J. Plant Biol. (2013) 56:187-197
DOI 10.1007/s12374-013-0213-4
The Role of Rice microRNAs in Abiotic Stress Responses
Dong-Hoon Jeong and Pamela J. Green*
Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware
19711
Received: May 30, 2013 / Accepted: June 11, 2013
© Korean Society of Plant Biologists 2013
Abstract microRNAs (miRNAs) are a class of small
noncoding RNAs that regulate gene expression at the post-
transcriptional level by mRNA cleavage or translation
inhibition. They play diverse roles in plant growth and
development as well as abiotic stress responses. In response
to abiotic stresses such as drought, salt, cold, heat, and
nutrient limitations, the expression levels of some miRNAs
change, resulting in a modulation of the expression patterns
of miRNA target genes that are associated with stress
adaptations. In rice, stress-responsive miRNAs have been
identified and characterized, and conserved regulation of
conserved miRNAs as well as new regulation by conserved
miRNAs and rice-specific miRNAs is evident. The regulatory
mechanisms controlling target gene expression by stress-
responsive miRNAs include both the coherent and incoherent
regulatory networks that are dynamic and complex. A better
understanding of the regulation of miRNAs and targets during
stress responses can contribute to rice breeding for
improving yield, quality and tolerance to abiotic stresses.
Here, we review current advances in the area of rice
miRNAs and target RNAs associated with abiotic stresses
and discuss how they relate to miRNA-mediated stress-
tolerance.
Keywords: miRNA, Abiotic stress, Target gene, Deep
sequencing, PARE, Rice
Overview of Plant miRNA Biogenesis, Function, and
Identification
MicroRNAs (miRNAs) are 20- to 24-nucleotides (nt) small
non-coding RNAs that repress target gene expression at the
post-transcriptional level (Bartel 2004; Voinnet 2009). They
are encoded by MIRNA genes that are transcribed into the
primary miRNAs (Pri-miRNAs) by RNA polymerase II (Xie
et al. 2005). Pri-miRNAs are processed to become miRNA
precursors (pre-miRNAs), forming self-complementary stem-
loop secondary structures that often have an imperfect
double-stranded character. Mature miRNAs are generated
from this stem-loop structure as a miRNA/miRNA* duplex
by a DICER-LIKE (DCL) protein activity. miRNA strands
of this duplex are then associated with ARGONAUTE
(AGO) proteins in a RNA-induced Silencing Complex (RISC)
while the miRNA*s are rapidly degraded (Park et al. 2002;
Kurihara and Watanabe 2004; Chen 2005).
The regulatory function of plant miRNAs is exerted by
several mechanisms. In general, most plant miRNAs guide
AGO proteins to recognize their target RNAs by near perfect
base-pairing and guide endonucleolytic target RNA
cleavage. Subsequently, target mRNAs are rapidly degraded
by the exosome and an exoribonuclease called XRN4. In
addition, some miRNAs inhibit target mRNA translation,
thereby limiting protein production (Brodersen et al. 2008;
Dugas and Bartel 2008; Lanet et al. 2009; Yu and Wang
2010). miRNAs can also guide cleavage of non-coding
RNAs such as in TAS loci to induce production of trans-
acting siRNAs (Peragine et al. 2004; Vazquez et al. 2004;
Allen et al. 2005) which, in turn, can direct cleavage of
complementary mRNAs. Recently, distinct 22-nt miRNAs
have been reported to facilitate generation of clusters of
phased 21-nt or 24-nt siRNAs (Johnson et al. 2009; Song et
al. 2012). Finally, 24-nt long miRNA, called lmiRNAs, have
been found in rice and direct DNA methylation at their
source and also function in trans on target genes to regulate
gene expression (Wu et al. 2010).
miRNAs have been identified through direct cloning,
sequencing, and analysis of small RNA libraries or through
computational prediction (Jones-Rhoades and Bartel 2004;
Lu et al. 2005). The recent advents of high-throughput
sequencing have greatly increased the rate of identification
of miRNAs. To date, 5,127 pre-miRNAs have been identified
*Corresponding author; Pamela J. Green
Tel : +1-302-831-6160
E-mail : [email protected]
REVIEW ARTICLE
188 J. Plant Biol. (2013) 56:187-197
from 67 plant species and have been registered in the
miRBase sequence Database (release 18.0;Kozomara and
Griffiths-Jones 2011). Of those, 591 pre-miRNAs encoding
708 miRNAs have been reported in rice. They have great
potential to regulate thousands of genes. These observations
emphasize the importance of gene regulatory mechanisms
mediated by miRNAs in addition to those mediated at the
transcriptional level by transcription factors.
Association of Plant miRNAs with Abiotic Stresses
Abiotic stresses, such as drought, salt, cold, heat, and
nutrient deficiency, are major limiting factors in agriculture
and this is certainly the case for rice yield. To adapt and
survive under abiotic stress conditions, sessile plants need an
efficient strategy such as modulation of gene expression.
