced sensitivity to exogenous ABA treatment and are consistent with the results
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of Earley et al (2010).
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In addition, we investigated the response of 35S:MIR168a and ago1-27 lines to salt treat-
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ment. The germination efficiencies of 35S:MIR168a and ago1-27 seeds were much lower on
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1
medium containing 50 or 100 mM NaCl compared to wild-type seeds (Figure 2D). Under the
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treatment of 100 mM NaCl, the germination efficiencies were 70% for wild type, 42% and
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37% for 35S:MIR168a #4 and #6, respectively, while only 18% for ago1-27. These results
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suggest that 35S:MIR168a and ago1-27 plants are hypersensitive to salt stress. After germi-
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nation, the difference in growth between these lines and the wild-type became less apparent.
6
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MIR168a overexpression plants and ago1-27 mutant display an enhanced resistance to
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drought
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Guard cells form the stoma on the leaf epidermis and respond to environmental factors by
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changing stomatal aperture. Drought stress is able to promote the synthesis of ABA in plants,
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leading to a quick closure of stoma to prevent water loss by transpiration (Blatt, 2000). Pre-
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vious transcriptome and proteomic analysis detected AGO1 transcripts and proteins in Ara-
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bidopsis guard cells (Leonhardt et al., 2004; Zhao et al., 2008). Consistent with this, we ob-
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served MIR168a expression in guard cells in transgenic lines carrying pMIR168a:GUS (Fig-
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ure S3) in this study.
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Given the drought-inducible expression of MIR168a as well as the expression of MIR168a
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and AGO1 in guard cells, we hypothesized that miR168a and AGO1 may play a role in plant
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drought tolerance. To address this point, we performed a drought tolerance experiment and
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found that both 35S:MIR168a and ago1-27 plants displayed higher survival rates under water
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deficit conditions. Three-week-old plants were treated with drought for 15 days before being
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rewatered. Two days after the rewatering, only 46 out of 90 wild-type plants survived, whe-
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reas all of 35S:MIR168a and ago1-27 plants recovered (Figure 3A). We hypothesized that the
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enhanced drought tolerance of 35S:MIR168a and ago1-27 plants might have resulted, at least
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partially, from their lower transpiration rate. To test this prediction, we examined the transpi-
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ration rate and stomatal aperture of 35S:MIR168a and ago1-27 plants. The water loss rates of
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35S:MIR168a and ago1-27 lines were 71% to 77% of those of the wild-type plants (Figure
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3B). We also measured the stomatal opening of 35S:MIR168a and ago1-27 plants under
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drought condition. The stomatal apertures of 35S:MIR168a and ago1-27 plants were 78% to
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1
84% of the wild type. The stomatal closure was significantly enhanced in 35S:MIR168a and
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ago1-27 lines under drought condition (Figure 3C). These data suggest that overexpression of
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MIR168a or mutation in AGO1 promotes stomatal closure, resulting in drought tolerance.
4
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mir168a-2 mutant displays ABA hyposensitivity and drought hypersensitivity
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To determine whether a loss-of-function mutant of MIR168a exhibits the opposite pheno-
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type to the MIR168a overexpression plant in ABA and stress responses, we analyzed the
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mir168a-2 mutant, which had been shown to have reduced levels of miR168 and increased
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AGO1 transcripts (Vaucheret, 2009). Under standard conditions, mir168a-2 plants had more
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lateral roots than wild-type plants, but otherwise grew normally. Under ABA treatment,
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wild-type plants showed inhibitions of germination and root growth, but these inhibitions
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were not as serious in the mir168a-2 mutants (Figures 4A and 4B). Under 0.3 µM ABA
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treatment, the germination efficiency of wild type decreased more than 90%, while
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mir168a-2 decreased only about 60%. Consistently, when the seedlings were treated with 10
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µM ABA, the root growth rate of wild type reduced nearly 50%, while that of mir168a-2 dis-
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played much less change. These results suggest that the mir168a-2 mutant has reduced ABA
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sensitivity. When measured by the fresh weight loss of detached rosette leaves, the water loss
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rate of mir168a-2 mutant was 30% higher than that of the wild-type plants (Figure 4C). At
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the 6th day of water withholding, the stomatal aperture index of mir168a-2 leaves was 0.24,
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which was 18% greater than that of the wild type (Figure 4D). In contrast to that of
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35S:MIR168a and ago1-27 plants, the stomatal closure of mir168a-2 mutant was significant-
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ly reduced under water limitation condition. Additionally, when wild-type and mir168a-2
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plants were subjected to 11 days of water-withholding condition, as shown in Figure 4E, two
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days after the rewatering all of the wild-type plants recovered, whereas only 45 among 87
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mir168a-2 mutant lines survived, indicating the mir168a-2 mutation led to a reduction in
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plant tolerance to drought stress.
