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Distinctive expression patterns and roles of the

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Distinctive expression patterns and roles of the miRNA393TIR1

homolog module in regulating flag leaf inclination and primary
and crown root growth in rice (Oryza sativa)
Hongwu Bian1*, Yakun Xie1*, Fu Guo1, Ning Han1, Shengyun Ma1, Zhanghui Zeng1, Junhui Wang1,
Yinong Yang1,2 and Muyuan Zhu1
1
Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Institute of Genetics, College of Life Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China; 2Department of
Plant Pathology and Huck Institute of Life Sciences, Pennsylvania State University, University Park, PA 16802, USA

Summary
Author for correspondence: MicroRNA (miRNA)-mediated regulation of auxin signaling components plays a critical role
Muyuan Zhu in plant development. miRNA expression and functional diversity contribute to the complexity
Tel: +86 571 88206536 of regulatory networks of miRNA target modules.
Email: myzhu@zju.edu.cn
This study functionally characterizes two members of the rice (Oryza sativa) miR393 family
Received: 6 May 2012 and their target genes, OsTIR1 and OsAFB2 (AUXIN SIGNALING F-BOX), the two closest
Accepted: 21 June 2012 homologs of Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 (TIR1).
We found that the miR393 family members possess distinctive expression patterns, with
New Phytologist (2012) 196: 149161 miR393a expressed mainly in the crown and lateral root primordia, as well as the coleoptile
doi: 10.1111/j.1469-8137.2012.04248.x tip, and miR393b expressed in the shoot apical meristem. Transgenic plants overexpressing
miR393a b displayed a severe phenotype with hallmarks of altered auxin signaling, mainly
including enlarged flag leaf inclination and altered primary and crown root growth. Further-
Key words: crown root growth, flag leaf
inclination, miRNA393, primary root, rice more, OsAFB2- and OsTIR1-suppressed lines exhibited increased inclination of flag leaves at
(Oryza sativa). the booting stage, resembling miR393-overexpressing plants. Moreover, yeast two-hybrid
and bimolecular fluorescence complementation assays showed that OsTIR1 and OsAFB2
interact with OsIAA1.
Expression diversification of miRNA393 implies the potential role of miRNA regulation
during species evolution. The conserved mechanisms of the miR393 target module indicate
the fundamental importance of the miR393-mediated regulation of auxin signal transduction
in rice.

This interaction results in Aux IAA ubiquitination and subsequent

Introduction
The plant hormone auxin influences virtually every aspect of
growth and development in plants, including stem elongation,
phototropic and gravitropic responses, apical dominance, and
lateral and adventitious root formation (Vanneste & Friml,
2009). Auxin promotes the degradation of auxin indole-3-acetic
acid (Aux IAA) transcriptional repressors, thereby allowing
AUXIN RESPONSE FACTORS (ARFs) to activate the tran-
scription of auxin-responsive genes (Worley et al., 2000; Gray
et al., 2001; Tiwari et al., 2001). Degradation of Aux IAA tran-
scriptional repressors requires TRANSPORT INHIBITOR
RESPONSE PROTEIN 1 (TIR1), an auxin receptor (Dharmasiri
et al., 2005; Kepinski & Leyser, 2005). Auxin binding to TIR1,
the F-box subunit of the ubiquitin ligase complex SCF (TIR1),
stabilizes the interaction between TIR1 and the Aux IAA proteins.
degradation (Gray et al., 2001). Auxin signaling components
have been conserved throughout land plant evolution, and have
proliferated and specialized to control specific developmental
processes (Chapman & Estelle, 2009). Research on monocots
and eudicots has shown similar mechanisms for auxin signaling
in these divergent species. However, monocots have a very dif-
fer ent anatomy from dicots (McSteen, 2010), and many of
the characteristics that distinguish monocots and dicots involve
structures whose development is controlled by auxin, such as
cotyledons, the root system and the leaf vasculature. It is not yet
clear whether auxin controls the differences in morphology seen
in dicots vs monocots. So far, both conservation and diversi-
fication of mechanisms of auxin signal transduction have been
discovered. There are 31 members in the Aux IAA gene family
in rice (Jain et al., 2006), 24 of which are regulated by auxin
(Song et al., 2009a) and three of which have been functionally
characterized. OsIAA1 3 transcription is induced by auxin and