Proper regulation of stress-responsive genes is important for
both recognition and response to stress conditions. The
ability of plants to employ miRNAs to repress stress-
responsive gene expression post-transcriptionally provides a
considerable advantage compared with regulation by
transcription factors alone. The importance of miRNAs in
abiotic stress responses has been implicated by the fact that
several mutants such as hyponastic leaveas-1 (hyl1), hua
enhancer-1 (hen1), and dcl1, which are defective in miRNA
metabolism, are hypersensitive to the stress hormone
Abscisic acid (ABA) or abiotic stresses in Arabidopsis (Lu
and Fedoroff 2000; Zhang et al. 2008). In addition, the
association of miRNAs with abiotic stress responses has
become clear from the fact that many miRNAs from diverse
species are responsive to abiotic stress (Sunkar and Zhu
2004; Jeong et al. 2011). Induction and/or repression of
miRNAs in response to abiotic stress can modulate the
expression of target genes that may be involved in a
particular stress response and/or tolerance. More importantly,
phenotypic analysis of mutants or transgenic plants, in which
the expression of either stress-responsive miRNAs or their
target genes was manipulated, has proven the role of
miRNAs during the corresponding stress conditions. Below,
we have focused on rice miRNAs that have been implicated
in abiotic stress responses because of the importance of rice
as a major food crop and as a model for other crops.
Drought and Salt Stress-responsive miRNAs in Rice
Drought is the most serious stress to threaten the world’s
food supply. In the case of rice, about half of the land used
for growing rice is affected by drought (Zhang 2007).
Variation in annual rainfall patterns, uneven distribution of
rainfall during the rice growing season, and insufficient
rainfall in many areas contribute to drought stress in rice.
Salt stress is another major agricultural problem effecting
crop yield, especially for rain-fed agriculture in coastal areas
and for irrigated agriculture with poor water quality.
Generally, rice is considered to be relatively more tolerant to
salt stress at germination (Walia et al. 2005). Among cereals,
however, rice is the most salt-sensitive crop at the young
seedling stage (Gongl et al. 2006; Faiyue et al. 2012). Since
both drought and salt stress cause ionic and osmotic stress in
plant cells, these stresses are closely related and share many
common signal transduction mechanisms and gene expression
profiles (Zhu 2002).
In an early study based on microarray experiments,
miR393 and miR169 were identified as up-regulated under
drought stress in rice (Zhao et al. 2007). Reports indicate
miR393 is commonly and consistently up-regulated during
drought stress in many plants such as Arabidopsis,
Medicago, common bean, and rice (Sunkar and Zhu 2004;
Zhao et al. 2007; Arenas-Huertero et al. 2009; Wang et al.
2011). miR393 plays an important role in normal
development, defense against pathogenic bacteria, and the
nitrate response by targeting genes encoding the TIR1
(Transport Inhibitor Response Protein 1)/AFB (Auxin
Signaling F-box Protein) family of F-box auxin receptors
(Navarro et al. 2006; Vidal et al. 2010; Chen et al. 2011). In
rice, miR393 targets two rice auxin receptor gene homologs:
OsTIR1 and OsAFB2, and guides cleavage of their
transcripts (Wu et al. 2009; Li et al. 2010; Zhou et al. 2010;
Xia et al. 2012). Recently, it was reported that over-
expression of miR393 in rice caused them to exhibit
increased tillers and early flowering as well as reduced
tolerance to salt and drought and hyposensitivity to auxin
(Xia et al. 2012). This implies that drought- inducible
miR393-mediated suppression of auxin signaling could
affect the response to drought in plants.
Drought-inducible miR169 was later also reported to be
induced by high salinity in rice (Zhao et al. 2009). However,
a discrepant result was reported in Arabidopsis, in which
miR169 was down-regulated by drought stress treatment (Li
et al. 2008) in contrast to the situation in rice. The down-
regulation of miR169 partially effected drought-induced
expression of its target, NFYA5. Further, the knockout of
nfya5 as well as overexpression of miR169 rendered
Arabidopsis plants more sensitive to drought, whereas NFY5
over-expressing plants exhibited a drought tolerant phenotype
(Li et al. 2008). Further functional analysis of miR169
during drought and salt stress responses in rice may explain
these discrepant results.