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ABRE motifs within the MIR168a promoter are responsible for ABA responsive
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1
2
MIR168a expression
To further elucidate the regulatory mechanism of MIR168a in ABA response, we screened
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the
4
(http://www.dna.affrc.go.jp/PLACE/ signalscan.html) and identified 5 putative ABRE motifs
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(M1 to M5), all of which contain a core sequence of “ACGT”. M1 to M5 are located in the
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MIR168a promoter at -981 to -978, -875 to -871, -301 to -297, -275 to -271, and -126 to -122,
7
respectively. The ABRE motif is a major class of ABA responsive cis-elements and is found
8
in most of the genes regulated by ABA (Kim, 2006). To test which of the ABRE motifs in the
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MIR168a promoter is mainly responsible for ABA response, a series of truncated fragments
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of the MIR168a promoter were fused to the GUS (Figure 5A), and the resulting constructs
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were transformed into wild-type plants. Transgenic plants containing an 800-bp upstream re-
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gion from the transcription start site of the MIR168a promoter (pMIR168aΔ1:GUS) had sim-
13
ilar GUS activities to plants with the 1000-bp MIR168a promoter (pMIR168aΔ0:GUS) with
14
or without ABA treatment, suggesting that the M1 and M2 motifs are not essential for
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MIR168a expression in response to ABA (Figure 5B). When M3 and M4 were deleted, the
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pMIR168aΔ2:GUS plants showed reduced GUS activity, but still could be significantly in-
17
duced by the ABA treatment. When M5 was removed (pMIR168aΔ3:GUS), a decrease of the
18
MIR168a promoter activity was observed with or without ABA treatment, suggesting that M5
19
is crucial for the basal expression and response of MIR168a to ABA. Furthermore, when the
20
M3, M4, and M5 motifs were eliminated simultaneously (pMIR168aΔ4:GUS), the MIR168a
21
promoter activity was reduced and almost no ABA response was detected. These results in-
22
dicate that the three ABREs near the transcription start site of MIR168a mainly contribute to
23
the MIR168a expression and ABA response.
promoter
region
of
MIR168a
using
the
PLACE
prediction
software
24
25
ABF1, ABF2, ABF3 and ABF4 directly interact with the ABRE motif in the MIR168a
26
promoter
27
The ABF family members, also referred as AREB (ABA-responsive element binding pro-
28
tein), have been shown to bind ABRE in yeast one-hybrid screen analysis (Choi et al., 2000).
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1
ABF/AREB proteins belong to the bZIP class of transcription factors and are required for
2
ABA-mediated stress response by binding to the G-ABRE motif. In Arabidopsis, the
3
ABF/AREB family consists of 13 members, eight of which have been reported to be involved
4
in ABA signal transduction (Bensmihen et al., 2002; Jakoby et al., 2002). These eight mem-
5
bers can be further divided into two classes. One class includes ABF1, ABF2/AREB1,
6
ABF3/AtDPBF5 and ABF4/AREB2, which are ubiquitously expressed and induced by ABA
7
and various abiotic stresses (Jakoby et al., 2002; Kang et al., 2002; Kim et al., 2004). The
8
other class includes ABA INSENSITIVE 5 (ABI5) AREB3, DC3 PROMOTER-BINDING
9
FACTOR (AtDPBF2), and EEL, which are mainly expressed in embryos and involved in seed
10
maturation, germination and early seedling development (Bensmihen et al., 2002; Jakoby et
11
al., 2002). Given that members of the ABF class share a similar expression pattern with
12
MIR168a (Kang et al., 2002; Kim et al., 2004; Gazzani et al., 2009; Vaucheret, 2009), we
13
speculated that ABFs may be associated with MIR168a expression.
，
14
To test our prediction, we first used the yeast one hybrid assay to test the binding activity
15
of ABFs to the M5 motif within the MIR168a promoter (Figure 5C). Yeast cells carrying
16
plasmids containing the cDNAs of ABF1, ABF2, ABF3 and ABF4 were able to grow on a
17
medium lacking histidine in the presence of 60 mM 3-aminotriazole (3-AT), but cells carry-
18
ing plasmids containing the cDNAs of ABI5 (Bensmihen et al., 2002) and the negative con-
19
trol, Tapetum Degeneration Retardation (TDR) (Li et al., 2006) did not. All of the
20
3-AT-resistant yeast strains had induced lacZ activity and formed blue colonies on filter pa-
21
pers containing 5-bromo-4-chloro-3-indolyl β-D-galactoside. These data indicate that cDNAs
22
of ABF1, ABF2, ABF3 and ABF4 encode proteins that specifically bind to the M5 motif of
23
the MIR168a promoter and activate the transcription of the reporter genes in yeast.
24
To further investigate whether ABF1, ABF2, ABF3 and ABF4 are able to physically inte-
25
ract with the MIR168a promoter in planta, chromatin immunoprecipitation (ChIP)-PCR was
26
performed (Zhang et al., 2010). Transgenic T2 lines of 35S:ABF1-Myc, 35S:ABF2-Myc,
27
35S:ABF3-HA and 35S:ABF4-HA treated with 100 μM ABA or drought for 6 h were used for
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
ChIP assays. Three primer pairs were designed to scan the MIR168a promoter (F1 to F3)
2
(Figure 5D). ChIP-PCR enrichment revealed that ABF1, ABF2, ABF3 and ABF4 were
3
mainly associated with the F3 region, which contains M3, M4 and M5 (Figure 5E, 5F), sug-
4
gesting that ABF1, ABF2, ABF3 and ABF4 directly bind to the MIR168a promoter in vivo in
5
response to ABA or drought treatment.
6
7
The expression of MIR168a is positively regulated by ABF1, ABF2, ABF3 and ABF4
8
To further determine whether ABF1, ABF2, ABF3 and ABF4 can regulate the expression
9
of MIR168a in vivo, levels of mature miR168 and pre-miR168a were analyzed in 2-week-old
10
lines overexpressing ABF1, ABF2, ABF3 and ABF4 (Figure S4). Without ABA treatment, the
11
mature miR168 level changed less in the lines overexpressing ABF1 and ABF2, and slightly
12
increased in ABF3 and ABF4 overexpressing lines (Figure 6A). Likewise, levels of
13
pre-miR168a were also slightly elevated in ABF2, ABF3 and ABF4 overexpressing lines.