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suppressed by light (Thakur et al., 2001), and the protein has a
short half-life and is degraded by the proteosome (Thakur et al.,
2005), similar to Aux IAA proteins in Arabidopsis. Overex-
pression of OsIAA1 causes defects in root development and an
additional defect of enlarged leaf angle (Song et al., 2009b). The
F-box gene family, including homologs of the TIR1 gene family,
has been catalogued in rice (Jain et al., 2007). However, it
remains unclear whether the TIR1 and TIR1-like auxin receptor
interaction with Aux IAA proteins in Arabidopsis is conserved in
other plants, particularly monocots.
Conserved microRNAs (miRNAs) target conserved homolo-
gous genes in diverse plant species (Jones-Rhoades et al.,
2006). miR393 is a conserved miRNA family discovered in
many plants. In Arabidopsis, four F-box genes, TIR1, AFB1,
AFB2 and AFB3 (AUXIN SIGNALING F-BOX), have been
validated as targets of miR393 (Jones-Rhoades & Bartel, 2004;
Navarro et al., 2006; Parry et al., 2009). Loss of TIR1 has a
modest effect on auxin responses and plant development; how-
ever, quadruple tir1 afb mutants exhibit a severe embryonic
phenotype and a variety of growth defects, indicating that
these genes have overlapping functions (Dharmasiri et al.,
2005). Recent studies have demonstrated that the miR393 target
regulatory module plays a role in root system architecture during
nitrate responses (Vidal et al., 2010), leaf development
(Si-Ammour et al., 2011), auxin response (Parry et al., 2009;
Chen et al., 2011) and antibacterial resistance in response to
pathogen attack (Navarro et al., 2006). In rice, the miR393 fam-
ily is encoded by two loci, MIR393a and MIR393b (Jones-Rho-
ades & Bartel, 2004; Archak & Nagaraju, 2007). Recently,
conserved miRNA targets OsTIR1 (Os05g05800) and OsAFB2
(Os04g32460), which encode putative TIR1-like proteins, were
identified by degradome sequencing (Li et al., 2010; Zhou et al.,
2010) and 5-rapid amplification of cDNA ends (5-RACE) (Xia
et al., 2012). Genome-wide analyses have revealed that Oryza
sativa and Arabidopsis thaliana possess apparently divergent pat-
terns of miRNA regulatory systems (Zhang et al., 2011). To
date, however, the expression patterns of miR393 in monocots
remains virtually unknown. Therefore, it is necessary to
examine whether diversified expression has occurred in the rice
miR393 family and how it affects growth and development in
monocots.
In this study, we report the characterization of rice miR393
and knockdown of its two target genes, OsTIR1 and OsAFB2,
the two closest homologs of Arabidopsis TIR1. Our results
reveal conserved roles of the miR393 target module in auxin
signal transduction between Arabidopsis and rice, but distinc-
tive expression patterns and functions in growth and develop-
ment in rice.
Materials and Methods
Plant materials and growth conditions
Rice (Oryza sativa L. ssp. japonica cv. Nipponbare) was used in
this study. Plants were cultivated in an experimental field during
natural growing seasons. For root measurements in seedlings,
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sterilized seeds were germinated in the dark for 23 d and then
transferred to nutrient solution culture (Yoshida et al., 1976) at
pH 5.06.0 in the glasshouse at 28C with 16 h of light and 8 h
of dark. For expression pattern analyses, various tissues were
collected at different stages when the seedlings were grown in
normal rice solution culture.
Gene constructs and rice transformation
To generate the 35S:miR393 constructs, 583- and 747-bp frag-
ments surrounding the miRNA sequence including the fold-back
structure were amplified from genomic DNA with the specific
primers for 35S:miR393a and 35S:miR393b, respectively. The
amplified fragments were sequenced and subcloned into KpnI
and BamHI sites downstream of the Cauliflower mosaic virus
(CaMV) 35S promoter in pCAMBIA13011 (a derivative of
pCAMBIA1301 carrying the 35S promoter and the RBS termina-
tor).
For the promoter:b-glucuronidase (promoter:GUS) con-
structs, 2.0- and 2.5-kb fragments upstream from the predicted
fold-back of miR393 were amplified for ProMIR393a:GUS and
ProMIR393b:GUS, respectively. The fragments were then trans-
ferred into the pENTR D-TOPO vector (Invitrogen, Carlsbad,
CA, USA) and subsequently into the destination vector
pHGWFS7 containing an Egfp: uidA gene fusion by LR clonase
reactions according to the manufacturers instructions.
For RNA interference (RNAi) constructs, 885- and 751-bp
fragments were amplified from OsTIR1 and OsAFB2, respec-
tively. The fragments were inserted into the pENTR D-TOPO
vector (Invitrogen) and subsequently cloned into pH7GWIWG2
(I) by LR clonase reactions.
MIM393 was generated as a target-mimic construct. The
24-nucleotide, miR399-complementary motif in IPS1 (Franco-
Zorrilla et al., 2007) was exchanged for a sequence complemen-
tary to miR393. The construct was subcloned into KpnI and
BamHI sites under the control of the 35S promoter in pCAM-
BIA13011.
Rice transformation was performed by the Agrobacterium
tumefaciens-mediated co-cultivation approach. Transformed calli
were selected on hygromycin-containing medium. Homozygous
T3 seeds were used for further study.
All primers used in this work are listed in Supporting Informa-
tion Table S1. The gene constructs used for rice transformation
were verified by sequencing.
DNA gel blot analysis
For DNA gel blot analysis, total DNA was isolated from
approximately 1 g of leaf tissues from transformed plants, as
described previously. DNA (20 lg) was digested with EcoRI
at 37C overnight, separated on 1% (w v) agarose gel and
then transferred to a nylon membrane. Subsequent hybrid-
ization with a digoxigenin-labeled HPT-specific probe was
performed using the DIG High Prime DNA Labeling
and Detection Starter Kit II (Roche) according to standard
protocols.
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Real-time reverse transcription-polymerase chain reaction
(RT-PCR) assays