Two rice siRNAs, which were previously annotated as
miR441 and miR446 but are currently considered as siRNAs
due to inconsistency with the recent criteria of the
recommended for plant miRNAs (Meyers et al. 2008), were
J. Plant Biol. (2013) 56:187-197 189
reported to be down-regulated by drought stress treatment
(Yan et al. 2011a). They originate from stem-loop precursors
of the miniature inverted-repeat transposable element
(MITE) Stowaway1 and are processed by DCL3a
independent of OsRDR2. Interestingly, while these two
siRNAs, renamed as siR441 and siR446 later, were down-
regulated, their primary transcripts were induced under
drought stress conditions. This implies that processing of
these siRNA precursors was inhibited. Over-expression of
the precursors reduced siR441 and siR446 accumulation and
the corresponding transgenic plants were less sensitive to
ABA and more sensitive to drought stress. It was proposed
that siR441 and siR446 play a positive role in the response
to drought stress by up-regulating MAIF1, which encodes an
F-box protein and is a predicted target of these two siRNAs
(Yan et al. 2011a; Yan et al. 2011b).
miR820 is a rice-specific miRNA and that is down-
regulated by drought stress treatment (Jeong et al. 2011). It
targets OsDRM2, which encodes a DNA methyltransferase
that is involved in RNA-directed de novo DNA methylation
(Pang et al. 2013). Although miR820-mediated OsDRM2
mRNA cleavage was empirically proven (Lu et al. 2008), the
expression of OsDRM2 was also down-regulated when
miR820 decreased during drought stress (Lu et al. 2008;
Jeong et al. 2011). Since the expression levels of a given
miRNA and its target are typically inversely correlated, this
incoherent relationship of their expression patterns implies
that miR820 may fine-tune the spatiotemporal expression of
OsDRM2 in response to drought rather than play a switch
role in its expression. miR820 consists of a 21-nt canonical
miRNA and a 24-nt variant considered to be a long miRNA
(lmiRNAs), which are processed from the same precursor by
DCL1 and DCL3a activities, respectively (Wu et al. 2010).
Canonical 21-nt miR820 is incorporated into the AGO1
protein and regulates OsDRM2 expression primarily through
mRNA cleavage, while 24-nt miR820 is loaded into AGO4
clade proteins and directs DNA methylation of miR820 loci
in cis as well as at OsDRM2 in trans (Wu et al. 2010). Since
DNA methylation by 24-nt miR820 may require OsDRM2
activity, both miR820 and OsDRM2 may cooperate for
negative-feedback regulation of themselves. This implies a
complex regulatory network, including incoherent regulation,
negative feedback, and antagonistic activities.
miRNAs Responsive to Temperature Stress in Rice
Extreme temperatures, such as cold or heat, affect not only
the growth duration, but also the growth pattern and
productivity of rice crops. The critical temperatures for the
development of the rice plant at different growth stages
varies (Nguyen 2002). For example, cold stress has a higher
impact at the germination and reproductive anthesis stages
compared to the vegetative growth stages. High temperature
stress more seriously affects the reproductive anthesis and
seed ripening stages, resulting in damage to grain fertility.
Cold-stress-responsive rice miRNAs have been reported
based on genome-wide profiles of miRNA expression
determined using microarray or deep sequencing methods
(Jian et al. 2010; Lv et al. 2010; Jeong et al. 2011). Of these,
miR167 and miR319 were shown to be down-regulated by
cold stress in rice seedlings (Lv et al. 2010). While their
precursors are down-regulated after 3-h, the mature miRNAs
are decreased after 12h of cold stress. Among the predicted
targets of miR319, two genes that encode TEOSINTE
BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription
factors and one gene that encodes a MYB transcription
factor were up-regulated after cold treatment. Intriguingly,
both miR167 and miR319 were reported to be up-regulated
by the cold stress in Arabidopsis (Sunkar and Zhu 2004;
Zhou et al. 2008). This is another example of a discrepancy
between the miRNA expression patterns between
Arabidopsis and rice. Such differences may be due to
variations in either experimental conditions or the methods
used to measure miRNA expression changes. However, it is
also possible that these miRNAs play a species-specific role
in cold responses.
Deep sequencing data and the following validation
experiment showed that the expression of miR1425 was
suppressed during cold stress in rice panicle tissues.
miR1425 targets a set of genes encoding Pentatricopeptide
repeat (PPR) proteins that are RNA-binding proteins involved
in post-transcriptional processes such as RNA splicing, RNA
editing, RNA stability and translation in organelles such as
mitochondria and chloroplasts (Delannoy et al. 2007; Schmitz-
Linneweber and Small 2008). A group of PPR genes are
known to be targeted by multiple miRNA families such as
miR161 and miR400, or by TAS1 and TAS2 trans-acting
siRNAs that are generated after cleavage of the TAS transcripts
is mediated by miR173 in Arabidopsis (Rhoades et al. 2002;
Sunkar and Zhu 2004; Allen et al. 2005). miR1425 is a rice-
specific miRNA that targets PPR genes (Lu et al. 2008).
