Developmental Biology 289 (2006) 3 – 16

www.elsevier.com/locate/ydbio

Review

Plant microRNA: A small regulatory molecule with big impact

Baohong Zhang *, Xiaoping Pan, George P. Cobb, Todd A. Anderson *
The Institute of Environmental and Human Health (TIEHH), and Department of Environmental Toxicology, Texas Tech University, Lubbock, TX 79409-1163, USA

Received for publication 31 July 2005, revised 10 October 2005, accepted 17 October 2005

Abstract

MicroRNAs (miRNAs) are an abundant new class of non-coding ¨20 – 24 nt small RNAs. To date, 872 miRNAs, belonging to 42 families,
have been identified in 71 plant species by genetic screening, direct cloning after isolation of small RNAs, computational strategy, and expressed
sequence tag (EST) analysis. Many plant miRNAs are evolutionarily conserved from species to species, some from angiosperms to mosses.
miRNAs may originate from inverted duplications of target gene sequences in plants. Although miRNA precursors display high variability, their
mature sequences display extensive sequence complementarity to their target mRNA sequences. miRNAs play important roles in plant post-
transcriptional gene regulation by targeting mRNAs for cleavage or repressing translation. miRNAs are involved in plant development, signal
transduction, protein degradation, response to environmental stress and pathogen invasion, and regulate their own biogenesis. miRNAs regulate
the expression of many important genes; a majority of these genes are transcriptional factors.
D 2005 Elsevier Inc. All rights reserved.

Keywords: microRNA; Plant; Gene regulation; Development; Environmental stress; Signal transduction

Introduction usually consisting of ¨20 –22 nucleotides for animals and

MicroRNAs are a class of recently discovered non-coding
endogenous small RNAs (Bartel, 2004). Although these small
RNAs were first discovered a decade ago (Lee et al., 1993;
Wightman et al., 1993), they were not recognized until 2001
(Lee and Ambros, 2001; Lau et al., 2001; Lagos-Quintana et
al., 2001). Since then, miRNAs have attracted a huge interest
from scientists (Bartel, 2004). Several laboratory and compu-
tational approaches have shown that this new class of small
RNAs has diverse and important roles in plant growth and
development (Bartel, 2004). This paper highlights research
progress on plant miRNAs and their various functions on plant
growth, development, and stress responses.
Identification of plant microRNAs
Methods for identifying miRNAs are based on their major
characteristics: (1) all miRNAs are small non-coding RNAs,
* Corresponding authors.
E-mail addresses: baohong.zhang@ttu.edu (B. Zhang),
todd.anderson@tiehh.ttu.edu (T.A. Anderson).
0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2005.10.036
¨20 –24 nt for plants (Reinhart et al., 2002; Bartel, 2004). (2)
All miRNA precursors have a well-predicted stem loop hairpin
structure, and this fold-back hairpin structure has a low free
energy (Reinhart et al., 2002; Bartel, 2004; Bonnet et al.,
2004b) as predicted by MFOLD (Zuker, 2003; Mathews et al.,
1999) or other computational programs. A majority of miRNA
precursors have considerably lower free energy ( 32– 57 kcal/
mol) than transfer RNAs (tRNAs) ( 26– 29 kcal/mol) or
ribosomal RNAs (rRNAs) ( 33 kcal/mol) as demonstrated by
Bonnet et al. (2004b) using statistical analysis. (3) Many
miRNAs are evolutionarily conserved, some from worm to
human (Weber, 2005; Berezikov et al., 2005; Altuvia et al.,
2005), or from ferns to core eudicots or monocots in plants
(Reinhart et al., 2002; Llave et al., 2002; Floyd and Bowman,
2004; Axtell and Bartel, 2005; Zhang et al., 2005). Plant
miRNAs are less conserved than animal miRNAs. Usually,
only the mature miRNAs are conserved in plants instead of
miRNA precursors that are usually conserved in animals
(Bartel, 2004). However, some of these characteristics are not
unique to miRNAs (Ambros et al., 2003). For example, tRNAs
also have stem loop structures. Therefore, when a small RNA is
considered a miRNA, all of these major characteristics should
be included. To avoid designating other small RNAs or
4 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16
fragments of other RNAs as miRNAs, Ambros et al. (2003)
developed combined criteria to identify new miRNAs. These
combined criteria (5) include both biogenesis and expression
criteria, neither of which on its own is sufficient for identifying
a candidate gene as a new miRNA (Ambros et al., 2003). So
far, all newly predicted or identified miRNAs have conformed
to these rules.
Currently, there are four approaches for identifying miR-
NAs: genetic screening (Lee et al., 1993; Wightman et al.,
1993), direct cloning after isolation of small RNAs (Lu et al.,
2005a), computational strategy (Brown and Sanseau, 2005),
and expressed sequence tags (ESTs) analysis (Zhang et al.,
2005). In 1993, two research groups led by Ambros and
Ruvkun found that a small 22 nt RNA derived from a gene
called lin-4 suppressed the expression of lin-14, which controls
the timing of Caenorhabditis elegans larval development (Lee
et al., 1993; Wightman et al., 1993). lin-4 is recognized as the
founding member of a new class of small RNAs called
miRNAs (Lee and Ambros, 2001; Lau et al., 2001; Lagos-
Quintana et al., 2001; Bartel, 2004). Since lin-4 was recognized
as a miRNA in 2001, thousands of miRNAs have been
identified from various organisms including C. elegans,
Drosophila melanogaster, fish, mouse, rat, and human (Lai,
2003).
Compared with animal miRNAs, plant miRNAs were
identified 10 years later. In 2002, several groups found plant
miRNAs by cloning small RNAs (Llave et al., 2002; Reinhart
et al., 2002; Park et al., 2002). Since then, different approaches
have been employed to identify new miRNAs. To date, 872
miRNAs belonging to 42 families have been identified in 71
plant species. Of these, 391 have been deposited in the miRNA
Registry Database (version Rfam 6.0 released in April 2005)
(Griffiths-Jones, 2004). In the past 3 years, the total number of
miRNAs has dramatically increased as the approaches for
identifying species-specific miRNAs are enhanced.
Initially, miRNAs were identified by genetic screening (Lee
et al., 1993; Wightman et al., 1993). This method was similar
to methods for identifying other traditional genes. Although
this method was useful for identifying some miRNAs, such as
lin-4 and lin-7, the application of this method was limited
because it is expensive, time consuming, and dominated by
chance.
To overcome some of the shortcomings of genetic screen-
ing, another experimental approach was recently described for
isolating and identifying new miRNAs. This approach involves
direct cloning after isolation of small RNAs (Lu et al., 2005a;
Fu et al., 2005). In this approach, small RNA molecules are
first isolated by size fractionation. Then these small RNAs are
ligated to RNA adapters at their 5V and 3V ends. Finally, they are
reverse transcribed into cDNA, which is then amplified and
sequenced (Lu et al., 2005a). Because only small RNAs are
isolated and screened by this method, it is a more efficient way
to obtain miRNAs than general genetic screening. The
approach has been used to identify new miRNAs in several
organisms including animals (Fu et al., 2005) and plants (Lu et
al., 2005a, Xie et al., 2005; Sunkar et al., 2005). Recently, Lu et
al. (2005a) further refined this method by combining it with
massively parallel signature sequencing (MPSS) to study
Arabidopsis miRNAs. This method is not only suitable for
identifying plant miRNAs but also can quantify miRNA
abundance at the same time. Using this approach, Lu et al.
(2005a) identified 77 of 92 available Arabidopsis miRNAs in
the miRNA database at the time of their study. This approach
should allow for more miRNA discoveries once adapted for the
study of other plant species.
The third approach is the traditional computational ap-
proach. This approach is based on a genome sequence (Lim et
al., 2003a,b; Lai et al., 2003; Grad et al., 2003; Wang et al.,
2004; Bonnet et al., 2004a,b; Jones-Rhoades and Bartel, 2004;
Bentwich et al., 2005; Brown and Sanseau, 2005; Sunkar et al.,
2005; Li et al., 2005; Berezikov et al., 2005; Adai et al., 2005;
Wang et al., 2005). Several laboratories have designed
computational programs, such as MIRscan (http://genes.
mit.edu/mirscan/) (Lim et al., 2003a,b) and MiRAlign (http://
bioinfo.au.tsinghua.edu.cn/miralign/) (Wang et al., 2005), and
successfully predicted miRNA genes in Arabidopsis (Wang et
al., 2004; Bonnet et al., 2004a,b; Jones-Rhoades and Bartel,
2004; Adai et al., 2005), rice (Bonnet et al., 2004a,b; Li et al.,
2005), human (Lim et al., 2003a; Wang et al., 2005; Bentwich
et al., 2005), C. elegans (Grad et al., 2003; Lim et al., 2003b),
and other animals (Lim et al., 2003a; Brown and Sanseau,
2005). At present, a majority of the miRNAs deposited in
miRNA databases {The miRNA Registry (http://www.sanger.
ac.uk/Software/Rfam/mirna/index.shtml) and The Arabidopsis
Small RNA Project (http://asrp.cgrb.oregonstate.edu/)} were
identified by computational approaches. However, the actual
computational approach used was slightly inefficient and
certainly not comprehensive. The predicted miRNAs need to
be confirmed by experiments such as cloning or Northern
blotting.
The fourth approach is an expressed sequence tag (EST)
analysis approach. It is well recognized that several miRNAs
are evolutionarily conserved from species to species (Llave et
al., 2002; Reinhart et al., 2002; Floyd and Bowman, 2004;
Weber, 2005; Berezikov et al., 2005; Altuvia et al., 2005;
Axtell and Bartel, 2005; Zhang et al., 2005). This suggests a
powerful approach to predict homologies or orthologues of
previously known miRNAs. Weber (2005) used this principle
to find 35 new human and 45 new mouse putative miRNA
genes. More importantly, this approach is very useful for
predicting miRNAs in multiple species, especially in species
whose genomes are unknown. Recently, we developed an EST
analysis approach to identify miRNA homologies. After
mining a repository of ESTs publicly available, we identified
481 homologues of miRNAs previously known in one plant
species (Arabidopsis) but not in other species. This suggests
that EST analysis is a good alternative method for identifying
miRNAs, especially for species whose genome is poorly
understood. However, this method can only identify conserved
miRNAs. Most miRNAs are more likely nonconserved, so it is
impossible to find these genes based on the EST approach.
Compared with other more mature research areas of gene
prediction, predicting and annotating miRNAs still need more
work. The four existing approaches have different advantages
 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16 5