Total RNA was isolated with the TRIzol RNA Extraction Kit
(Invitrogen), and treated with RNAse-free DNAse I (TaKaRa,
Dalian, China). Real-time PCR was performed by the Master-
cycler ep realplex system (Eppendorf, Hamburg, Germany) with
the SYBR PrimeScript RT-PCR Kit (Perfect Real Time; TaKa-
Ra). The gene-specific primers are listed in Table S1. Primers for
OsTIR1 and OsAFB2 were designed from each side of the
miR393 cleavage site, and relative transcript levels were normal-
ized using OsActin as a standard.
Small RNA blot analysis
Total RNA was extracted from roots and leaves at various stages,
flag leaves and panicles at the booting stage using TRIzol reagent
(Invitrogen). Thirty micrograms of total RNA were fractionated
on 17% polyacrylamide gels under denaturing conditions (7 M
urea). Blots were hybridized using digoxigenin end-labeled LNA
oligonucleotides (Exiqon, Vedbaek, Denmark) complementary
to the miR393a sequence.
Histochemical detection of GUS activity
Histochemical localization of GUS staining was performed by
incubating tissues and organs in a solution of 1 mg ml)1
5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid (Sigma), 1 mM
potassium ferricyanide, 0.1% Triton X-100, 0.1 M sodium
phosphate buffer, pH 7.0, and 10 mM EDTA overnight at
37C, followed by clearing with 70% ethanol.
Auxin resistance analysis
Rice seeds were dehusked and sterilized with 70% ethanol for 5
min and then with 20% sodium hypochlorite (v v) for 30 min,
rinsed five times with sterile water, and placed on N6 medium
supplemented with or without 100 nM 2,4-dichlorophenoxy-
acetic acid (2,4-D; Sigma), and incubated at 28 1C in a
growth chamber (12 12-h photoperiod and 250 lmol s)1 m)2
light intensity). After 2 wk, the seedlings were observed and mea-
sured. Photographs were taken with a Nikon D80 camera
(Nikon, Ayutthaya, Thailand).
Alternatively, sterilized rice seeds were germinated in the dark
for 23 d and then transferred to moistened filter paper. To eval-
uate auxin resistance, seedlings with a root length of approxi-
mately 3 cm were grown hydroponically, supplemented with
various concentrations of 2,4-D (0, 50, 100 and 200 nM). The
root length was measured at different time periods as indicated.
More than 20 seedlings were used in each treatment. ANOVA
followed by Tukey post-tests was performed to determine the sig-
nificant differences.
Subcellular localization
The OsTIR1:GFP and OsAFB2:YFP fusions were obtained by
Gateway cloning (Invitrogen). The full-length cDNAs of stop
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codon-less OsTIR1 and OsAFB2 were amplified using KOD FX
DNA Polymerase enzyme (Toyobo, Kyoto, Japan). The cDNAs
were transferred into pENTR D-TOPO vector (Invitrogen) and
subsequently cloned into pH7FWG2 and pB7YWG2 (Karimi
et al., 2002), respectively, by LR clonase reactions. The fusion
constructs were transformed into onion epidermal cells by parti-
cle bombardment. After transformation, onion epidermal cells
were incubated at 22C on Murashige and Skoog salt (MS) plates
in the dark for 16 h before examination. Enhanced green fluores-
cent protein (EGFP), yellow fluorescent protein (YFP) and cyan
fluorescent protein (CFP) fluorescence and differential interfer-
ence contrast (DIC) images were visualized by a Zeiss Leica TCS
SP5 confocal microscope (Mannheim, Germany).
Yeast two-hybrid assay
The vectors and strains used for the yeast two-hybrid assay
were provided in the Matchmaker GAL4 Two-Hybrid
System 3 (Clontech, CA, USA). The yeast two-hybrid assay and
a-galactosidase activity measurement were performed according
to the Yeast Protocol Handbook (Clontech, CA, USA). The
BD-OsTIR1, BD-OsAFB2 and AD-OsIAA1 plasmids were con-
structed by inserting PCR fragments of full-length cDNAs into
the appropriate plasmids. The PCR fragment of OsTIR1 was
amplified with the specific primers containing an EcoRI or SalI
site. The resulting fragment was digested with EcoRI and SalI,
and cloned into pGBKT7 to generate plasmid BD-OsTIR1. The
PCR fragment of OsAFB2 was cloned into pGBKT7 to generate
plasmid BD-OsAFB2. The PCR fragment of OsIAA1 was ampli-
fied with the primers containing an NdeI or XhoI site, and cloned
into pGADT7 to generate plasmid AD-OsIAA1. The plasmids
were transformed into yeast strain AH109. All primers used for
yeast two-hybrid assays are listed in Table S1.
Bimolecular fluorescence complementation (BiFC) assays
The cDNAs of OsTIR1 OsAFB2 and OsIAA1 were inserted into
the pENTR D-TOPO vector and subsequently cloned into
pUGW2-nYFP and pUWG2-cYFP (Nakagawa et al., 2007),
respectively, by LR clonase reactions (Invitrogen). As a result,
OsTIR1 OsAFB2 were fused to the split N-terminal YFP frag-
ment and OsIAA1 to the C-terminal YFP fragment. The fusion
constructs were transformed into onion epidermal cells by particle
bombardment. Fluorescence and DIC images were visualized by
a Zeiss Leica TCS SP5 confocal microscope.
Results
Distinctive expression patterns of miR393 family members
The rice genome contains two miRNA393 genes: OsmiR393a
and OsmiR393b (Jones-Rhoades & Bartel, 2004; Navarro et al.,
2006; Parry et al., 2009). The mature OsmiR393a and
OsmiR393b are 21- and 22-nucleotide RNAs, respectively, and
their sequences are identical, except that OsmiR393b has an extra
U nucleotide at its 3 end (Fig. S1). To characterize the
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expression pattern of the two miR393 family members, we ana-
lyzed miR393 expression via small RNA blots in various organs
and tissues. Interestingly, the miRNA expression varied consider-
ably between miR393a and miR393b in wild-type plants
(Fig. 1a). Mature miR393a was detected in the roots of young
rice seedlings at the one-leaf and three-leaf stages (1L-R and
3L-R), but was undetectable in leaves (1L and 3L). By contrast,
miR393b expression was high in aerial organs, but undetectable
in roots. miR393b accumulated in young leaves (1L and 3L), flag
leaves (FL) and inflorescence tissues (BP), with the highest level
in flag leaves. Thus, the accumulation patterns were different