Fertility restorer (Rf-1) gene, which encodes a PPR protein
and that is a target of miR1425, is known to lead to increased
cold tolerance in hybrid rice at the booting stage by increasing
the number of potentially fertile pollen grains (Komori and
Imaseki 2005). Rf-1 was up-regulated during cold stress when
miR1425 is down-regulated, implying that miR1425 plays
role in modulating the expression of Rf-1 under cold
conditions (Jeong et al. 2011).
miR812q was reported to be extremely up-regulated by
cold stress at the booting stage of rice plants (Jeong et al.
2011). It is a 24-nt miRNA that originates from a stem-loop
precursor of MITE Stowaway1. The MIR812 family is
190 J. Plant Biol. (2013) 56:187-197
comprised of 17 precursors that share sequence similarities.
However, miR812q is generated from a sequence-diverged
region and has a unique miRNA sequence compared to the
other family members. In addition, miR812q is unique
among family members because it responds to cold stress,
suggesting miR812q is a new evolving miRNA in rice.
Calcineurin B-like (CBL) protein interacting protein kinase
10 (CIPK10) is targeted for cleavage by cold-inducible
miR812q, and thus down-regulated during the cold stress
(Jeong et al. 2011). CIPK genes are regulated by various
abiotic stresses and mediate environmental stress tolerance in
the Ca2+-dependent CBL-CIPK signaling pathway (Xiang et
al. 2007; Batistic and Kudla 2009).
High-temperature-responsive miRNAs have not been
studied well in plants in comparison to other stress-
responsive miRNAs, and have only been reported in
Populus, Brassica rapa, wheat, and rice (Lu et al. 2008; Xin
et al. 2010; Jeong et al. 2011; Yu et al. 2012). In rice,
miR397b.2 is induced by heat stress. Previously, miR397 in
Arabidopsis was reported to be up-regulated by salt stress
treatment (Sunkar and Zhu 2004) but, the expression of rice
miR397 was not changed by cold stress. Intriguingly, a target
of miR397b.2, which encodes an L-ascorbate oxidase, is
down-regulated during the heat stress while miR397b.2 is
increased, and is induced by salt stress in rice (Jeong et al.
2011). This indicates that the rice miR397 family has
evolved to modulate the expression of L-ascorbate oxidase
under heat stress rather than salt stress.
miRNAs Associated with Nutrient Homeostasis in Rice
Macronutrients, such as phosphorus, sulfur, nitrogen, and
potassium, are essential and required for normal growth and
development of plants. They are so limited in soil that their
acquisition, assimilation, and metabolism are tightly regulated
in plants. Deprivation of specific macronutrients commonly
affects the quality and production of crop plants such as rice.
Recent studies have implicated the involvement of miRNAs
in nutrient homeostasis.
The association of the phosphorus-deprivation response
with miRNAs is well studied for miR399 and miR827. The
expression of both miRNAs is specifically induced when the
plants are deprived of phosphate (Pi) which is the naturally
occurring form of phosphorus. Up-regulation of miR399 is
well-conserved in many plants such as Arabidopsis, tomato,
common bean, and rice (Fujii et al. 2005; Aung et al. 2006;
Bari et al. 2006; Chiou et al. 2006; Valdés-López et al.
2008). PHR1, a MYB transcription factor, is known to
induce the expression of miR399 in response to Pi starvation
in Arabidopsis (Fujii et al. 2005). In rice, OsPHR2, the
homolog of AtPHR1, is implicated as a significant regulator
of miR399 expression under Pi starvation conditions (Zhou
et al. 2008) . Up-regulation of miR399 results in down-
regulation of its target, PHO2 which encodes ubiquitin-
conjugating E2 enzyme (UBC24). PHO2 negatively
regulates Pi uptake and translocation by degrading target
proteins such as PHO1 which has been implicated in Pi
loading to xylem (Liu et al. 2012). Thus, miR399-mediated
PHO2 down-regulation under Pi starvation conditions
facilitates Pi uptake and translocation. Over-expression of
miR399 in Arabidopsis induces more accumulation of Pi
than wild type, and transgenic plants expressing a PHO2
mRNA engineered to be insensitive to miR399 showed less
inhibition of primary root growth during Pi deprivation than
those of wild type plants (Fujii et al. 2005; Aung et al. 2006).
In rice, a PHO2 ortholog was identified by map-based
cloning of the leaf tip necrosis1 (ltn1) mutant (Hu et al.
2011). The ltn1 mutant exhibited increased Pi uptake and
translocation even under Pi-sufficient conditions. The
resulting excessive Pi accumulation leads to toxicity and
causes growth defects and leaf tip necrosis. Transgenic rice
plants constitutively over-expressing Osa-MiR399 displayed
a phenotype similar to that of the ltn1 mutant and exhibited
down-regulation of LTN1 transcripts. This suggests the role
of the rice miR399-LTN1 module in the Pi signaling is
similar to that of Arabidopsis miR399-PHO2 (Hu et al.
2011).