and shortcomings (Table 1). By combining these methods, regions although other regions have much lower nucleotide
more miRNAs will be quickly discovered. conservation. Both miRNA conservation and miRNA target
conservation indicate that plant miRNAs have a very deep
Origin, conservation, and diversity of miRNAs in plants origin in plant phylogeny, at least since the last common
ancestor of bryophytes and seed plants (Floyd and Bowman,
In animals, miRNAs are evolutionarily conserved; many 2004; Zhang et al., 2005). Gene regulation by miRNA is an
miRNAs, such as miR7 and miR18, are conserved from worm ancient evolutionary mechanism to control gene expression. It
to human (Weber, 2005; Lagos-Quintana et al., 2001). is one part of the global gene regulation mechanisms. This
Pasquinelli et al. (2000) considered that animal miRNAs suggests that miRNA-mediated gene regulation existed more
existed at least 420 million years ago, with a common ancestor than 425 million years ago in the plant kingdom. The age of
of metazoans. miRNA conservation has also been observed in plant miRNA is comparable to the age of miRNA regulation in
plants (Llave et al., 2002; Reinhart et al., 2002; Sunkar and metazoans, however, no evidence yet shows that plant miRNAs
Zhu, 2004; Wang et al., 2004; Jones-Rhoades and Bartel, 2004; and animal miRNAs have a common ancestor.
Bonnet et al., 2004a,b; Adai et al., 2005; Axtell and Bartel, A total of 117 miRNAs have been found in Arabidopsis
2005), however, only mature plant miRNAs are conserved, and deposited in the miRNA database (Griffiths-Jones, 2004).
unlike the conservation of both mature miRNAs and miRNA These miRNAs can be grouped into 42 miRNA families
precursors in animals (Reinhart et al., 2002; Zhang et al., according to their nucleotide sequence similarity. A majority
2005). Experimental and computational analysis has indicated of the miRNA families have also been found in other species,
that many plant miRNAs and their targets are conserved such as rice, maize, and sorghum (Griffiths-Jones, 2004). Of
between monocots and dicots, based on limited data for the 42 plant miRNA families, some are highly evolutionarily
Arabidopsis and rice (Bonnet et al., 2004a; Jones-Rhoades conserved; there are only a few nucleotide changes among a
and Bartel, 2004; Sunkar and Zhu, 2004; Wang et al., 2004; wide variety of plant species (Zhang et al., 2005). Axtell and
Adai et al., 2005). Monocots and dicots diverged about 125 Bartel (2005) found that the miR159/319 family existed in 10
million years ago, thus, miRNAs should have existed at that plant species. miRNA families 156/157 and 165/166 exist in
time when they diverged from the same ancestor. Recently, 9 plant species, ranging all plant lineages. Zhang et al. (2005)
Axtell and Bartel (2005) employed microarray technology to reported that about 30% of miRNA families existed in at least
analyze the expression of several miRNAs in different plant 10 different plant species. Of these miRNA families, miR156
species. They found that some miRNAs existed not only in was found in 31 different plant species (Zhang et al., 2005).
dicots and monocots but also in ferns, lycipods, and mosses. In Based on conservation, miRNAs are classified into 4 groups:
addition, our EST analysis demonstrated that miRNAs exist in highly, moderately, lowly, and non-conserved miRNAs
more than 71 plant species including distantly related plant (Jones-Rhoades and Bartel, 2004; Zhang et al., 2005). For
species from mosses to higher flowering plants (Zhang et al., some miRNAs, for example, miR163 and miR158, Jones-
2005). Recently, a study by Floyd and Bowman (2004) showed Rhoades and Bartel (2004) considered as non-conserved
that the class-III homeodomain-leucin zipper (HD-Zip) genes, miRNAs. However, after dbEST analysis, 5 and 4 plant
one of the targets of miR166, have conserved miR166 target species were found to have homologues of these two
miRNAs, respectively. Thus, they should probably be re-
classified as lowly conserved miRNA families (Zhang et al.,
Table 1 2005). It is expected that some nonconserved or lowly
Comparison of four approaches for identifying miRNAs
conserved miRNAs may be re-classified as more miRNAs
Genetic Direct cloning Computational EST are discovered. Conservation is an indicator of miRNA
screening after isolation method analysis
of small RNAs
function; conserved miRNAs play an important role in
conserved gene regulation, such as leaf and flower morphol-
Specific software No No Yes No
Require genome No No Yes No
ogy or signal transduction. Non-conserved miRNAs may play
sequence more specific roles in specific plant species, such as the
Cost High High, but less Moderate Low differentiation and elongation of cotton fibers.
than genetic Many mechanisms are involved in new gene regulatory
screening origins and their networks; one of these mechanisms is that
Efficiency Low High Low High
False positive Low Low High Moderate
new gene regulatory networks originate from duplication of
possibility protein-coding sequences (Levine and Tjian, 2003; Teichmann
Need experimental No No Yes Moderate and Babu, 2004; Allen et al., 2004). After analyzing several
confirmation miRNA-related sequences in Arabidopsis, Allen et al. (2004)
Possibility for new High High High Low presented a model for miRNA origin. In their model, plant pre-
miRNAs
Suitable to wide Yes Yes No Yes
miRNAs originated from their target genes by formation of
variety of species inverted duplications which have been transcribed, but not
Comprehensive Yes Yes Moderate Yes modified further (Allen et al., 2004). miRNAs may originate
MiRNA quantitative No Yes No Somewhat anywhere within the plant genome; many genomic regions
information were found to be sites of miRNAs although these regions were
6 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16
previously considered featureless (Lu et al., 2005a,b). These
findings suggest that miRNA origin may be more complicated
than previously thought, involving many mechanisms such as
inversion and duplication.
Biogenesis of plant microRNAs
miRNA biogenesis requires multiple steps in order to form
mature miRNAs from miRNA genes (Bartel, 2004; Kurihara
and Watanabe, 2004). First, a miRNA gene is transcribed to a
primary miRNA (pri-miRNA), which is usually a long
sequence of more than several hundred nucleotides (Fig. 1).
This step is controlled by Pol II enzymes (Bartel, 2004;
Kurihara and Watanabe, 2004; Lee et al., 2004). Second, the
pri-miRNA is cleaved to a stem loop intermediate called
miRNA precursor or pre-miRNA. This step is controlled by
the Drosha RNase III endonuclease in animals (Lee et al.,
2002; Bartel, 2004) or by Dicer-like 1 enzyme (DCL1) in
plants (Tang et al., 2003; Kurihara and Watanabe, 2004).
Loss-of-function dcl1 mutants have low levels of miRNA
synthesis (Schauer et al., 2002). In animals, pre-miRNAs are
then transported by exportin 5 from the nucleus into the
cytoplasm (Yi et al., 2003; Lund et al., 2004; Zeng and
Cullen, 2004), followed by formation of miRNA:miRNA*
duplex and mature miRNAs by another RNase III-like enzyme
called Dicer (Bernstein et al., 2001; Bartel, 2004). In this step,
however, plant miRNAs differ from animals. Plant miRNAs
are cleaved into miRNA:miRNA* duplex possibly by dicer-
like enzyme 1 (DCL1) in the nucleus rather than in the
cytoplasm (Papp et al., 2003; Bartel, 2004), then the duplex is
translocated into the cytoplasm by HASTY, the plant
orthologue of exportin 5 (Park et al., 2005). In the cytoplasm,
both plant and animal miRNAs are unwound into single
strand mature miRNAs by helicase (Bartel, 2004). Finally, the
mature miRNAs enter a ribonucleoprotein complex known as
the RNA-induced silencing complex (RISC) (Hammond et al.,
2000; Martinez et al., 2002; Schwarz et al., 2002) where they
regulate targeted gene expression (Bartel, 2004). This suggests
that miRNA biogenesis is complicated; several enzymes are
required for processing long pri-miRNA to ¨20– 24 nt mature
miRNAs.
Molecular mechanisms of plant gene regulation by microRNAs
In the RISC complex, miRNAs bind to target messenger
RNA (mRNA) and inhibit gene expression through perfect or
near-perfect complementarity between the miRNA and the
mRNA (Bartel, 2004). This causes gene silencing (Almeida
and Allshire, 2005) termed RNA interference (RNAi) in
animals (Hannon, 2002), quelling in fungi (Cogoni et al.,
1996), and post-transcriptional gene silencing (PTGS) in
plants (Baulcombe, 2004). Animal miRNAs usually bind to
target mRNAs through imperfect complementarity at multiple
sites located at the 3V untranslated regions (UTR), hamper
ribosome movement along the mRNA, and repress gene
expression (Carrington and Ambros, 2003). Plants have
similar mechanisms in gene expression regulation. However,
in plants, most target mRNAs only contain one single miRNA
complementary site, and most corresponding miRNAs typi-
cally perfectly complement to these sites and cleave the target
mRNAs (Bartel, 2004). Unlike animal miRNA targets, the
complementary sites in plants can exist anywhere along the
target mRNA rather than at the 3-UTR. Recently, another
mechanism was identified in plant miRNA regulation.
Although some miRNAs, such as miR172, can perfectly
complement to target mRNAs, they regulate gene expression
by repressing gene translation possibly through inhibition of
ribosome movement (Aukerman and Sakai, 2003; Chen,
2004). However, a recent study observed that miR172 can
also efficiently guide cleavage of its target transcripts
(Schwab et al., 2005). This suggests that miRNAs may be
involved in more complicated mechanisms to control gene
expression in plants than in animals. Plant miRNAs regulate
gene expression at the post-transcriptional level not only by
repression of mRNA translation but also by direct cleavage of
mRNAs.
To provide new insights into the mechanism of plant
miRNA function, Schwab et al. (2005) employed genome-
wide expression profiling to analyze parameters for miRNA-
guided mRNA cleavage. Transgenic technology was used to
overexpress several plant miRNAs (including miR156,
miR159, miR164, and miR319/JAW) in order to study the
effect of several parameters on mRNA cleavage. These
parameters included the number of mismatches, sequence
complementarity between miRNA and the corresponding
mRNA, and free energy of miRNA paired with potential
target sites. Based on their results, Schwab et al. (2005)
deduced a set of empirical parameters for miRNA target
recognition and concluded that plant miRNAs have higher
specificity than siRNAs. Their study also demonstrated that
feedback regulation makes the effects of miRNAs on steady-
state levels of target mRNAs more complicated. Feedback
regulation of miRNA targets may provide a sensitive
mechanism for fine tuning the expression of target genes
(Schwab et al., 2005).
Diversity of plant microRNA functions
Although miRNAs are relatively small, they play an
important role in gene expression. miRNA is considered as
one of the most important post-transcriptional gene regulators
(Carrington and Ambros, 2003) since it was originally
recognized in 2001 (Lee and Ambros, 2001; Lau et al., 2001;
Lagos-Quintana et al., 2001). In animals, miRNAs have proven
to be involved in many functional processes such as
development (Wienholds et al., 2005; Giraldez et al., 2005),
stress response (Ambros, 2003), and carcinogenesis (Iorio et
al., 2005; Croce and Calin, 2005). Although investigations of
plant miRNAs are behind animal miRNAs, the versatile
functions of plant miRNAs are also becoming clearer (Kidner
and Martienssen, 2005a). A majority of miRNA functions are
currently investigated by overexpression or lowered expression
of miRNAs. The following section summarizes several miRNA
functions in plants.
 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16 7