between miR393a and miR393b, especially in aerial and under-
ground organs in rice.
(a)
(b)
(c)
Fig. 1 Expression analysis of miR393 in wild-type rice (Oryza sativa)
plants. (a) RNA gel blot analysis of accumulation of miR393 in wild-type
plants. Twenty-one- and twenty-two-nucleotide small RNAs represent
miR393a and miR393b, respectively. 1L, first leaf; 1L-R, root of one-leaf
stage seedlings; 3L, third leaf; 3L-S and 3L-R, shoot and root of three-leaf
stage seedlings; 10L, tenth leaf; 10L-R, 10-leaf stage root; FL, flag leaf; BP,
booting panicle. (b, c) Quantitative reverse transcription-polymerase chain
reaction (RT-PCR) analyses of the transcripts of two MIR393 precursors in
various tissues during different developmental stages as indicated in (a).
Mean values were obtained from three independent samples. Error bars
represent standard deviation.
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To quantitatively investigate the transcription levels of the
miR393 family members, we examined MIR393a and MIR393b
precursors in various tissues by quantitative RT-PCR. Quantita-
tive RT-PCR analysis showed that the transcripts of the
MIR393a precursor were present at high levels in the roots of
seedlings (Fig. 1b), whereas the MIR393b precursor was detected
at a high level in young leaves (2L) of seedlings, and steadily
decreased throughout plant growth (Fig. 1c). Therefore, the
levels of MIR393a and MIR393b precursors were consistent with
the accumulation of their mature miRNAs in various tissues. The
transcription patterns demonstrate that MIR393a is actively
expressed in roots, whereas MIR393b is expressed mainly in the
aerial organs.
To examine the spatial expression patterns of the MIR393
genes in detail, we generated the reporter gene fusions
ProMIR393a:GUS and ProMIR393b:GUS, in which 22.5-kb
fragments upstream of the miRNA stemloop regions were iso-
lated and fused with GUS. These reporter constructs were intro-
duced into rice by Agrobacterium-mediated transformation. GUS
staining showed that miR393a was strongly expressed in emerg-
ing crown adventitious roots, but not in the primary root
(Fig. 2a,b). In emerging crown roots, the GUS expression
domain stopped 0.5 mm from the root tip (Fig. 2c). In the ini-
tial stage of lateral root primordium formation, GUS staining
was observed in pericycle cells, and gradually disappeared in the
developing primordium (Fig. 2d). Interestingly, we found that
GUS activity was very strong in the coleoptile tip of imbibed seed
(Fig. 2e), and gradually appeared in the scutellar node of the
embryo in germinating seed (Fig. 2f). GUS staining was also
observed in the immature anther at early stages of floral develop-
ment (Fig. 2g). By contrast, MIR393b was not detected in the
roots, immature anthers or coleoptiles, but was expressed in the
shoot apical meristem (Fig. 2h). This supports the notion that
the MIR393a and MIR393b genes are transcriptionally regulated
in a tissue-specific manner. Compared with the expression of
MIR393b, MIR393a expression is restricted to more localized
regions with high auxin concentrations, such as coleoptile tips and
lateral root primordia. Together, all of the above results suggest that
the miR393 family members possess distinctive, tissue-specific
expression patterns in rice.
Overexpression of miR393 dramatically influences the
growth and development of rice
The differential expression patterns of OsmiR393 family mem-
bers prompted us to investigate whether functional diversification
occurred in these two miR393 members. We generated trans-
genic plants expressing the stemloop precursors of miR393a
and miR393b under the control of the CaMV 35S promoter.
Lines containing a single copy of the transgene were selected from
independent T0 generation lines based on DNA gel blot analysis
(Fig. S2). Homozygous T3 transgenic seeds were obtained for
further investigation. Phenotypic observation of the transgenic
plants showed that the overexpression of miR393 dramatically
enlarged the lamina joint angle of flag leaves (Fig. 3a). The angle
of the lamina joint of miR393-overexpressing plants was > 90,
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(a) (b)
*
*
* *
(e) (f)
Fig. 2 Spatial expression patterns of MIR393a and MIR393b. (ag) b-Glucuronidase (GUS) activity in the ProMIR393a:GUS transgenic rice line. (a) Roots.
An arrow indicates the primary root, and asterisks indicate the crown roots. (b) Crown roots. (c) Emerging crown roots. (d) Region of lateral root initiation.
An arrow indicates the crown root, and asterisks indicate the lateral roots. (e) Coleoptiles of 2-d-old seed after imbibition. (f) Embryo during seed
germination. (g) Immature anther. (h) GUS activity in the shoot apical meristem in the ProMIR393b:GUS transgenic line. Bars: (a, b, e, f) 2 mm; (c, d, g)
0.5 mm; (h) 3 mm.
whereas that of the wild-type was < 10 at the booting stage
(Fig. 3b,c). In addition, overexpression of miR393 reduced plant
height (Fig. 3c).
In addition to altered flag leaf angle and plant height,
35S:miR393a and 35S:miR393b seedlings had longer primary
roots than the wild-type control after 2 wk of solution culture
(Fig. 4a). The number of crown roots was reduced in 4-wk-old
transgenic seedlings compared with the wild-type (Fig. 4b).
These results demonstrate that both miR393a- and miR393b-
overexpressing plants displayed typical phenotypes associated
with altered auxin signaling.
In addition, the miR393-overexpressing plants displayed other
changes throughout seed development. For example, the outer
glume of the spikelet had an abnormal shape and failed to close
(Fig. 5a). Furthermore, the size of the transgenic seeds was
reduced compared with wild-type seeds (Fig. 5b). Small RNA
blot analysis of miR393 expression showed that the transgenic
plants with strong phenotypic aberrations had high expression
levels of miR393 (Fig. 5c). A clear parallel between the severity
of phenotypes and the level of miR393 expression suggests that
the observed phenotypes were indeed caused by the ectopic