Interestingly, the activity of miR399 is known to be
regulated by the TPSI1/Mt4 gene family of noncoding
RNAs. In Arabidopsis, the At4/IPS1 (INDUCED BY
PHOSPHATE STARVATION1) gene in this family functions
to encode target mimics that modulates the activity of
miR399 by sequestering it from binding to PHO2 mRNA
(Franco-Zorrilla et al. 2007). In this model, both At4 and
miR399 are up-regulated under Pi starvation conditions
(Shin et al. 2006). Thus, PHO2 expression is maintained
under Pi starvation despite the presence of an abundant level
of miR399 (Aung et al. 2006; Bari et al. 2006). Since
homologs of At4/IPS1 genes are present in many plant
species (Rymarquis et al. 2008), target mimicry-based
regulation of miR399 could be a conserved mechanism. In
rice, At4 homolog, OSPI1 (Oryza sativa phosphate-
limitation inducible gene 1) was identified and up-regulated
during the Pi starvation (Wasaki et al. 2003).
In addition to miR399, miR827 was also identified as a
well-conserved and Pi-starvation inducible miRNA both in
Arabidopsis (Hsieh et al. 2009; Pant et al. 2009) and rice
(Lin et al. 2010; Jeong et al. 2011) . In Arabidopsis, it targets
the NLA (Nitrogen Limitation Adaptation) gene which
encodes a RING-type ubiquitin ligase with an SPX (SYG1/
Pho81/XPR1) domain involved in the response to nitrogen
deficiency (Peng et al. 2007). This implies that miR827 may
play role in the cross talk between the signaling pathways
J. Plant Biol. (2013) 56:187-197 191
responding to Pi limitation and nitrogen limitation. Indeed,
NLA was recently reported to participate in the regulation of
Pi homeostasis in a nitrate-dependent manner by regulating
the expression of a high-affinity phosphate transporter
PHT1.1 and its traffic facilitator PHF1 (Kant et al. 2011).
Instead of targeting OsNLA, rice miR827 targets two distinct
genes: OsSPX-MFS1 and OsSPX-MFS2, which encode
proteins with SPX and MFS (Major Facilitator Superfamily)
domains. Although both genes are negatively regulated by
miR827-mediated mRNA cleavage events, they show
opposite expression patterns in response to Pi starvation. The
expression of OsSPX-MFS1 is reduced upon Pi starvation,
indicating that a direct relationship between induction of
miR399 and down-regulation of OsSPX-MFS1.In contrast,
the expression of OsSPX-MFS2 is increased during Pi
starvation, suggesting the involvement of complex regulation
of miR827 and its target genes in rice. Both a TOS17
transposon insertion mutant of OsSPX-MFS1( mfs1) and a
transgenic plant over-expressing miR827 showed impaired
Pi homeostasis and over-accumulated Pi (Wang et al. 2012).
During the limitation of sulfate, which is a major source of
sulfur, the expression of miR395 is significantly up-regulated
in Arabidopsis (Jones-Rhoades and Bartel 2004) and rice
(Jeong et al. 2011). In both plants, miR395 targets two
families of genes, ATP sulfurylase (APS) and Sulfate transporter
(SULTR), which are involved in sulfate accumulation and
allocation, respectively. In Arabidopsis, the abundance of
APS1 transcripts decreases when miR395 increases during
sulfate starvation which is the expected negative correlation
between miRNA and its target (Jones-Rhoades and Bartel
2004). However, another target, SULTR2;1, is up-regulated
during sulfate starvation when miR395 is up-regulated.
These discrepant expression patterns might be explained by
their precise spatial expression patterns. miR395 is mainly
expressed in phloem companion cells (Kawashima et al.
2009) while SULTR2;1 is mainly expressed in xylem
parenchyma cells (Takahashi et al. 1997; Takahashi et al.
2000). Thus, it is proposed that the function of miR395 is to
suppress SULTR2;1 expression in phloem companion cells
under conditions of sulfate limitation to restrict SULTR2;1
expression to the xylem. Similar results were also observed
in rice (Jeong et al. 2011). During sulfate starvation, one
miR395 target, SULTR1, is down-regulated, whereas the
other targets, SULTR2 and APS1, are up-regulated when
miR395 is induced. The precise spatial expression patterns of
these targets have yet to be determined in rice. Thus, more
experiments are needed to clarify the role of miR395 in rice
during sulfate starvation.
The association of miRNAs and nitrogen signaling has
been well studied within the context of auxin-mediated
lateral root growth in Arabidopsis. Under nitrogen limiting
conditions, miR167 is specifically down-regulated while its
target, Auxin responsive factor 8 (ARF8), increases, resulting
in lateral root initiation (Gifford et al. 2008). In addition, the
miR399/AFB3 target RNA module was also reported to play
a role in nitrogen-auxin crosstalk involved lateral growth
development in Arabidopsis (Vidal et al. 2010). Nitrate
treatment induces both miR393 and its target, AFB3 which
encodes an auxin receptor. This incoherent feed-forward
mechanism affects primary and lateral root growth responses
to nitrate. Whether miR167/ARF8 and miR393/AFB3
regulatory modules also play a role in nitrogen signaling in
rice has not yet been reported.