Fig. 1. A simplified model of miRNA biogenesis and function in plants.

miRNAs regulate plant development
Dicer-like enzyme 1 (DCL1) is an important enzyme that
processes pri-miRNA to pre-miRNA then to miRNA:miRNA*
duplex (Papp et al., 2003; Bartel, 2004). Normal expression of
the dcl1 gene is essential to synthesize and accumulate mature
miRNA (Papp et al., 2003). Loss-of-function of the dcl1 gene
reduced the level of mature miRNA and consequently caused
many developmental abnormalities, such as altered leaf shape,
delayed floral transition, arrested embryos at early stages, and
female sterility (Park et al., 2002; Reinhart et al., 2002; Dugas
and Bartel, 2004). HASTY is an orthologue of exportin 5,
which is the protein transporting miRNA:miRNA* duplex
from the nucleus to the cytoplasm in Arabidopsis (Park et al.,
2005). Without HASTY, it is impossible to synthesize mature
miRNAs in Arabidopsis. Bollman et al. (2003) demonstrated
that loss-of-function of hasty gene caused many pleiotropic
phenotypes, including disrupting leaf shape and flower
morphology, reducing fertility, and accelerating vegetative
phase change and disrupting the phyllotaxis of the inflores-
cence (Bollman et al., 2003). These findings alone indicate that
miRNAs are involved in a variety of developmental processes
in plants.
Several experiments have demonstrated that many miRNAs
regulate various plant development processes, including leaf
morphogenesis and polarity (Emery et al., 2003; Mallory et al.,
2004a; Juarez et al., 2004; Zhong and Ye, 2004; Bowman,
2004; Bao et al., 2004; Kim et al., 2005), floral differentiation
and development (Chen, 2004; Aukerman and Sakai, 2003),
root initiation and development (Mallory et al., 2004b; Laufs et
8 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16
al., 2004; Guo et al., 2005), vascular development (Floyd and
Bowman, 2004; Kim et al., 2005), and transition of plant
growth from vegetative growth to reproductive growth (Achard
et al., 2004; Lauter et al., 2005). A majority of these miRNAs
affect plant traits by regulating the expression of transcription
factors that influence cell fate determination (Rhoades et al.,
2002; Mallory et al., 2004b). At present, about 50% of the
miRNA targets that have been identified are transcription
factors (Bartel, 2004; Kidner and Martienssen, 2005a). Of these
miRNAs, at least 12 miRNA families target mRNAs encoding
transcriptional factors that regulate plant development (Allen et
al., 2004). Combined, these studies have illustrated that a
majority of miRNAs regulate plant development by controlling
the levels of transcription factors that are important in
development. Detailed effects of miRNA on different tissues
are discussed in the following sections.
miRNA and leaf development
Plant leaves exhibit an asymmetry pattern along the adaxial/
abaxial (upper/lower) axis. This pattern is controlled by the
polarized expression of class-III homeodomain leucine zipper
(HD-ZIP) transcription factor genes (Juarez et al., 2004).
PHABULOSA (PHB), PHAVOLUTA (PHV), and REVO-
LUTA (REV) are three closely related Arabidopsis HD-ZIP
transcription factors. Dominant mutations in any of these three
transcription factor genes (phb, phv, and rev) results in
radialization and adaxialization of leaf and vascular bundles
in the stem (Emery et al., 2003; McConnell et al., 2001).
Recently, several experiments have demonstrated that all of
these transcription factors are the targets of miR165 and
miR166, and are regulated by these two miRNAs (Emery et al.,
2003; Mallory et al., 2004a; Juarez et al., 2004; Zhong and Ye,
2004; Bowman, 2004; Bao et al., 2004; Kim et al., 2005).
Mallory et al. (2004a) constructed a phb mutation that changed
the complementary sites of miR165 and miR166, but did not
change the PHB protein sequence. They observed that
disrupted PHB mRNA pairing with miRNA resulted in
developmental defects in phb-d mutants (Mallory et al.,
2004a). They also found that the 5V portion of miRNA is
crucial for miRNA-mediated gene regulation (Mallory et al.,
2004a). Results of in-situ hybridization indicated that the
expressed pattern of miR165/166 (Kidner and Martienssen,
2004) is similar to that of its target PHB mRNA (McConnell et
al., 2001).
The HD-ZIP gene family is conserved in all lineages of land
plants, including mosses, ferns, gymnosperms, and angios-
perms (Floyd and Bowman, 2004). The miRNAs, miR165 and
miR166, targeting the HD-ZIP gene family, are also evolu-
tionarily conserved in all lineages of land plants (Zhang et al.,
2005). The developmental abnormality caused by loss-of-
function HD-ZIP genes in Arabidopsis was also observed in
other plant species. In corn, rolled leaf 1 (rld1) is a homologue
of the Arabidopsis HD-ZIP genes. Juarez et al. (2004) found
that miR166 directly cleaves rld1 mRNA and alters leaf
polarity in maize. They also observed that miR166 may be a
mobile signal in leaf development based on in situ hybridiza-
tion. In their experiments, miRNA 166 exhibited a progres-
sively expanding expression pattern in leaves and phloem
during leaf development.
In addition to miR166 and miR165, miR159/JAW also
regulates leaf development by targeting a subset of TCP
transcription factor genes (Palatnik et al., 2003). Overexpres-
sion of miRJAW resulted in low levels of all tested TCP
mRNAs and caused jaw-D phenotypes, including uneven leaf
shape and curvature (Palatnik et al., 2003). In contrast,
overexpression of miRJAW-resistant TCP mutants indicated
that miRJAW-guided mRNA cleavage was sufficient to restrict
TCP function (Palatnik et al., 2003). miR165/166 and miR159/
JAW are essential for controlling the pattern and development
of leaves by direct regulation of two classes of transcription
factor genes (HD-ZIP and TCP).
miRNA and floral development and vegetative phase change
Flower development is one of the most important stages of
plant development, especially for flowering plants. Mature
flowers consist of carpels, stamens, petals, and sepals. The
origin of the flower can be well explained by the ABC model
(reviewed by Lohmann and Weigel, 2002; Jack, 2004).
According to this model, four flower organs are controlled
by the combinatorial actions of three classes (A, B, and C) of
transcription factors (Lohmann and Weigel, 2002). APETALA
2 (AP2) is one of the class A genes that play an important role
in flowering time and flower morphology. AP2 and AP2-like
proteins are required for plant flowering and floral organ
identity. Recently, two independent laboratories have demon-
strated that AP2 is one of the targets of miR172 (Chen, 2004;
Aukerman and Sakai, 2003). Overexpression of miR172