expression of miR393. Taken together, our results demonstrate
that overexpression of miR393 dramatically influences plant
growth and development in rice.
Characterization of target genes regulated by miR393 and
their expression patterns
The rice miR393 family is predicted to target two TIR1-like
genes, Os04g32460 and Os05g05800, which were identified by
degradome sequencing (Li et al., 2010; Zhou et al., 2010). Each
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(c) (d)
*
*
(g) (h)
has a sequence that is partially complementary to that of miR393
with three mismatched nucleotides (Fig. S1). In Arabidopsis,
miR393 targets the auxin receptors AFB1, AFB2, AFB3 and
TIR1. Phylogenetic analysis of TIR1 AFB genes has shown that
Os04g32460 and Os05g05800 are likely to be orthologous to
Arabidopsis AFB2 and TIR1 (Jones-Rhoades & Bartel, 2004;
Navarro et al., 2006; Parry et al., 2009). Recently, Os04g32460
and Os05g05800 were named as OsAFB2 and OsTIR1, respec-
tively (Xia et al., 2012).
Target mimicry, in which an uncleavable target is expressed in
the plant, has been used to sequester specific miRNAs in nonpro-
ductive interactions, thus increasing the levels of the endogenous
target transcripts (Franco-Zorrilla et al., 2007). 35S:MIM393
was generated as such a target-mimic construct for miRNA393.
We measured the transcripts of OsAFB2 and OsTIR1 in
35S:MIM393, 35S:miR393a and 35S:miR393b transgenic plants
by quantitative RT-PCR analyses with primers flanking the
miR393 target sites. We observed that the transcript levels of
OsTIR1 and OsAFB2 increased in the flag leaves of 35S:MIM393
transgenic line compared with wild-type plants (Fig. 6a). The
increased levels of miR393 caused a dramatic reduction in the
transcript levels of OsAFB2 and OsTIR1 in miR393-overexpress-
ing plants. Our results confirmed that OsTIR1 and OsAFB2, neg-
atively regulated by miR393, are miR393 targets in rice.
To determine the expression pattern of miR393 target genes,
real-time RT-PCR was performed with total RNA isolated from
roots, leaves and shoots at various growth stages. The pattern of
OsTIR1 expression is similar to that of OsAFB2, with the highest
level of expression in leaf two (2L) from young seedlings
(Fig. 6b,c). The expression of the two genes decreased dramati-
cally in leaf three (3L), and then steadily increased from leaf three
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(a)
(b)
WT 35S:miR393a 35S:miR393b
(c)
* *
* *
Fig. 3 Overexpression of miR393 increases the inclination of flag leaves
and decreases the rice plant height. (a) Overexpression of miR393 in trans-
genic rice plants results in increased laminar angles of flag leaves at the
booting stage. Wild-type (WT) (left), 35S:miR393a (middle) and
35S:miR393b (right) transgenic lines are shown. Arrows indicate the flag
leaves. Bar, 10 cm. (b) Close-up view of individual lamina joints of the WT
and 35S:miR393a and 35S:miR393b transgenic lines shown in (a).
(c) Quantification of the lamina joint angles of the flag leaves (left) at the
booting stage, and plant heights (right) at the seed maturation stage. Data
represent means standard deviation (n > 20). Asterisks show significant
(ANOVA; *, P < 0.05) difference between overexpressing lines and WT.
to leaf ten (10L) and flag leaves (FL). In roots, the expression levels
of OsAFB2 and OsTIR1 were highest in young roots from
one-leaf (1L-R) and two-leaf (2L-R) stage seedlings, respectively.
Overall, the mRNA levels of OsTIR1 and OsAFB2 were largely
similar in different tissues.
Overexpression of miR393 results in 2,4-D resistance
Considering that miR393 targets auxin receptor homologs, we
examined whether the overexpression of miR393 affects the auxin
response in rice. Wild-type and miR393-overexpressing trans-
genic seeds were germinated on N6 medium with or without the
synthetic auxin 2,4-D. After 2 wk of growth, wild-type seedlings
displayed dramatic inhibition of primary root elongation and
increased numbers of crown roots in the 100 nM 2,4-D treat-
ment (Fig. 7a). However, both 35S:miR393a and 35S:miR393b
plants had longer primary root lengths and fewer crown roots
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(a) 35S:miR393a 35S:miR393b WT
(b) * * (c)
** **
Fig. 4 Overexpression of miR393 influences root growth.
(a) Two-week-old 35S:miR393a and 35S:miR393b transgenic rice
seedlings compared with the wild-type (WT). Arrows indicate primary
roots. Bar, 5 mm. (b) Quantification of the primary root length of miR393-
overexpressing plants and WT grown in solution for 2 wk. (c) Quantification
of the crown root number of miR393-overexpressing plants and WT
grown in solution for 4 wk. Data represent mean values standard
deviation (n > 18). The asterisks show significant (ANOVA; *, P < 0.05;
**, P < 0.01) differences between overexpressing lines and WT.
than the wild-type. The shoot lengths were not obviously dif-
ferent between the overexpressing plants and the control. Inter-
estingly, when cultured on callus-inducing medium with a
high auxin concentration (10 lM 2,4-D), seeds of miR393-
overexpressing lines still exhibited shoot and root hair growth
instead of callus, whereas seeds of the wild-type displayed callus
growth after 2 wk (Fig. S3). This indicates that the overexpres-
sion of miR393 results in 2,4-D resistance, implying that the
miR393 targets function in the auxin response.
As root elongation is very sensitive to auxin, reduced inhibition
of primary root elongation is a typical auxin-resistant phenotype
(Chhun et al., 2003). To illustrate further the decreased auxin
sensitivity of the transgenic plants, we examined primary root
growth in different concentrations of auxin. After 2 wk of growth
in solution medium containing 0, 50, 100 or 200 nM 2,4-D, the
primary root lengths of the transgenic and wild-type plants were
measured (Fig. 7b). The primary root length of wild-type plants
treated with 200 nM 2,4-D was significantly shorter than the
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(a) (b) (a)