Comprehensive expression profiles of miRNAs have
revealed many nitrogen starvation-responsive miRNAs in
Arabidopsis (Pant et al. 2009; Liang et al. 2012), rapeseed
(Pant et al. 2009), maize (Zhao et al. 2012), and rice (Jeong
et al. 2011; Nischal et al. 2012). Among these, miR169 is
usually reported to be down-regulated under conditions of
nitrogen limitation in Arabidopsis, maize, and rice. Further
study in Arabidopsis revealed that target genes of miR169,
specifically, the NFYA family transcription factors, were
induced during nitrogen starvation when miR169 is repressed
(Pant et al. 2009). Moreover, transgenic Arabidopsis plants
over-expressing MIR169a accumulated less nitrogen and
were more sensitive to nitrogen stress than the wild type
(Zhao et al. 2011). It is notable that miR169 and the NFYA
genes were also reported to be involved in drought stress
responses (Li et al. 2008), implying that low expression of
miR169 during nitrogen limitation could contribute to
drought tolerance of N-limited plants.
Rice-specific miR3979 was reported to be down-regulated
under nitrogen starvation conditions (Jeong et al. 2011). It is
intriguing that miR3979 is a root–preferential miRNA and
consequently, its regulation in response to nitrogen supply is
limited only to roots, where nitrogen is mainly absorbed.
miR3979 is predicted to target a gene encoding an anthranilate
phosphoribosyl-transferase which is involved in tryptophan
(Trp) biosynthesis. It is known that the Trp biosynthetic pathway
in plants leads to the production of not only Trp but also
secondary metabolites such as the phytohormone auxin and the
phytoalexin camalexin (Kutchan 1995; Radwanski and Last
1995). Trp biosynthetic genes are generally induced by amino
acid starvation as well as abiotic and biotic stresses (Zhao et al.
1998). Thus, it is possible that, during nitrogen limitation, down-
regulation of miR3979 induces Trp biosynthesis followed by
increased auxin and camalexin, resulting in lateral root initiation
and stress resistance, respectively.
Mechanisms of Target Gene Regulation by Stress-
Responsive miRNAs
Efforts to profile rice miRNAs have identified many
192 J. Plant Biol. (2013) 56:187-197
stress-responsive miRNAs which are either induced or
repressed during abiotic stress. Given that miRNAs negatively
regulate the expression of their target genes by target RNA
cleavage or translation repression (Voinnet 2009; Huntzinger
and Izaurralde 2011), the expression patterns of miRNA
target genes generally show an inverse correlation with those
of miRNAs. Therefore, for the majority of cases that involve
target cleavage, the simple expectation is that, when
miRNAs are induced by stress treatments, their target
mRNAs are reduced and vice versa (Fig. 1A). Furthermore,
this negative correlation between target mRNA and miRNA
accumulation is often considered proof of miRNA targeting.
Indeed, in many cases, the expression of the stress-regulated
miRNA and its targets are mutually exclusive, such as an
example observed in miR399 and its target PHO2 suggesting
that the stress-responsive miRNA is responsible for
switching off the expression of target genes under stress
conditions. However, many other cases also represent an
opposite trend such that miRNAs and their targets are co-
expressed in the same tissue or under the same stress
conditions. One striking, well-characterized example is the
case of miR168 targeting of AGO1 in Arabidopsis. The
miRNA is expressed everywhere in the plant where its target
is required, thus implying the involvement of fine tuning
mechanisms to modulate gene expression through a miRNA-
mediated feedback loop (Vaucheret et al. 2004; Vaucheret et
al. 2006). Another example of co-expression of a miRNA
and its target was observed for miR395 and SULT2;1 where
both are up-regulated in response to sulfate starvation. As
mentioned earlier, the differential expression patterns of
miR395 and its target SULT2;1 in a cell-type specific
manner gave a clue to explain the discrepancy of their
expression. A general illustration of such spatial regulation is
shown in Fig. 1B. A miRNA that is highly expressed in a
cell type restricts the expression of its target where it is
expressed. Conversely, the target gene is specifically
expressed in a cell type in which the miRNA is lowly
expressed. In this situation, examination of the stress-
responsive expression patterns in whole organs would
obscure the complex and subtle changes that could be
detected with cell type-level resolution (Dinneny et al. 2008;
Iyer-Pascuzzi et al. 2011).