inhibited the translation of the AP2 gene and AP2-like genes
(target of eat 1, TOE1) and resulted in early flowering and
disrupting the specification of floral organ identity similar to
loss-of-function ap2 gene mutants (Chen, 2004; Aukerman and
Sakai, 2003). miR172 regulates ap2 gene expression through
translational inhibition rather than through mRNA cleavage
(the usual mechanism for most miRNAs) (Chen, 2004;
Aukerman and Sakai, 2003).
The scarecrow-like (SCL) (GRAS domain) family is a class
of plant-specific transcription factors (Llave et al., 2002;
Reinhart et al., 2002). Three SCL genes (SCL6-II, SCL6-III,
and SCL6-IV) in Arabidopsis and 4 in rice have perfectly
complementary sequences with miR171 (Llave et al., 2002;
Reinhart et al., 2002). This indicates that these SCL genes are the
targets of miR171. miR171 is predominantly expressed in
inflorescence and flower tissues; very little miR171 can be
detected in leaf or stem tissues (Llave et al., 2002). This suggests
that miR171 may have some role in floral development.
One important characteristic of floral development is phase
change. The emergence of floral meristems symbolizes the
phase change from vegetative growth to reproductive growth.
This transition (including the transition from a juvenile to adult
leaf) is regulated by the AP2-like gene glossy15 (gl15) in
maize (Lauter et al., 2005). gl15 is usually expressed in maize
juvenile leaves. Maize transitions from vegetative growth to
 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16
reproductive growth by decreasing gl15 gene expression.
Lauter et al. (2005) demonstrated that gl15 is one of the
targets of miR172. Overexpression of miR172 resulted in
decreasing gl15 expression through gl15 mRNA cleavage and
resulted in delayed phase change from vegetative to reproduc-
tive (Lauter et al., 2005).
GAMYB is a positive regulator of LEAFY, which is an
important factor in floral development (Gocal et al., 2001;
Kaneko et al., 2004). GAMYB is one target of miR159. One
study indicated that miR159 directly guides the cleavage of
mRNA encoding GAMYB-related proteins and further affects
the GA-promoted activation of LEAFY (Achard et al., 2004).
Overexpression of miR159 resulted in a reduction of LEAFY
transcript levels and further perturbed anther development and
delayed flowering in short-day photoperiods (Achard et al.,
2004). This indicates that miR159-guided GAMYB cleavage
regulates plant anther development and flowering time.
miR156 is also involved in floral development and phase
change by targeting members of Squamosa promoter binding
protein like (SPL) plant-specific transcription factors. The SPL
family has 16 members; some (such as SPL3) are involved in
floral transition and regulating plant flowering (Cardon et al.,
1997). Recent results indicated that overexpression of miR156
affects phase transition from vegetative growth to reproductive
growth, including the quickly initiation of rosette leaves, a
severe decrease in apical dominance, and a moderate delay in
flowering (Schwab et al., 2005).
miRNA and shoot and root development
Cup-shaped cotyledon 1 (CUC1) and CUC2 are two
important transcription factors of the NAM/ATAF/CUC
(NAC)-domain transcription factor family which is restricted
to plants (Riechmann et al., 2000). They play important roles in
both embryogenic and floral development (Aida et al., 1997;
Takada et al., 2000; Hibara et al., 2003). Loss-of-function of
these genes resulted in abnormal floral and shoot development
(Aida et al., 1997; Takada et al., 2000; Hibara et al., 2003;
Mallory et al., 2004b). Recently, five members (CUC1, CUC2,
NAM, NAC1, At5g07680, and At5g61430) of the NAC-
domain gene family in Arabidopsis were identified that have
complementary sites with miR164 and are targets of miR164
(Rhoades et al., 2002; Mallory et al., 2004b; Laufs et al., 2004;
Guo et al., 2005). Of these, NAC1 is involved in lateral root
development (Xie et al., 2002), whereas CUC1 and CUC2
regulate meristem development and separation of aerial organs
(Aida et al., 1997). Overexpression of miR164 resulted in the
fusion of vegetative and floral organs, unbalanced floral organ
numbers, and reduced lateral root emergence (Mallory et al.,
2004b; Laufs et al., 2004; Guo et al., 2005). In contrast,
miR164-resistant mutants of CUC1 and CUC2 resulted in
Arabidopsis embryonic, vegetative, and floral development
defects, including misshaping of rosette leaves, altering flower
structure, and changing cotyledon orientation (Laufs et al.,
2004; Mallory et al., 2004b). miR164 also controlled organ
boundaries and root formation by regulating NAC1 and CUC2
expression (Mallory et al., 2004b; Laufs et al., 2004; Guo et al.,
9
2005). Loss-of-function miR164 mutants accumulated NAC
mRNA, resulting in more lateral root formation (Guo et al.,
2005). In contrast, overexpression of miR164 in wild-type
Arabidopsis resulted in decreasing NAC1 mRNA levels and
decreased lateral root emergence (Guo et al., 2005). miR164
plays an important role in controlling shoot and root
development by regulating several NAC transcription factors.
It may be possible to increase crop yields and resistance to
environmental stress by appropriately controlling miR164
expression.
miRNA and vascular development
HD-ZIP proteins also regulate vascular development as well
as lateral organ polarity and meristem formation. ATHB15, a
member of the HD-ZIP family, is predominantly expressed in
vascular tissues, suggesting that it may play some role in plant
vascular development (Ohashi-Ito and Fukuda, 2003; Kim et
al., 2005). ATHB15 is one target of miR166 (Rhoades et al.,
2002). Overexpression of miR166a resulted in decreasing
ATHB15 mRNA levels and caused accelerated vascular cell
differentiation from cambial/procambial cells and consequently
produced an altered vascular system with expanded xylem
tissue and an interfascicular region (Kim et al., 2005). This
regulation mechanism may exist in all vascular plant species
(Floyd and Bowman, 2004; Kim et al., 2005). In addition,
some miRNAs may function in the biosynthesis of cell wall
metabolites (Lu et al., 2005b) or cotton fiber development
(Zhang et al., 2005).
miRNAs regulate miRNA and siRNA biogenesis and function
DCL1 is one of the key enzymes in mature miRNA
formation (Papp et al., 2003; Bartel, 2004). DCL1 mRNA is
the target of miR162 (Xie et al., 2003). Xie et al. (2003)
demonstrated that the level of DCL1 mRNAs was much higher
in miRNA-defective hen1 mutant plants than in normal
Arabidopsis plants. To further test that DCL1 mRNA is
negatively regulated by miRNA-guided mRNA cleavage, Xie
and co-workers employed transgenic Arabidopsis plants,