*
WT *

35S:miR393a
#1

* * *
35S:miR393b *
#4

(c) 35S:miR393a 35S:miR393b

WT (b)
#10 #9 #6 #1 #3 #4 #5 #7
miR393

U6

Fig. 5 Overexpression of miR393 influences seed development.

(a) Compared with wild-type (WT) rice plants, seeds of miR393-overex-
pressing lines failed to close. (b) The dehusked seeds of miR393-overex-
pressing lines were smaller than those of WT. (c) RNA gel blot analysis of
miR393 levels in WT and 35S:miR393a and 35S:miR393b transgenic lines.

root lengths under 0, 50 and 100 nM treatments (ANOVA,

(c)
P < 0.05). However, both 35S:miR393a and 35S:miR393b
plants were able to maintain primary root elongation in the 200
nM 2,4-D treatment for 2 wk, showing an auxin-resistant pheno-
type. This result further demonstrates that miR393 overexpres-
sion results in an increase in auxin resistance.

Knockdown of OsTIR1 or OsAFB2 alters flag leaf

inclination
To investigate the role of OsTIR1 AFB2 genes in plant growth
and development, we generated OsTIR1- and OsAFB2-RNAi
transgenic lines in a wild-type background. We characterized the
phenotype of 12 and 16 independent transgenic lines obtained
for the knockdown of OsTIR1 or OsAFB2, respectively. Interest-
ingly, droopy flag leaves appeared in AFB2-RNAi lines at the
booting stage (Fig. 8a), resembling the trait observed in plants
overexpressing miR393. RT-PCR analysis showed that the level
of endogenous AFB2 expression was reduced in the AFB2-RNAi
line (Fig. 8b). A similar flag leaf inclination phenotype was also
observed in TIR1-RNAi lines, although the change in angle was
relatively small in comparison with that in AFB2-RNAi
lines (Fig. S4). However, except for the angle of the flag leaf,
other morphological characteristics in OsTIR1- and OsAFB2-
suppressing lines were similar in appearance to those of wild-type
plants. These results indicate that the reduced expression of either
OsAFB2 or OsTIR1 increases the inclination of flag leaves, but
the reduced expression of either OsTIR1 or OsAFB2 alone has
only a modest effect on plant growth and development in rice.
OsTIR1 and OsAFB2 interact with OsIAA1
Molecular genetic studies in Arabidopsis have illustrated that the
TIR1 AFBs interact with Aux IAAs, controlling auxin signaling.
It is known that OsIAA1 protein is nuclear localized and plays an
important role in auxin signaling pathways and plant morpho-
genesis in rice (Song et al., 2009b). To determine the subcellular
Fig. 6 Expression analyses of miR393 target genes in transgenic and
wild-type (WT) rice plants. (a) RNA was isolated from the flag leaf of WT,
two lines overexpressing miR393 (35S:miR393a and 35S:miR393b) and
the target-mimic line (35S: MIM393). A primer pair spanning the miR393
target site was used to quantify expression of the uncleaved target
mRNAs. Quantitative reverse transcription-polymerase chain reaction
(RT-PCR) with primers specific to OsTIR1 and OsAFB2 was performed and
the levels in WT plants are set to unity. Mean values were obtained from
three independent samples. Error bars represent standard deviation. Aster-
isks show significant (ANOVA; *, P < 0.05) difference between transgenic
lines and WT. (b, c) Quantitative RT-PCR analyses of OsTIR1 and OsAFB2
transcripts in various tissues. For each gene, relative expression is shown
by using the expression level detected in the flag leaf as the reference.
Mean values were obtained from three independent samples. Error bars
represent standard deviation of the expression ratio. 1L-R, 2L-R, 4L-R,
10L-R, roots of one-leaf, two-leaf, four-leaf and ten-leaf stage seedlings,
respectively; 1L, first leaf; 2L, second leaf; 4L, fourth leaf; 10L, tenth leaf;
FL, flag leaf; BP, booting panicle; 3L-S, shoot of three-leaf stage seedling.
localization of OsAFB2 and OsTIR1, the full-length cDNA of
each gene was fused to the YFP and GFP reporter genes, respec-
tively, and placed under the control of the 35S promoter. The
IAA1:CFP fusion gene driven by the 35S promoter was used as a
control. These constructs were co-transformed into onion epider-
mal cells, and the transformed cells were checked for the
GFP CFP signal. GFP and YFP were detected in the nuclei of
the onion cells for OsTIR1:GFP and OsAFB2:YFP fusion con-
structs, respectively (Fig. 9a). Meanwhile, the CFP signal for the

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(a)
WT 35S:miR393a 35S:miR393b
(b)
a
a
b
b b b
a b
b *
b b
c Fig. 8 Knockdown of an miR393 target gene results in an increased lamina
Fig. 7 Overexpression of miR393 increases auxin resistance. (a) Wild-type
(WT) and miR393-overexpressing rice lines grown for 2 wk in N6 medium
supplied with (right) or without (left) 100 nM 2,4-dichlorophenoxyacetic
acid (2,4-D). Arrows indicate primary roots. (b) Primary root lengths of
WT (black) and miR393-overexpressing lines (35S:miR393a, light gray;
35S:miR393b, dark gray) grown for 3 wk in solution medium supplied with
various concentrations of 2,4-D as indicated. Data represent
means standard deviation (n > 18). For WT, primary root elongation
was significantly inhibited at 200 nM 2,4-D (ANOVA, P < 0.05) vs at 0,
50 and 100 nM 2,4-D. For miR393-overexpressing lines, primary
root length showed no significant difference at 50, 100 and 200 nM
concentrations of 2,4-D (ANOVA, P < 0.05).Values marked with dif-
ferent letters are significantly (P < 0.05) different between various
concentrations.
OsIAA1:CFP fusion protein was also located in the nuclei of the
cells. Merged images demonstrated that the GFP YFP and CFP
signals overlapped (Fig. 9a). The results thus indicate that
OsAFB2 and OsTIR1 are nuclear proteins.
To determine whether OsTIR1 and OsAFB2 interact with
Aux IAAs, we performed a yeast two-hybrid assay for interacting
proteins using the full-length OsTIR1 and OsAFB2 proteins as
baits and OsIAA1 as prey. Three strains of yeast containing each
combination of bait (BD) and prey (AD) vectors were grown on
medium with a-gal. The results showed that both OsTIR1 and
OsAFB2 interact with OsIAA1 in yeast (Fig. 9b). To verify the
interaction between OsTIR1 OsAFB2 and OsIAA1 in planta,
BiFC assays were performed in onion epidermal cells (Fig. 9c).
OsTIR1 or OsAFB2 was fused to the split N-terminal YFP frag-
ment and OsIAA1 to the C-terminal YFP fragment. Bright
YFP fluorescence was observed when OsTIR1-nYFP and
OsIAA1-cYFP or OsAFB2-nYFP and OsIAA1-cYFP were
co-bombarded into the onion cells. The cells transformed with
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(a)
WT AFB2-RNAi-07
(b)
bending angle of the flag leaf at the booting stage. (a) Comparison of
angles of the flag leaf at the booting stage between wild-type (WT) rice
(left) and the OsAFB2-RNAi transgenic line (right). Arrows indicate the flag
leaves. (b) Expression levels of endogenous OsAFB2 in WT and the
OsAFB2-RNAi transgenic line. Mean values were obtained from three
independent samples. Error bars represent the standard deviation. An
asterisk shows a significant (Students t-test; *, P < 0.05) difference.
IAA1-cYFP, TIR1-nYFP or AFB2- nYFP alone did not show any
fluorescence. These results demonstrate that in vivo interactions
occur between OsTIR1 OsAFB2 and OsIAA1 in plant cells, sug-
gesting conserved interactions between TIR1 homologs and IAAs
in both rice and Arabidopsis.
Discussion
Expression diversification in the rice miRNA393 family
High-throughput sequencing analysis suggests that a minority
of annotated miRNA gene families are conserved between
plant families, whereas the majority are family or species specific
(Griffiths-Jones et al., 2008; Sunkar et al., 2008). The evolution
of miRNA processing and functional diversity has led to the
identification of the complex regulatory networks of miRNA tar-
get modules that control plant development and nutritional
responses, together with other important processes (Cuperus
et al., 2011). Therefore, it is necessary that the diversified func-
tions of miRNAs be studied in a range of species. Based on the
miRBase data (Release 17), the miR393 family is present in all
angiosperm lineages (Kozomara & Griffiths-Jones, 2011). In
Arabidopsis GUS-reporter lines, the expression patterns of
miR393a and miR393b are similar, with both expressed ubiqui-
tously in all tissues, such as roots, stems, rosette leaves, cauline
leaves and inflorescences (Parry et al., 2009; Chen et al., 2011).
This suggests that the expression levels of AtMIR393a and
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(a)