The relationship of expression patterns between a miRNA
and its target RNAs is more dynamic and complex in a
regulatory network where transcriptional induction/
repression by transcription factors and post-transcriptional
repression by miRNAs co-exist. Under a stress-condition,
transcription of both stress-responsive miRNAs and their
target genes can be independently regulated. Their regulation
can be either in the same or the opposite direction. In other
words, transcriptional regulation of a miRNA and its target,
during a stress, can be induction/repression (Fig. 2A),
repression-induction (Fig. 2B), induction of both (Fig. 2C),
or repression of both (Fig. 2D). The first two examples are
in a coherent network where the effects of transcriptional and
posttranscriptional regulatory pathways on miRNA-target
gene expression are the same (Fig. 2A and B). The other two
examples show in an incoherent network where two
pathways have opposite effects (Fig. 2C and D). In coherent
regulation, miRNAs play cooperative switch-like roles along
with transcription factors. For example, under stress, both the
miRNA and the transcriptional repressor suppress target
gene expression. Thus, these two regulatory pathways
cooperate to repress the target gene rapidly (Fig. 2A). In the
other example of coherent regulation, target gene expression
is rapidly increased by the combination of miRNA
repression and the function of a transcriptional activator (Fig.
2B). In this case, another role of the miRNA is to repress
target gene expression under normal conditions, minimizing
the basal level of target expression. In the case of incoherent
regulation, miRNAs function to fine-tune target gene
expression rather than switch it on and off. For instance,
under a given stress condition, both a transcriptional
activator and a miRNA may be induced (Fig. 2C) as was
observed in Arabidopsis miR393/AFB3 under conditions of
Fig. 1. Spatiotemporal effects of stress-regulated miRNAs ontarget gene expression. A. Temporal regulation of target geneexpression by stress-regulated miRNAs. Stress-induced miRNAsdown-regulate the expression of their target genes during a givenstress condition, while stress-repressed miRNAs up-regulateexpression of their target genes during the stress condition. B.Spatial regulation of target gene expression by stress-regulatedmiRNAs. Stress-responsive miRNAs that are regulated in a cell-type-specific manner can spatially restrict the expression of theirtargets in a particular cell or cell type.
J. Plant Biol. (2013) 56:187-197 193
nitrogen limitation. In this situation mentioned earlier, up-
regulation of AFB3 is modulated by miR393 induction in an
incoherent negative feed-forward loop. Stress-inducible
target gene expression patterns may take the form of a
saturated curve or a pulse depending on the strength of
miRNA repression. The other example of incoherent
regulation was observed in the rice miR820/OsDRM2
module where both the miRNA and the target gene were
repressed by drought stress (Fig. 2D). In this case, the
fundamental role of the miRNA is to keep the expression of
the target gene at basal levels under normal conditions.
However, as drought stress down-regulates the expression of
OsDRM2 at the transcriptional level, and the miR820-
mediated repressive pathway is transcriptionally dampened,
slow repression of target gene expression ensues. Taken
together, these scenarios illustrate that miRNA functions can
be fully understood only by addressing regulatory interactions
within networks.
Conclusions and Future Prospects
In this review, we have discussed our current understanding
of the role of rice miRNAs in response to abiotic stresses
including drought, salt, cold, high temperature, and the
limitation of nutrients such as Pi and sulfate. In particular, we
have provided the evidence that stress-regulated rice
miRNAs play a role in modulating expression levels of their
target genes which are involved in stress responses and
adaptation. Although the stress responsiveness of some rice
miRNAs is well conserved in other plants, new regulation of
conserved miRNAs as well as stress regulation of rice-
specific miRNAs was also reported, highlighting the
potential for identification of more stress-responsive
miRNAs in rice. Moreover, we highlighted the diverse
regulatory mechanisms and patterns of target gene
expression by stress-responsive miRNAs. In addition to
coherent regulation by miRNAs, incoherent relationships
between miRNAs and their target gene expression levels
provide another layer of gene regulatory networks in a stress-
responsive pathway.
Despite many reports introduced in this review, there are
still limitations to our understanding on the role of rice
miRNAs in abiotic stress responses. First, many regulated
miRNA candidates that were originally identified by high-
throughput analysis using microarrays or deep sequencing
lack validation experiments to confirm their expression
patterns. It has been reported that a technical bias, introduced
by hybridization, ligation efficiency, PCR amplification, or
sequencing methods, often leads to results being inaccurate
(Zhang et al. 2011). Thus, each individual miRNA
expression pattern during the stress conditions must be
carefully examined by another validation assay such an RNA
blot, a quantitative RT-PCR, or the splinted-ligation mediated
miRNA detection method, before follow up studies. While
the first two experiments are widely used for quantification,
the splinted-ligation mediated detection method is primarily
proposed to discriminate among miRNA variants including
members of miRNA families and isomiRs (Jeong and Green
2012). Second, detailed information about spatial and
temporal expression patterns of stress-regulated miRNAs
Fig. 2. The effects of stress-responsive miRNAs and transcriptionalregulators on expression of target RNA. Stress-inducible orrepressible miRNAs cooperate with transcriptional regulators tocontrol the expression pattern of miRNA-target genes during stressconditions. The expression patterns of miRNAs and their targetscan be either coherent or incoherent as illustrated in the followingexamples. A. Coherent repression of a target expression by astress-inducible miRNA and a transcriptional repressor. B.Coherent induction of a target expression by a stress-repressiblemiRNAs and a transcriptional activator. C. Incoherent induction ofa target gene by a stress-inducible miRNA and a transcriptionalactivator. D. Incoherent repression of a target gene by a stress-repressible miRNA and a transcriptional repressor. Circled A,Activator; circled R, Repressor.