expressing P1/HC-Pro protein which is a virus-encoded
suppressor of RNA silencing and inhibits miRNA-guided
targeted mRNA degradation (Kasschau et al., 2003), to study
the function of miR162. Their results showed that DCL1
mRNAs were overexpressed in their transgenic plants. These
data indicate that DCL1 mRNA is negatively regulated by
miR162.
Regardless of the regulation mechanism (mRNA cleavage
or translational repression), miRNA-guided gene regulation
requires that miRNA form a complex with RISC. RISC
contains many components; loss-of-function of each compo-
nent affects the function of RISC and also affects the function
of miRNAs. ARGONAUTE proteins, also known as PPT
proteins, are a core component of RISC (Hammond et al.,
2001; Caudy et al., 2003; Vaucheret et al., 2004). ARGO-
NAUTE proteins contain a single-stranded RNA binding site
(Yan et al., 2003). This characteristic allows ARGONAUTE
10 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16
proteins to directly associate with miRNAs before and after
RISC recognizes their mRNA targets (Vaucheret et al., 2004).
There are 10 proteins in the Arabidopsis ARGONAUTE
protein family (Morel et al., 2002). Several experiments have
indicated that ARGONAUTE1 (AGO1) is required for stem
cell function and organ polarity (Kidner and Martienssen,
2005b). Computational analysis and 5VRACE experiments
demonstrated that only ARGONAUTE 1 (AGO1) mRNA is
the target of miR168 (Rhoades et al., 2002; Vazquez et al.,
2004; Kidner and Martienssen, 2005a). miR168-guided cleav-
age of AGO1 mRNA is crucial for the post-transcriptional gene
regulation of the ago1 gene (Vazquez et al., 2004). Vaucheret et
al. (2004) demonstrated that changing the nucleotides of AGO1
mRNA and making them less complementary with miR168
resulted in increasing levels of AGO1 mRNA (Vaucheret et al.,
2004) and consequently caused developmental defects similar
to dcl1 or hen1 mutants (Xie et al., 2003; Vaucheret et al.,
2004; Kidner and Martienssen, 2005a,b). Increased target
mRNA suggested that non-functional RISCs were formed in
the mutant plants (Vaucheret et al., 2004). These defects were
rescued by expressing a compensatory miRNA that is
complementary to the mutant AGO1 mRNA (Vaucheret et
al., 2004).
Recently, five ta-siRNA-generating transcripts were found
to be complementary to miR173 or miR390 and were also
identified as targets of these two miRNAs (Allen et al., 2005;
Bartel, 2005). Allen et al. (2005) found that miR173 guided in-
phase processing of four ta-siRNA primary transcripts, whereas
miR390 guided in-phase processing of trans-acting siRNA3
(TAS3) ta-siRNA primary transcripts. Based on these findings,
Allen and colleagues proposed a model for miRNA-directed
initiation of ta-siRNA biogenesis. In their model, a 5V or 3V
terminus within the pre-ta-siRNA transcripts was formed with
the help of miRNAs, then RDR6-dependent formation of
dsRNA and Dicer-like processing, finally yielded phased ta-
siRNAs that negatively regulate other genes (Allen et al.,
2005). miRNA not only regulates the expression of genes
controlling plant development but also regulates its own
biogenesis and/or function. To date, at least five miRNAs
(miR162, miR168, miR173, miR390, and miR398) are known
to regulate miRNA biogenesis or function.
miRNAs involved in signal transduction
Recently, several independent laboratories observed that
miRNAs regulate key components of hormone signaling
pathways and further regulate hormone homeostasis and
related developmental processes (Sorin et al., 2005; Mallory
et al., 2005; Guo et al., 2005). Several miRNAs affect signal
transduction, especially hormone signaling pathways. Plant
hormones, especially auxin, are important regulators of plant
growth and development, particularly plant cell division,
elongation, and differentiation (Palme et al., 1991; Woodward
and Bartel, 2005). Plant hormones also play important roles in
organ formation and response to various environmental stresses
(Palme et al., 1991; Woodward and Bartel, 2005). After
analysis of a large number of ESTs, Zhang et al. (2005) found
that several miRNAs (miR159, miR160, miR164, and miR167)
were obtained from tissues induced by abscisic acid (ABA),
GA, jasmonic acid (JA), salicylic acid (SA), and other
phytohormones. miR159 is a modulator of GAMYB through
the miRNA-guided cleavage of mRNA encoding GAMYB-
related proteins that are transcription factors regulating LEAFY
(Achard et al., 2004). Achard et al. (2004) demonstrated that
miR159 was positively regulated by the plant hormone
gibberellin (GA). Overexpression of miR159 resulted in
decreased LEAFY mRNAs, delayed flowering time, and
altered floral development (Achard et al., 2004).
Auxin (principally indole-3-acetic acid) is an important
hormone in plants. It affects many aspects of plant growth
and development by influencing AUXIN RESPONSE
FACTORS (ARF), a plant-specific family of DNA binding
proteins (Hagen and Guilfoyle, 2002; Weijers and Jurgens,
2005). ARFs regulate the expression of auxin-inducible
genes, such as GH3 and auxin/indole-3-acetic acid (Aux/
IAA) by binding auxin response promoters (AREs) (Hagen
and Guilfoyle, 2002). A total of at least 23 ARFs have been
identified in Arabidopsis thus far. Of these, at least 5 ARFs
have complementary sites with miRNAs. ARF10, ARF16,
and ARF17 are potential targets of miR160, and ARF6 and
ARF8 are potential targets of miR167 (Rhoades et al., 2002;
Mallory et al., 2005). Mallory et al. (2005) demonstrated
that disrupting miR160 function increased ARF10, ARF16,
and ARF17 mRNA levels, altered GH3-like gene expres-
sion, and led to severe developmental defects. It has also
been observed that miR160-resistant ARF plants have
dramatic developmental abnormalities (Mallory et al.,
2005; Sorin et al., 2005).
NAC1 is a transcriptional activator that transduces auxin
signals for lateral root initiation by regulating transport
inhibitor response 1 (TIR1) (Xie et al., 2002; Guo et al.,
2005). Guo et al. (2005) demonstrated that loss-of-function
miR164 mutants blocked the auxin signaling pathway, result-
ing in increased NAC1 mRNA levels and more lateral root
production. In contrast, overexpression of miR164 resulted in
downregulation of NAC1 gene expression and reduced lateral
root initiation (Guo et al., 2005). The expression of miR164
and NAC1 can be regulated by auxin through a signaling
pathway (Guo et al., 2005). The induction of miR164 at least
partially by auxin indicated an autoregulatory loop in which
miRNA-guided NAC1 mRNA cleavage attenuated and termi-
nated auxin signaling (Guo et al., 2005). miR164-guided NAC
mRNA cleavage downregulates auxin signals for lateral root