14
Surprisingly, AGO1 transcript was increased also in the lines overexpressing ABF1, ABF2,
15
ABF3 and ABF4 (Figure 6B). Previous studies show that ABA treatment enhances the activi-
16
ty of over-expressed ABF proteins to activate the downstream genes (Finkelstein et al., 2005;
17
Fujita et al., 2005). In accordance, under 6-hour and 12-hour treatment of 100 µM ABA, a
18
much stronger induction of pre-miR168a and mature miR168 was detected in all of the ABF
19
overexpression lines (Figure 6A). Furthermore, the AGO1 mRNA levels were significantly
20
lower after 12-hour treatment, although still higher than those in 12-hour treated wild type
21
plants (Figure 6B). Collectively, ABF1, ABF2, ABF3 and ABF4 may directly activate the
22
expression of MIR168a, but how they activate the expression of AGO1 remains to be eluci-
23
dated. In response to ABA, these four ABFs induce miR168, which partially contributes to
24
the reduction of the AGO1 transcript. We did not detect significant changes in the expression
25
of MIR168a in the individual abf mutant, which may be explained by the functional redun-
26
dancy of ABF family members (Yoshida et al., 2010).
27
28
The ABRE motif is conserved in plant MIR168 promoters
14
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
The increase of miR168 accumulation during ABA treatment was also observed in poplar
2
(Populus trichocarpa) and tobacco (Nicotiana tabacum) (Jia et al., 2009; Jia et al., 2010).
3
Homologs of miR168 were found in 15 plant species (Table S1) (miRBase, version16.0,
4
http://www.mirbase.org/index.shtml). We also detected ABA induced expression of miR168
5
in rice and maize after 6-hour treatment of 100 µM ABA (Figure S5). To understand whether
6
these diverse plant species share similar regulatory mechanism for miR168 accumulation to
7
Arabidopsis, we scanned MIR168 upstream sequences in 10 dicot and monocot species (Ara-
8
bidopsis thaliana, Brassica napus, Glycine max, Medicago truncatula, Oryza sativa, Populus
9
trichocarpa, Ricinus communis, Sorghum bicolor, Vitis vinifera, Zea mays) (Table S2), using
10
"profit" from EMBOSS (Rice et al., 2000). The promoter regions of these MIR168 genes
11
were predicted by defining the transcription start site using the transcription start site identi-
12
fication
13
ic=tssp&group=programs& subgroup=promoter). We found that most of the transcription
14
start sites were located within the 150-bp upstream region of the predicted hairpins of
15
MIR168 genes (Table S3). For each upstream sequence, a list of candidate ABRE sites was
16
generated by "profit" with percentage scores representing their similarities to the known
17
ABRE motifs (Gomez-Porras et al., 2007). Using an arbitrary 90% threshold of similarity
18
score, we found 24 putative ABRE motifs in 10 MIR168 upstream sequences from 8 species
19
(Figure 7 and Table S3). Some of the ABRE motifs share high similarity to M3, M4 and M5
20
in Arabidopsis MIR168. The putative ABRE motifs displayed a high occurrence in the
21
500-bp upstream region started from the beginning of miR168 hairpin precursors (Figure
22
7A). WebLogo analysis http://weblogo.berkeley.edu) and frequency matrix of the 24 puta-
23
tive ABRE motifs of the eight species showed that the core sequence “ACGTG” is conserved
24
within the promoter region of MIR168a homologs (Figure 7B and 7C). As the negative con-
25
trol, we checked ABRE occurrences within every known promoter in the genome of Arabi-
26
dopsis on the POBO website (http://ekhidna.biocenter.helsinki.fi/poxo/pobo/pobo). POBO
27
analysis showed that the distribution of ABRE in MIR168 upstream sequences was signifi-
program
for
plants
(TSSP)
(http://linux1.softberry.com/berry.phtml?
top-
(
15
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
cantly higher than the background set (Figure S6). These observations indicate that ABA re-
2
gulated MIR168 expression is conserved in diverse plant species.
3
4
DISCUSSION
5
Interference with AGO1 function alters ABA and several abiotic stress responses
6
A number of reports on the identification and functional analysis of stress responsible
7
miRNAs and siRNAs suggest that small RNAs may play an essential role in stress response
8
(Zhou et al., 2007; Sunkar, 2010). In addition, mutations in some genes involved in small
9
RNA biogenesis cause altered ABA and stress response, confirming the regulatory role of the
10
small RNA pathway in plant stress responses (Hugouvieux et al., 2001; Vazquez et al., 2004;
11
Kim et al., 2008; Zhang et al., 2008).
12
Our data showed that not only proteins responsible for miRNA biogenesis but also the
13
slicer required for miRNA action is employed in the post-transcriptional regulation of plant
14
abiotic stress. The reduced AGO1 level in both the hypomorphic allele ago1-27 and the
15
MIR168a overexpression lines leads to significantly enhanced sensitivity to ABA and mul-
16
tiple abiotic stresses, such as increased seed dormancy, inhibition of seed germination and
17
root elongation, and whole-plant drought tolerance. In contrast, mir168a-2 mutant, which
18
contains an increased level of AGO1 (Vaucheret, 2009), displayed ABA hyposensitivity and
19
drought hypersensitivity. These results together indicate that AGO1 is a negative modulator
20
of ABA signaling and stress responses in Arabidopsis.
21
Hypomorphic alleles of AGO1 mutation do not significantly affect the stability of most
22
miRNAs but reduce the cleavage efficiency of AGO1 (Vaucheret et al., 2004; Kurihara et al.,
23
2009). Previous microarray data reveal overaccumulation of a large portion of miRNA target
24
transcripts in ago1-9, ago1-11 and ago1-25 (Ronemus et al., 2006; Kurihara et al., 2009).