194 J. Plant Biol. (2013) 56:187-197
and their targets are often lacking in rice. Most reported
studies have analyzed expression profiles in whole plants
subjected to a specific stress at a single time point and most
cases were done during the early seedling stage. Given that
differential regulation of genes in different tissues is
important for response to stress, dynamic data including
various tissues and time points can provide valuable
information to better understand the underlying stress
responses that might be missed by using data that only
focuses on whole organs at a single time point. Moreover,
systemic analysis of relationships between expression
patterns of miRNAs and their target genes during time
courses and from diverse tissues or cell types could provide
better understanding of gene regulatory network in stress
response pathways. The third limitation to our knowledge of
the role of stress-responsive rice miRNAs is the
identification of authentic target RNAs. Although the is
general consensus that the relevance of regulated miRNAs in
stress responses will be mainly defined by their activity on
target mRNAs, the exploration of rice miRNA targets has
mainly relied on their computational prediction. Mapping the
miRNA-mediated cleavage site on a target mRNA is the
most direct empirical evidence for plant miRNA target
identification. The analysis of Parallel Analysis of RNA
Ends (PARE), also called degradome analysis, has been
proposed for genome-wide identification of miRNA targets
(Addo-Quaye et al. 2008; German et al. 2008; Gregory et al.
2008). PARE was reported in rice to be successful in
identifying and characterizing many rice miRNA targets
from tissues that were grown under healthy conditions (Wu
et al. 2009; Li et al. 2010; Zhou et al. 2010; Jeong and Green
2012). The next challenge will be to analyze PARE data
from the stress-treated tissues for a better understanding of
stress-regulated miRNA-mediated target gene regulation.
Finally, we would like to point out the current large gap
between the identification of stress-regulated miRNAs and
the confirmation of their function. The fact that a miRNA is
differentially regulated in response to an environmental
stress does not necessarily mean that the miRNA is involved
in stress responses. Thus, the use of transgenic or mutant
approaches is essential to better understand the biological
function of miRNAs. Due to the redundancy of members of
miRNA families and the relatively small size of MIR genes,
it is generally difficult to obtain insertional knockout mutants
of stress-responsive miRNAs. Thus, the miRNA-overexpressing
transgenic plants are commonly used for studying miRNA
functions. In addition, gain-of-function mutants, in which
miRNA-resistant target genes are ectopically expressed, can
demonstrate functions of the miRNA that phenotype occurs
when miRNA-mediated regulation is blocked. Moreover, it
was recently proposed that transgenic plants overexpressing
an artificial target mimic transcript can provide insight into
miRNA function by sequestering miRNAs of interest
(Todesco et al. 2010; Yan et al. 2012).
Abiotic stress tolerance is a complex trait that is one of the
important quantitative traits for rice breeding. To improve
this trait, most QTL studies have focused on the isolation of
protein coding genes that are responsible. It is now clear that
we also need to consider post-transcriptional regulation by
noncoding RNAs such as miRNAs. In Arabidopsis, a
naturally occurring single nucleotide polymorphism in the
MIR164A gene alters the efficiency of miRNA processing,
affecting leaf shape and shoot architecture in Arabidopsis
(Todesco et al. 2012). In rice, a single nucleotide
polymorphism in the miR156 and miR529 target site of
OsSPL14 elevates expression of the target resulting in fewer
tillers, more panicle branching, and higher yields in rice (Jiao
et al. 2010; Miura et al. 2010; Jeong et al. 2011). These
findings emphasize that stress-regulated miRNAs and
miRNA target sites need to be seriously considered during
QTL analysis to maximize future crop improvement efforts.
Acknowledgments
We thank Bryan von Hagel for editorial assistance. Our work in thisarea was primarily supported by the Agriculture and Food ResearchInitiative Competitive Grants Program grant no. 2011-67013-30036from the USDA National Institute of Food and Agriculture (NIFA),with additional support from USDA-NIFA grant # 2007-35100-18268 and DOE grant # DE-FG02-07ER64450.
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