development.
In addition to miR160, miR164, and miR167, other
miRNAs, such as miR393, may also be involved in signaling
pathways by regulating TIR1. TIR1 is an important component
of a SCF E3 ubiquitin ligase which degrades Aux/IAA proteins
in response to auxin (Gray et al., 2001; Mallory et al., 2005).
Currently, TIR1 and its three most closely related F-box
proteins are predicted to be targets of miR393 (Sunkar and
Zhu, 2004; Jones-Rhoades and Bartel, 2004). This suggests
that miRNA also can target F-box proteins and affect the
activity of the E3 enzyme.
 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16
All of these studies indicate that miRNAs are involved in
hormone-mediated signal transduction. miRNAs can either
directly target ARF and NAC1 or be induced by plant
hormones.
miRNAs involved in plant disease
Pathogen infection is an important biological factor that
extensively affects plant growth and development (Brown,
2002; Gurr and Rushton, 2005). Pathogen infection causes
about a 30% yield loss for a majority of crops and fruits (Zhang
et al., 2000; Pande et al., 2005). In the long-term, plants have
evolved complicated mechanisms to resist infection. One of
these mechanisms is pathogen-induced post-transcriptional
gene silencing (PTGS) (Ding, 2000). Recently, more and more
evidence has shown that miRNAs are involved in plant
diseases caused by different pathogens; some of them may
also be involved in virus-induced gene silencing.
Helper component-proteinase (HC-Pro), p19, p21, and p69,
are unrelated viral suppressors of gene silencing (Plisson et al.,
2003; Chapman et al., 2004). They play important roles in the
virus response to plant antiviral silencing response. These
suppressors are usually called pathogenicity factors, and they
cause disease and various developmental abnormalities (Chap-
man et al., 2004). Recently, several studies have shown that
these suppressors are involved in the regulation of several
miRNA activities (Kasschau et al., 2003; Chen et al., 2004;
Chapman et al., 2004; Llave, 2004). HC-Pro decreased miRNA
levels, interfered with miR171 activity, and caused miR171-
related developmental defects (Kasschau et al., 2003). When
expressing HC-Pro gene in plants, 8 of 10 miR171-guided
cleavage target mRNAs accumulated to elevated levels, and
caused TuMV- and other virus-induced diseases (Kasschau et
al., 2003). One possible mechanism is that HC-Pro, p19, and
p21 inhibited the formation and/or activity of miRNA-contain-
ing RISC complex and resulted in failure of PTGS in infected
plants (Kasschau et al., 2003; Chapman et al., 2004). In
contrast, another viral suppressor, p69, encoded by turnip
yellow mosaic virus (TYMV), increased the transcription
levels of a Dicer mRNA and miRNAs, and enhanced the
miRNA-guided cleavage of host mRNAs (Chen et al., 2004).
This suggests that p69 may enhance miRNA-mediated gene
silencing and consequently enhance plant resistance to patho-
gens. Further clarification of this may provide tools for
improving crop resistance.
miRNAs involved in environmental stress responses
Plants have evolved complicated mechanisms to overcome a
variety of environmental stresses. miRNAs may also play a role
in these mechanisms. In our study using EST analyses, 25.8%
of ESTs containing miRNAs were found in stress-induced plant
tissues (Zhang et al., 2005). Although no experiments have
confirmed that these miRNAs were only obtained from stress-
induced tissues, the large percentage of ESTs containing
miRNAs is indicative that miRNAs may play some role in
plant responses to environmental stress (Zhang et al., 2005).
11
Several recent studies have provided supportive evidence for
this hypothesis. One study demonstrated that miR395 was
overexpressed under sulfate starvation conditions (Jones-
Rhoades and Bartel, 2004). Drought, cold, and salinity are
other environmental stresses for plants; all of these conditions
strongly induced miR402 overexpression (Sunkar and Zhu,
2004). Other miRNAs, such as miR319, are induced by either
cold or other stress (Sunkar and Zhu, 2004). At low-sulfur
conditions, the ATP sulfurylase APS4 and the sulfate trans-
porter AST68 are accumulated. Both are targets of miR395
(Jones-Rhoades and Bartel, 2004; Allen et al., 2005). Recently,
Lu et al. (2005b) identified 48 miRNA sequences from the
Populus genome and found that a majority of these Populus
miRNAs target developmental- and stress/defense-related
genes. They also found that plant miRNAs can be induced
by mechanical stress and may function in critical defense
systems for structural and mechanical fitness.
Abscisic acid (ABA) and gibberellin (GA) are two kinds of
phytohormones which are involved in plant responses to
different environmental stresses. Recently, two research groups
independently found that either ABA or GA treatment
regulated miR159 expression (Sunkar and Zhu, 2004; Achard
et al., 2004) and further controlled floral organ development
(Achard et al., 2004).
miRNAs appear to be involved in plant responses to a
variety of biotic and abiotic environmental stresses. Environ-
mental stress induces certain miRNAs to be over- or under-
expressed, or plants may have evolved some mechanism to
synthesize certain miRNAs to help cope.
Conclusions and future perspectives
This review summarizes recent findings regarding plant
miRNAs and their versatile roles in plant development (Table
2). To date, hundreds of plant miRNAs have been identified by
genetic screening, computational approaches, and EST analy-
sis. However, the identification of plant miRNAs continues to
lag behind the more mature areas of gene research.
Identification of new microRNAs
Several computational approaches have estimated that
organisms probably contain about 1% miRNA genes of the
total protein-coding genes (Lai et al., 2003; Lim et al.,
2003a,b). Recently, bioinformatics studies indicated that the
proportion of genes in the genome under miRNA regulation
may be much larger than previously thought. For example,
Lewis et al. (2005) proposed that about 30% of all human
genes may be regulated by miRNAs. However, only 391 plant
miRNAs have been deposited to date in the miRNA database
(http://microrna.sanger.ac.uk) (Griffiths-Jones, 2004) although
more than 872 miRNAs have been identified in 71 plant
species. This is far from the predicted miRNA number.
Arabidopsis is a model plant species for modern molecular
studies. Its genomic sequence was encoded and became
publicly available in 2000 (The Arabidopsis Genome Initiative,
2000), and it is one of the plant species from which the greatest
12 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16