25
Among the list there are a number of targets of stress-responsive miRNAs, including the well
26
characterized COPPER/ZINC SUPEROXIDE DISMUTASE 1/2 (CSD1/2), NFYA5, ATP
27
SULFURYLASE 4 (APS4), AUXIN RESPONSE FACTOR 8 (ARF8) and UBIQUI-
28
TIN-CONJUGATING ENZYME 24 (UBC24). Targets of miR398, CSD1 and CSD2 encode
16
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
reactive oxygen species (ROS) scavengers that prevent the deleterious effects of oxidants.
2
Arabidopsis plants overexpressing a miR398-resistant version of CSD2 exhibit enhanced re-
3
sistance to oxidative stresses (Sunkar et al., 2006). NFYA5, target of miR169, which is the
4
positive regulator of drought resistance, is up-regulated by ABA and drought (Li et al., 2008).
5
APS4 is involved in miR395 mediated sulphate assimilation (Hatzfeld et al., 2000). The cir-
6
cuit between miR167a and ARF8 mediates nitrogen induced lateral root initiation and emer-
7
gence (Birnbaum et al., 2008). Down-regulation of UBC24 by miR399 is required for prima-
8
ry root inhibition and phosphate transporters induction in response to Pi starvation (Fujii et al.,
9
2005). Upregulation of these targets affects the response of the plant to adverse environments.
10
Indeed, we observed that the transcripts of CSD1/2, NFYA5, APS4 were upregulated in
11
ago1-27 and 35S:MIR168a lines (Figure S7).
12
13
miR168 modulates AGO1 homeostasis during ABA and several abiotic stress treatments
14
In wild type Arabidopsis, AGO1 homeostasis is maintained by an autoregulatory feedback
15
loop that involves coexpression of MIR168 and AGO1 as well as their interaction at the
16
post-transcriptional level (Vaucheret et al., 2006; Vaucheret, 2009; Varallyay et al., 2010).
17
AGO1 mRNA abundance is also controlled by siRNAs derived from its own transcripts
18
(Mallory and Vaucheret, 2009). In addition, AGO1 activity is further regulated negatively by
19
AGO10 and positively by a cyclophilin protein, SQUINT (SQN) (Mallory et al., 2009; Smith
20
et al., 2009).
21
Here we show that a coordinate transcriptional regulation of MIR168 and AGO1 also plays
22
an important role in modulating plant stress responses. Previous studies have demonstrated
23
that miR168 is induced by various stresses and ABA treatment (Liu et al., 2008; Jia et al.,
24
2009; Jia et al., 2010). Our results further demonstrate that the increase of miR168 level is at
25
least partially caused by the transcriptional activation of MIR168a, a ubiquitously expressed
26
miR168 gene. On the contrary, no significant change of AGO1 mRNA level was detected af-
27
ter 12-hour treatment of ABA, salt and cold. Simultaneously, AGO1 protein level displays
28
only subtle changes in response to ABA and cold, and is slightly induced by drought (Figure
17
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
S8). Less repression of AGO1 abundance might result from distinct regulatory mechanisms at
2
different regulatory levels. Consistent with this hypothesis, we observed that the promoter
3
activity of the transgenic plants harboring pAGO1:GUS construct, which is free from miR168
4
inhibition, is induced by ABA treatment. The opposite effects of ABA on AGO1 at transcrip-
5
tional and post-transcriptional levels might reflect an arms race between the forces transmit-
6
ting or attenuating ABA signals.
7
AGO1 is one of the core factors crucial for miRNA and siRNA-mediated gene silencing.
8
The fluctuation in AGO1 level affects normal development and responses to environmental
9
stimuli in plants (Morel et al., 2002; Ellendorff et al., 2009; Varallyay et al., 2010). Thus, it is
10
not surprising that plants evolve complicated mechanisms to fine-tune AGO1 levels under
11
stress conditions. Recently, miR168 was reported to be hijacked by plant viruses to alleviate
12
plant anti-viral function through inhibiting translational capacity of AGO1 mRNA (Varallyay
13
et al., 2010), indicating that miR168 acts as a rheostat exploited by different regulatory loops
14
to control AGO1 abundance. A novel negative regulator of AGO1, F-BOX WITH WD-40 2
15
(FBW2), was also reported to be responsible for fine-tuning ABA signaling by controlling
16
AGO1 protein level (Earley et al., 2010). Similar to mir168a-2, mutations in FBW2 has near-
17
ly no effect on plant morphology, but affects the sensitivity of plants to ABA (Earley et al.,
18
2010). These observations reveal that AGO1 level is modulated by multiple mechanisms in
19
response to external stimuli. The observation that AGO1 is transcriptionally upregulated by
20
ABA suggests that there might be other yet unknown mechanism involved in transcriptional
21
regulation of AGO1 in response to ABA.
22
23
miR168 is a component of the ABF transcriptional cassette and mediates ABA- depen-
24
dent stress signaling
25
A wealth of evidence reveals that miRNAs play a crucial role in controlling gene expres-
26
sion at the post-transcriptional level by targeting mRNA cleavage or translational repression.