(b)

(c)

Fig. 9 OsTIR1 and OsAFB2 interact with OsIAA1. (a) OsTIR1:GFP, OsAFB2:YFP and OsIAA1-CFP fusion proteins were localized in the nucleus of onion
epidermal cells. CFP, cyan fluorescent protein; DIC, differential interference contrast; GFP, green fluorescent protein; YFP, yellow fluorescent protein.
(b) Yeast two-hybrid assay of the interaction (indicated by formation of the blue color) of OsTIR1 (bait) with OsIAA1 (prey), and OsAFB2 (bait) with
OsIAA1 (prey). Three clones of yeast containing each combination of bait (BD) and prey (AD) vectors were grown on SD Ade His Leu Trp X-a-Gal
medium. pGADT7-T and pGBKT7-53, positive control. pGADT7-T and pGBKT7-Lam, negative control. (c) Bimolecular fluorescence complementation
(BiFC) visualization of OsTIR1 OsAFB2 and OsIAA1 interactions in onion epidermal cells.

AtMIR393b are similar in the whole plants. As the sequences of that the high levels of miR393 accumulation in aerial organs are
mature AtmiR393a and AtmiR393b are identical, it is difficult to reduced dramatically in the miR393b-1 mutant, whereas the low
distinguish between miR393a and miR393b at the transcript levels in roots are not affected (Si-Ammour et al., 2011). This
levels of mature miR393. However, recent findings have shown suggests that mature miR393 in aerial parts of the plant is