Table 2
Validated miRNA targets and their function
MiRNA Function Target genes Target protein class Regulated Reference
mechanism
miR156/157 Transcription factor SPL2, SPL3 and SPL10 SQUAMOSA-promoter binding mRNA cleavage Kasschau et al., 2003;
proteins Chen et al., 2004;
Vazquez et al., 2004;
Schwab et al., 2005
miR159/JAW Leaf development TCP2, CP3, TCP4, CP10 TCP, a bHLH transcription factor mRNA cleavage Palatnik et al., 2003
and cell division and TCP24
miR159 Hormone response GAMYB MYB mRNA cleavage Sunkar and Zhu, 2004;
Achard et al., 2004;
Schwab et al., 2005
miR160 Signaling transduction ARF10, ARF16 and ARF17 Auxin Response Factors mRNA cleavage Mallory et al., 2005;
Sorin et al., 2005
miR162 MiRNA biogenesis DCL1 Dicer-like protein mRNA cleavage Xie et al., 2003;
Kasschau et al., 2003
miR164 Plant morphogenesis CUC1,CUC2, NAM, NAC1 NAC domain transcription factor mRNA cleavage Mallory et al., 2004
miR164 Auxin signaling and root NAC1 NAC domain transcription factor mRNA cleavage Guo et al., 2005;
development Schwab et al., 2005
miR165/166 Meristem formation PHB, PHV, REV, RLD1 HD-ZIP transcription factors mRNA cleavage Floyd and Bowman,
and others 2004; Juarez et al., 2004
miR166 Vascular development ATHB15 HD-ZIP transcription factors mRNA cleavage Kim et al., 2005
miR167 Signaling transduction ARF6 and ARF8 Auxin Response Factors mRNA cleavage Rhoades et al., 2002;
Mallory et al., 2005
miR168 MiRNA functions AGO1 ARGONAUTE protein mRNA cleavage Vaucheret et al., 2004;
Rhoades et al., 2002;
Vazquez et al., 2004
miR171 Developmental patterning SCL6-II, SCL6-III, Scarecrow-like (GRAS domain) mRNA cleavage Llave et al., 2002;
SCL6-IV transcription factor Reinhart et al., 2002;
Kasschau et al., 2003
miR172 Floral development AP2, TOE1, TOE2, TOE3, APETALA2-like transcriptional mRNA cleavage; Aukerman and Sakai,
and phage change GL15 factors translational 2003; Chen, 2004;
repression Lauter et al., 2005;
Schwab et al., 2005
miR173 siRNA biogenesis TAS1, TAS2 Trans-acting siRNA mRNA cleavage Allen et al., 2005
miR390 siRNA biogenesis TAS3 Trans-acting siRNA mRNA cleavage Allen et al., 2005
miR393 Hormone response TIR1 F-box protein, a class of bHLH mRNA cleavage Jones-Rhoades and
and others transcription factors Bartel, 2004
miR394 Hormone signaling F-box F-box mRNA cleavage Jones-Rhoades and
Bartel, 2004
miR395 Environmental stress APS4, AST68 ATP sulfurylase; sulfate Unknown Jones-Rhoades and
response transporter Bartel, 2004; Allen
et al., 2005
miR399 Ubiquitin conjugation At2g33770 E2-UBC mRNA cleavage Allen et al., 2005;
Sunkar and Zhu, 2004
miR403 Unknown AGO2 ARGONAUTE protein Unknown Allen et al., 2005