27
However, little is known about the transcriptional regulation of miRNA genes. Previously,
28
myeloid transcription factors, PU.1 and CAAT enhancer-binding protein a (C/EBPa) were
18
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
shown to directly regulate the transcription of pri-miR-223, and this regulatory mechanism
2
seems to be conserved in mouse and human (Fukao et al., 2007). In Arabidopsis, the MIR398
3
expression regulator SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 7 (SPL7) is
4
essential for copper deficiency response and is required for the degradation of mRNAs that
5
encode copper/zinc superoxide dismutases (Yamasaki et al., 2009). A negative feedback loop,
6
which involves direct induction of MIR172b transcription by the miR156 targets SPL9 and
7
SPL10, contributes to the stabilization of the transition from juvenile to adult phases (Wu et
8
al., 2009). Recently, the expression of MIR164 was shown to be directly induced by TEO-
9
SINTE BRANCHED 1 AND CYCLOIDEA AND PCF TRANSCRIPTION FACTOR 3
10
(TCP3) to repress the expression of CUP-SHAPED COTYLEDON (CUC) genes (Koyama et
11
al., 2010). The above observations indicate that many of these post-transcriptional regulators
12
are transcriptionally controlled by developmental or environmental cues.
13
Rapid accumulation of the MIR168a precursor after ABA and stress treatments indicates
14
that MIR168a is regulated by stress signaling at the transcriptional level. ABA is involved in
15
the activation of MIR168 since the upregulation of miR168 by drought treatment was atte-
16
nuated in an ABA-deficient mutant aba1-5. A previous survey for cis-acting elements in
17
stress-responsive miRNAs revealed the presence of multiple ABREs in the MIR168a promo-
18
ter (Liu et al., 2008), suggesting a possible link between MIR168a transcription and
19
ABRE-binding proteins. A small clade of basic leucine zipper transcription factors, called
20
AREB/ABF, has been shown to convey ABA-dependent stress signaling by binding to ABRE
21
motifs within promoters of downstream genes and inducing gene expression (Kim, 2006).
22
The AREB/ABF regulon is the most important transcriptional cassette regulating
23
ABA-dependent gene expression (Jakoby et al., 2002; Yoshida et al., 2010). AREB/ABF pro-
24
teins function at an early step of ABA signal transduction and regulate a large spectrum of
25
stress-responsive genes (Kim, 2006).
26
In this study, we present biochemical and genetic evidence for the direct activation of
27
MIR168a expression by four members of the AREB/ABF family. Surprisingly, elevated
28
AGO1 expression level was also observed in these ABF overexpression lines. This might be
19
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
resulted from an unknown feedback regulatory loop of AGO1 by ABA or the ABF family.
2
The fluctuation of MIR168 and AGO1 expression impacts global miRNA action (Vaucheret et
3
al., 2004; Kurihara et al., 2009). Therefore, it is efficient to employ an initial regulator of one
4
pathway to modulate the central modifier of another pathway, thus coordinating the crosstalk
5
of two pathways. In addition, the findings that miR168 is upregulated by ABA and abiotic
6
stresses as well as that the conserved ABRE motifs were found in the MIR168 upstream se-
7
quences in multiple species lead us to the conclusion that higher plants may share a common
8
regulatory mechanism to control miR168 expression in response to ABA and abiotic stimuli.
9
But we can not exclude the possibility that there may be some other transcriptional factors
10
except ABFs binding to the same or other cis-elements within the promoter of MIR168. Pro-
11
moter deletion analysis will help to uncover other players involved in stress regulated
12
miR168 accumulation.
13
14
MATERIALS AND METHODS
15
Plant Materials and Stress Treatments
16
Arabidopsis thaliana, ecotype Columbia (Col-0) and Landsberg erecta (Ler), were used as
17
the wild-type. ago1-27 and aba1-5 mutants in Col-0 background were kindly provided by Dr.
18
Hervé Vaucheret and Hao Yu, respectively. CS25625 (mir168a-2) in Ler background was ob-
19
tained from the Arabidopsis Biological Resource Center. Plants were grown in the controlled
20
chamber at 22°C, 70% relative humidity under continuous illumination. Two-week-old seedl-
21
ings were subjected to various treatments. For ABA treatment, plants were sprayed with 100
22
µM ABA and harvested at various time points. For salt treatment, seedlings were transferred
23
to solutions containing 300 mM NaCl. For drought treatment, seedlings were dehydrated on
24
filter paper. For cold stress treatment, seedlings were kept at 4°C.
25
26
Plasmid Construction
27
The upstream fragment of MIR168a, spanning nucleotides -2065 to -1 with respect to the
28
MIR168a transcription start site (Jiang et al., 2006), was amplified from Arabidopsis genomic
20
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
DNA and then fused in frame with the GUS coding region in pCAMBIA1301. To generate
2
internal deletion constructs, the ABREs located at -302 to 272 and -126 to 119 nucleotides
3
were replaced with restriction endonuclease binding sites (NcoI and BglII) by PCR. The fu-
4
sion between the promoter of AGO1 and GUS was constructed according to Vaucheret et al
5
(Vaucheret et al., 2006).
6
The Cauliflower Mosaic Virus (CaMV) 35S promoter-MIR168a construct was described
7
previously (Jiang et al., 2006). The coding region of ABF1 and ABF2 were retrieved from
8
SalI/BamHI digested pEASY-Blunt vector and ligated to SalI/BamHI digested pCAM-
9
BIA1300-221-Myc vector to generate 35S:ABF1-Myc and 35S:ABF2-Myc. 35S:ABF3-HA
10
and 35S:ABF4-HA were generated by inserting the fusion of ABF coding region and the he-
11
magglutinin (HA) tag to the pCAMBIA1300-221 vector. These gene fusions were controlled
12
by the CaMV35S promoter and used for transformation of Arabidopsis.