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158 Research
produced predominantly by AtMIR393B, which helps to regulate
the auxin-related development of leaves (Si-Ammour et al.,
2011). Owing to the different size of mature OsmiR393a (21 nu-
cleotides) and OsmiR393b (22 nucleotides, Fig. S1), the steady
state levels of OsmiR393a and OsmiR393b can be detected in
small RNA blots (Fig. 1a). In rice, we found that the two
OsmiR393 family members exhibit differential expression in
specific tissues and or growth stages. Strikingly, OsmiR393a is
expressed mainly in the roots, whereas OsmiR393b is expressed
in aerial tissues. Spatio-temporal expression analysis indicated
that OsmiR393a transcription is restricted to emerging crown
root tips and lateral root primordia, but not in the primary root.
However, both miR393a and miR393b of Arabidopsis are
expressed in primary root tips (Parry et al., 2009; Chen et al.,
2011). Based on a very different anatomy in root system between
dicots and monocots, we speculate that the root-specific expres-
sion of OsmiR393a might play a partial role in the initiation of
crown root and lateral root primordia in rice.
It is known that auxin accumulates at locations of organ initia-
tion (Heisler et al., 2005; Dubrovsky et al., 2008). Likewise, the
expression of miR393 also actively occurs in the sites of organ
initiation, implying its role in auxin accumulation and organ
initiation. Interestingly, strong GUS activity was found in the
coleoptile tips of germinated seeds in the ProMIR393a:GUS trans-
genic line (Fig. 2e). Monocot coleoptiles have been used to inves-
tigate IAA production, polar transport and tropisms since the
early days of Darwin. IAA is synthesized at the tip of coleoptiles,
mainly within the top 01-mm region (Nishimura et al., 2009).
Our results indicate that the coleoptile tip, the region of IAA bio-
synthesis and accumulation, is also a region of active miR393a
transcription. Accordingly, we speculate that miR393 expression
might be involved in the local production and accumulation of
auxin, especially in the region of a developing primordium.
Taken together, our data show that the OsmiR393 family is
expressed in restricted regions of plant organs that are actively
growing, whereas its two members display spatio-temporal
expression patterns. Furthermore, although miR393 sequences
are conserved between rice and Arabidopsis, expression patterns of
the miR393 family differ in the two species. Similarly, miR827s
also exhibit differences in tissue expression between the two
species and regulate different classes of genes sharing a common
SPX domain (Lin et al., 2010). The potential functional differen-
tiation of the miRNA precursors is mirrored by subfunctionaliza-
tion in expression patterns (Ru et al., 2006; Wu et al., 2006;
Gutierrez et al., 2009). These data suggest that expression diversi-
fication has occurred in some miRNA families of rice.
OsmiR393 overexpression alters flag leaf angles and
primary and crown root growth in rice
Increased flag leaf inclination was striking in miR393-
overexpressing rice at the booting stage. It is known that an
increased angle of the lamina joint is a typical brassinosteroid
(BR)-related phenotype. Lamina joint inclination assays were
thus recognized as an extremely sensitive system for the determi-
nation of the biological activity of natural or synthetic BRs and
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auxin (Nakamura et al., 2009). In addition to BR-induced effects
on rice lamina inclination, auxin (IAA) also affects lamina
joint inclination at high concentrations and has a synergistic
interaction with BR (Zhao et al., 2010). OsIAA1-overexpressing
transgenic rice exhibited an increased angle of the lamina joint
and dwarfism through a cross-talk pathway with BR (Song et al.,
2009b). Our results show that OsmiR393 overexpression causes
dramatically increased inclination of the flag leaf, displaying an
auxin-resistant phenotype. OsTIR1 and OsAFB2, the targets of
miR393, exhibit high levels of expression in flag leaves. Com-
bined with the enlarged inclination of flag leaves in OsTIR1 and
OsAFB2 suppression lines, this suggests that OsTIR1 and
OsAFB2, negatively regulated by OsmiR393, play an important
role in the development of flag leaves.
In addition to flag leaf inclination, primary root elongation
and crown root formation were clearly affected by miR393 over-
expression in rice. In contrast, it has been reported that the over-
expression of miR393 generates only a modest phenotype in
Arabidopsis (Parry et al., 2009; Chen et al., 2011). This is
perhaps a result of the differences in the formation of adventi-
tious roots. It is well known that Arabidopsis rarely forms adventi-
tious roots, whereas rice produces numerous adventitious or
crown roots (Hochholdinger et al., 2004; Inukai et al., 2005). In
agreement with the deficient crown root morphology of overex-
pressing plants, miR393a transcription is active in regions of
emerging crown roots in rice. The localized expression of
miR393a in roots corresponds well to the areas of crown and
lateral root initiation, suggesting that miR393a might be
involved in the initiation and emergence of these root systems. In
rice, it is assumed that the first cell divisions yielding the new
crown root primordium originate from the innermost ground
meristem cells adjacent to the peripheral cylinder of vascular bun-
dles in the stem (Itoh et al., 2005). Division of these cells eventu-
ally leads to a crown root meristem and could involve specific
regulation of polar auxin transport via the GNOM1 PIN system.
Crown rootless1 (Crl1) encodes a positive regulator for crown and
lateral root formation, and its expression is directly regulated by
an ARF in the auxin signaling pathway (Inukai et al. 2005). The
auxin responsive AP2 ERF (APETALA2/ETHYLENE
RESPONSE FACTOR) transcription factor CROWN ROOT-
LESS5 is involved in crown root initiation (Kitomi et al., 2011).
Moreover, OsARF12 is implicated in the regulation of root
elongation (Qi et al., 2012). In maize, Rum1 (an Aux IAA pro-
tein) is also required for the control of embryonic seminal and
post-embryonic lateral root initiation (von Behrens et al., 2011).
It is known that local accumulation of auxin acts as a trigger for
organogenesis (Dubrovsky et al., 2008). An auxin minimum
zone restricts the occurrence of founder cell specification and
lateral root initiation via the TIR1 AFB pathway (Dubrovsky
et al., 2011). Considering that the miR393 targets are homologs
of auxin receptors TIR1 AFB, we assume that miR393 affects
crown root initiation and seminal root development through
negative regulation of the TIR1 AFB-like genes in rice. Because
root architecture is a key determinant of nutrient and water use
efficiency in crops, the miR393-mediated regulation of adventi-
tious root formation in rice is of considerable interest.
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Increased tillers and early flowering are two phenotypes in
artificial miR393-overexpressing rice (Xia et al., 2012). We
also observed these phenotypes in miR393a- and miR393b-
overexpressing lines (data not shown). However, it is surprising
that the phenotypic changes in flag leaf angle and growth of
primary and crown root observed by the overexpression of
OsmiR393 in our study were not reported by Xia et al. (2012).
Conserved mechanism of miR393 and TIR1 AFB2 in auxin
response
Previous phylogenetic studies have indicated that the TIR1 AFB
proteins are conserved among all land plants. The TIR1 AFB2
clade divided into two distinctive clades (AFB1 and AFB2) before
the separation of monocot and eudicot plants (Parry et al.,
2009). In the rice genome, there are six TIR1 AFBs homologs,
classified into TIR1 (Os05g05800), AFB2 (Os04g32460 and
Os11g31620) and AFB4 (Os02g52230, Os11g27450 and
Os03g08850; Parry et al., 2009) clades. Our results demonstrate
that OsAFB2 and OsTIR1 are up-regulated in 35S: MIM393
plants, indicating that they are the targets of miR393 in rice (Li
et al., 2010; Zhou et al., 2010; Xia et al., 2012).
Suppression of the expression of OsTIR1 or OsAFB2 alone
increased the inclination of the flag leaf, as observed in the plants
overexpressing OsmiR393. These results indicate that both
OsTIR1 and OsAFB2 contribute to flag leaf inclination, suggest-
ing an overlapping function. However, neither OsTIR1- nor
OsAFB2-RNAi displayed any defects in root growth and seed
development, which were observed in the miR393-overexpressing
plants. Overexpression of miR393 causes a simultaneous dra-
matic decrease in its target mRNAs and leads to an increase in
auxin resistance. This is similar to the mutation of multiple
targets, such as in the quadruple tir1 afb1 afb2 afb3 mutant
in Arabidopsis (Parry et al., 2009). Compared with miR393-
overexpressing plants, TIR1- and AFB2-RNAi lines displayed
only a weak phenotype. It has been predicted that most miRNAs
have multiple targets. Therefore, functional redundancy may
mask phenotypes if only one of the targets is suppressed. Genetic
evidence in Arabidopsis indicates that TIR1 and three AFB F-box
proteins act redundantly to mediate auxin responses throughout
plant development (Dharmasiri et al., 2005; Kepinski & Leyser,
2005). Multiple miR156 7-targeted SPL genes, in concert with
nontargeted SPL8, are essential for correct cell proliferation in
early anther development (Xing et al., 2010). Thus, it is likely
that OsTIR1 and OsAFB2 have redundant functions, or that
other similar auxin receptors exist in rice.
Auxin homeostasis and related developmental processes
depend on miRNA-mediated regulation of key components of
auxin signaling (Rubio-Somoza et al., 2009). The highly con-
served miR167ARF6 ARF8 nodes modulate auxin early
response regulators, and the overexpression of miR167a causes
defects very similar to those seen in arf6 arf8 double mutants
(Wu et al., 2006). Here, we found that the overexpression of
either of the two miR393 family members caused a phenotype
that was suggestive of severe defects in auxin response. Our
results demonstrate that the regulation of TIR1 and AFB2
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auxin receptors by miR393 links auxin action to transcriptional
regulation.
In conclusion, the overexpression of miR393 negatively regu-
lates mRNAs of OsTIR1 and OsAFB2. OsTIR1 and OsAFB2
interact with OsIAA proteins, probably releasing the activities
of ARFs, and resulting in an increased resistance to auxin.
The change in auxin response consequently affects diverse
aspects of plant growth and development, such as flag leaf incli-
nation, primary root growth, crown root initiation and seed
development.
Acknowledgements
This work was supported by the National Science Foundation of
China (Grant No. 30972016, No. 31171615, No. 30571197),
and the Key Project of Science and Technology of Zhejiang Prov-
ince (2009C12072).
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Supporting Information
Additional supporting information may be found in the online
version of this article.
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Fig. S1 Sequence analysis of OsmiR393 and its two targets. Table S1 List of primers used in this study

Fig. S2 Determination of HPT transgene copy number in trans- Please note: Wiley-Blackwell are not responsible for the content
formed T0 plants by DNA gel blot hybridization analysis. or functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
Fig. S3 Overexpression of miR393 results in auxin resistance. directed to the New Phytologist Central Office.

Fig. S4 RNA interference (RNAi) of OsAFB2 or OsTIR1 results

in increased lamina bending angles of the flag leaf. Arrows indi-
cate the flag leaves of increased inclination.

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