13
14
Histochemical GUS Staining and Quantification of GUS activity
15
Rosette leaves of 3-week-old homozygous transgenic plants were harvested. Histochemical
16
localization of GUS activity was determined as described previously (Kang et al., 2002). For
17
GUS quantification, at least 10 independent transgenic lines for each construct were used and
18
15 two-week-old T2 seedlings from each line were combined to measure GUS activity. At
19
least four repeats were carried out for each line. Standard deviation between lines was calcu-
20
lated.
21
4-methylumbelliferyl-b-D-glucuronide (MUG) assay method (Jefferson, 1987). Each sample
22
was replicated four times.
Glucuronidase
activity
was
quantified
using
the
fluorometric
23
24
RNA Analysis
25
Total RNA was extracted from control or treated plants with Trizol reagent (Invitrogen,
26
USA). miR168 and pre-miR168a were detected by the probe complemented with miR168 and
27
pre-miR168a loop sequences which were modified with 3’ and 5’ biotin. 40 μg total RNA
21
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
was loaded in each lane, and U6 was used as a loading control (Li et al., 2007). After strin-
2
gent washes, signals on the membranes were detected according to the manufacturer’s in-
3
struction for Chemiluminescent Nucleic Acid Detection Module (Thermo, USA). The
4
qRT-PCR analyses were carried out with total RNA using Bio-Rad's real-time PCR amplifi-
5
cation systems (USA). Each gene was assayed on three biological replicates and normalized
6
using the TUBULIN4 cDNA level (Aldon et al., 2008).
7
8
Germination and Seedling Growth Assay
9
Seeds of wild type, ago1-27, 35S:MIR168a and mir168a-2 plants were collected at the
10
same time and stored in the same conditions. They were plated on ABA-free medium for 4
11
days at 4°C before switching to 22°C to germinate, then the germination (fully emerged ra-
12
dicle) efficiency was scored at various times. For germination under various NaCl doses,
13
seeds were plated on MS medium containing 0, 50, 100 and 150 mM NaCl for 4 days before
14
counting. For germination under various ABA doses, seeds were plated on MS medium con-
15
taining 0, 0.1, 0.3 and 0.5 µM ABA for one week. For the root growth assay, plants were
16
germinated on MS plates and transferred after 4 days to fresh plates containing ABA at the
17
final concentrations indicated in the text. Pictures of the elongated roots were taken 5 days
18
later.
19
20
Drought Treatment and Measurement of Transpiration Rate and Stomatal Aperture
21
For drought treatment, 3-week-old soil-grown plants were not watered for 11 to 15 days.
22
The photographs were taken 2 days after the rewatering. The transpiration rate of detached
23
leaves was measured by weighing freshly harvested leaves placed on open dishes on the la-
24
boratory bench, with the abaxial side up. Leaves of similar developmental stages from
25
4-week-old soil-grown plants were used (Kang et al., 2002; Kim et al., 2004).
26
Stomatal aperture analysis was conducted according to the previous description (Li et al.,
27
2008). Stomata apertures were measured 6 days after dewatering. For each treatment, 80
28
measurements were collected, and each experiment was repeated at least three times.
22
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
2
Yeast One-Hybrid Analysis
3
The coding regions of the transcription factors ABF1, ABF2, ABF3, ABF4 and ABI5 were
4
amplified from Arabidopsis Col-0 cDNA using gene specific primers. The PCR products
5
were then cloned into the pGADT7 prey vector (CLONTECH, USA), creating a translational
6
fusion between the GAL4 activation domain and the transcription factor. Three tandem re-
7
peats of the -135 to -110 MIR168a promoter region were synthesized and cloned into pHISi-1
8
vector (CLONTECH, USA) carrying the HIS3 reporter gene and pLacZi vector (CLON-
9
TECH, USA) containing the LacZ reporter gene respectively. Induction of HIS3 and LacZ
10
indicates positive interaction. Yeast one hybrid assay was performed according to the
11
CLONTECH Yeast Protocols Handbook.
12
13
Quantitative Chromatin Immunoprecipitation-PCR (qChIP-PCR) Analysis
14
The procedure for ChIP of ABFs-DNA complexes in the 35S:ABF1-Myc, 35S:ABF2-Myc,
15
35S:ABF3-HA and 35S:ABF4-HA transgenic Arabidopsis leaves was modified from previous
16
descriptions (Bowler et al., 2004). Chromatin was isolated from 1.5 g of formaldehyde
17
cross-linked tissue from leaves of 4-week-old transgenic plants sprayed with 100 μM ABA
18
for 6 hours and seedlings of 2-week-old transgenic plants treated with drought for 6 hours.
19
Preabsorption with protein A-Sepharose beads and immunoprecipitation with c-Myc or HA
20
monoclonal Antibody (SIGMA-ALDRICH, USA) were performed as described in Bowler et
21
al (2004). A small aliquot of untreated sonicated chromatin was reversely cross-linked and
22
used as the total input DNA control. Quantitative ChIP-PCRs were performed with primers
23
specific to different regions upstream of MIR168a. ChIP populations from immune or control
24
immunoprecipitations were used as templates and normalized by the amount of the DNA
25
fragments in corresponding input DNA populations. The difference between the normalized
26
mean cycle threshold (Ct) of immunized population and the pre-immunized population was
27
calculated to obtain the relative enrichment of each upstream fragment (Zhang et al., 2010).
28
The primers used in this article are listed in Supplemental Table 5.
23
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
2
Sequence Analysis of MIR168 Promoters in Plants
3
Sequences of miR168 from Arabidopsis thaliana, Brassica napus, Glycine max, Medicago
4
truncatula, Oryza sativa, Populus trichocarpa, Ricinus communis, Sorghum bicolor, Vitis vi-
5
nifera, Zea mays and their corresponding genome coordinates were obtained from the miR-
6
Base (version16.0, http://www.mirbase.org/index.shtml). Upstream sequences 2 kb relative to
7
the beginning of the predicted hairpins were extracted from available genome sequences re-
8
spectively according to their coordinates. These 2-kb upstream sequences were then scanned
9
to identify ABRE sites on both plus and minus strands using "profit" from EMBOSS (Rice et
10
al., 2000). A previously discovered matrix of ABRE was adopted as input matrix for "profit"
11
(Gomez-Porras et al., 2007). In addition, Plant Promoter Identification program (TSSP,
12
http://linux1.softberry.com/berry.phtml? topic=tssp&group=programs& subgroup=promoter)
13
was used to check the transcription start site of these sequences.
14
15
Western blot analysis
16
The AGO1 antibody was purchased from Agrisera (Sweden) was used at 1:3000 dilution in
17
the immunoblot analysis. Anti-β-Tubulin was purchased from Santa Cruz Biotechnology, Inc
18
(USA) was used at 1:1000 dilution in the experiment.
19
20
Accession numbers for the genes in this article are as follows: MIR168a (AT4G19395);
21
AGO1 (AT1G48410); TUBULIN4 (At5g44340); RD29A (AT5G5 2310); ABA1 (AT5G67030);
22
ABF1 (AT1G49720); ABF2 (AT1G45249); ABF3 (AT 4G34000); ABF4 (AT3G19290); ABI5
23
(AT2G36270); NFYA5 (AT1G54160); APS4 (AT5G43780); CSD1 (AT1G08830) and CSD2 (
24
AT2G28190).
25
26
ACKOWLEDGEMENTS
27
We thank Dr. Hervé Vaucheret for kindly providing the mutant ago1-27, Dr. Hao Yu for pro-
28
viding the mutant aba1-5, Dr. Jianping Hu for editing the manuscript, TAIR for supplying
24
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
1
mir168a-2 seeds. We also thank Dr. Yuke He for providing the protocol for small RNA blot.
2
3
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FIGURE LEGENDS
24
Figure 1. Expression analysis of mature miR168, pre-miR168a, AGO1 and GUS in
25
wild-type, aba1-5 and pAGO1:GUS transgenic plants under ABA and abiotic stress
26
treatments.
27
Northern blot analysis of mature miR168 and pre-miR168a (A) and qRT-PCR analysis of
28
AGO1 mRNA (B) in 2-week-old wild-type Arabidopsis seedlings (Col) with drought, 300
29
mM NaCl, cold and 100 μM ABA treatments. The significant induction of AGO1 mRNA by
30
12 h drought treatment is indicated with asterisks (n=3, P < 0.05, Student’s t test). (C) North-
31
ern blot analysis of miR168 and qRT-PCR analysis of AGO1 mRNA in 2-week-old wild-type
30
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1
and aba1-5 mutant plants (n=10) under standard and drought conditions. Numbers below the
2
blot figures in A and C show the relative abundance compared to the control U6, the level of
3
each target in non-treated wild type (Col) was set to 1. These experiments were repeated three
4
times and similar results were observed. (D) qRT-PCR analysis of GUS and AGO1 mRNA in
5
three independent 2-week-old pAGO1:GUS transgenic lines with the treatment of drought for
6
0 h, 6 h and 12 h. (E) qRT-PCR analysis of GUS and AGO1 mRNA in three independent
7
2-week-old pAGO1:GUS transgenic lines with the application of 100 μM ABA over time.
8
The significant difference between GUS and AGO1 is indicated with asterisks (n=3, P < 0.05,
9
Student’s t test). Error bars denote standard deviation (SD). Quantifications were normalized
10
to the expression of TUBULIN4. The level of AGO1 and GUS in wild type (Col) or
11
non-treated sample was set to 1.
12
13
Figure 2. ago1-27 and MIR168a overexpression plants display ABA and salt hypersensi-
14
tivity.
15
(A) 35S:MIR168a and ago1-27 seeds showing delayed germination compared with wild-type
16
seeds (Col) on ABA-free medium. Each data point represents the mean of triplicate experi-
17
ments (n=100 each), and the bars indicate standard errors. (B) Seven-day-old 35S:MIR168a
18
and ago1-27 seedlings on MS medium containing 0.3 µM ABA. The representative picture is
19
shown (C) ABA inhibition of primary root elongation of 35S:MIR168a (line 4 and 6) and
20
ago1-27 seedlings. Seeds were germinated on MS medium for 4 days, transferred to media
21
containing 30 μM ABA, and root length was measured after 5-day treatment. The representa-
22
tive picture is shown. (D) Seed germination on media containing 0, 50, 100, or 150 mM NaCl.
23
Experiments were performed in triplicate (n=100 each), and the bars indicate standard errors.
24
25
Figure 3. ago1-27 and MIR168a overexpression plants display enhanced drought toler-
26
ance.
27
(A) Three-week-old plants treated with drought for 15 days before being rewatered. The
28
photographs were taken 2 days after the rewatering. Number codes indicate number of sur-
31
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1
viving plants out of total number (three repeats, 90 plants in total). (B) Transpiration rate of
2
4-week-old plants. Rosette leaves (third and fourth leaves) at same developmental stages
3
were detached and weighed at various times. Each data point represents the mean of triplicate
4
experiments (n=10 each), and the bars show standard errors. (C) Stomatal aperture of leaves
5
from 3-week-old plants with drought treatment for 6 days. (n=80, P < 0.05, Student’s t test).
6
Error bars denote standard error.
7
8
Figure 4. mir168a-2 plants display ABA hyposensitivity and drought hypersensitivity.
9
(A) Germinations of 4-day-old wild type and mir168a-2 seedlings on MS medium containing
10
different concentrations (0, 0.1, 0.3, 0.5 μM) of ABA. Each data point represents the mean of
11
triplicate experiments (n=100 each), and the bars show standard errors. (B) ABA inhibition of
12
the primary root elongation of mir168a-2 seedlings. Seeds were germinated on MS medium
13
for 3 days, transferred to media containing 10 μM ABA, and root length was measured after
14
5-day treatment. The representative picture is shown. (C) Transpiration rate of 4-week-old
15
mir168a-2 leaves. Rosette leaves (the third and fourth leaves) at same developmental stages
16
were detached and weighed at various times. Each data point represents the mean of triplicate
17
experiments (n=10 each), and the bars show standard errors. (D) Stomatal aperture of
18
3-week-old leaves with drought treatment for 6 days (n=80, P < 0.05, Student’s t test). Error
19
bars denote standard error. (E) Three-week-old plants treated with drought for 11 days before
20
being rewatered. The photographs were taken 2 days after the rewatering. Number codes in-
21
dicate number of surviving plants out of total number (three repeats, 87 plants in total).
22
23
Figure 5. ABF1, ABF2, ABF3 and ABF4 bind the MIR168a promoter in vitro and in vivo.
24
(A) Different constructs of the MIR168a promoter. The blue boxes indicate ABRE; the white
25
boxes indicate mutated ABRE. The five ABREs were named M1 to M5, respectively.
26
pMIR168aΔ0:GUS contains a 1000-bp promoter region beginning from the transcription start
27
site (TSS). pMIR168aΔ1:GUS contains a 800bp promoter beginning from the TSS with M1
28
and M2 deleted. pMIR168aΔ2:GUS contains a 1000-bp promoter with mutated M3 and M4.
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1
pMIR168aΔ3:GUS contains a mutated M5. pMIR168aΔ4:GUS contains mutated M3, M4 and
2
M5. (B) GUS activity in 10 independent transgenic Arabidopsis plants (n=15) harboring var-
3
ious constructs shown in part A with or without 6 h 100 μM ABA treatment. Data show
4
mean± SD for four independent experiments (*P < 0.05, Student’s t test). (C) The bait DNA
5
sequence used in yeast one hybridization contained three tandem repeats of a 25-bp fragment
6
(5’CAAGAGAACACGTGTCGGAAAATGA3’) which includes the ABRE motif M5 within
7
the MIR168a promoter. Six plasmids containing insert DNA fragments of ABF1, ABF2,
8
ABF3, ABF4, ABI5 and TDR were retransformed into yeast strains YM4271 carrying the re-
9
porter genes HIS and lacZ under the control of the 75-bp fragment containing three M5 motif.
10
The transformants were examined for growth in the presence of 3-AT and β-galactosidase
11
(β-gal) activity. (D) Schematic diagram of the MIR168a promoter. The bent arrow indicates
12
the translational starting site. The blue boxes indicate ABRE motifs. F1, F2 and F3 represent
13
DNA fragments amplified in the quantitative ChIP-PCR assay. (E) ChIP enrichment test
14
showing the binding of ABF1-Myc, ABF2-Myc, ABF3-HA and ABF4-HA to the MIR168a
15
promoter after application of 100 μM ABA for 6 hours. (F) ChIP enrichment test showing the
16
binding of ABF1-Myc, ABF2-Myc, ABF3-HA and ABF3-HA to the MIR168a promoter un-
17
der drought condition for 6 hours. Error bars denote SD (n=5, P < 0.05, Student’s t test).
18
19
Figure 6. Expression analysis of miR168 and AGO1 in wild type and ABF1, ABF2, ABF3
20
and ABF4 overexpression lines with or without ABA treatment.
21
(A) Mature miR168 and pre-miR168a accumulated in the individual ABF1, ABF2, ABF3 and
22
ABF4 overexpression lines (ABF1OE, ABF2OE, ABF3OE and ABF4OE) with 0 h, 6 h and 12
23
h ABA treatment by Northern blot analysis. This experiment was repeated three times inde-
24
pendently and similar results were observed. Numbers below the blot figures show the rela-
25
tive abundance compared to the control U6, the level of each target in non-treated wild type
26
(Col) was set to 1. (B) AGO1 mRNA also induced in the individual ABF1, ABF2, ABF3 and
27
ABF4 overexpression lines with 0 h, 6 h and 12 h ABA treatment by qRT-PCR analysis. But
28
the scale of the inductions was decreased by ABA. The expression level of AGO1 mRNA in
33
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1
the wild-type plants under non-stressed conditions was defined as 1.0. Error bars denote SD
2
(n=3).
3
4
Figure 7. Plants have conserved ABRE motifs within the MIR168 promoters.
5
(A) The localizations of ABRE motifs in 2-kb upstream sequences of 10 plant MIR168 hair-
6
pin precursors in 8 species (ath: Arabidopsis thaliana, bna: Brassica napus, osa: Oryza sativa,
7
ptc: Populus trichocarpa, rco: Ricinus communis, vvi: Vitis vinifera, gma: Glycine max, zma:
8
Zea mays). The yellow box denotes ABRE motif in the sense strand (ABRE+); the red box
9
denotes ABRE motif in the antisense strand (ABRE-); the blue arrow denotes TSS; two-way
10
arrow denotes TATA box; the blue line denotes the hairpin precursor of MIR168 gene. (B)
11
Base composition of ABRE motifs which were found in the promoters of MIR168 from the
12
eight common species. (C) Frequency matrix of the ABRE motifs in the promoters of
13
MIR168 from the eight species.
14
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