number of miRNAs have been identified. The Arabidopsis
Information Resource (TAIR) has currently listed 21,316 genes
that code proteins. These genes do not include the genes
encoding proteins for which the subcellular location was
unclear and the chloroplast- and mitochondria-targeted pro-
teins. Thus, Arabidopsis should have more than 213 miRNAs.
However, only 114 miRNAs have been identified in the
Arabidopsis genome to date, far from the 1.0% of the predicted
protein-coding genes.
Traditional computational approaches have made great
progress in predicting new miRNAs; a majority of the
miRNAs have been predicted by these approaches. How-
ever, all of these computational approaches and the newly
identified EST analysis strategy are based on the evolu-
tionary conservation of miRNAs. A majority of miRNAs
are likely to be non-conserved and/or species-specific, such
as those that control cotton fiber differentiation and
elongation. This makes it impossible to adapt the current
approaches to predict these non-conserved miRNA genes.
How to identify these non-conserved miRNAs remains an
open question.
Identification and validation of microRNA targets
Studies on miRNA targets are lagging further behind the
identification of miRNA genes; a majority of miRNA targets
have not been found or the predicted targets have not been
validated by experiments. Identifying miRNA targets will
improve our understanding of the miRNA-mediated mechan-
isms of plant growth and development. This is important for
 B. Zhang et al. / Developmental Biology 289 (2006) 3 – 16
designing new miRNA-related strategies to enhance plant
resistance to environmental stresses and to increase crop yields.
Mechanisms of microRNA biogenesis
Factors that control miRNA biogenesis and how these
miRNAs are regulated are still unclear. It was proposed that
miRNAs originated more than 400 million years ago both in
plants and animals. To date, there is no evidence that plant
miRNAs and animal miRNAs have the same ancestor; they
appear to have evolved independently. As we know, Dicer is an
important enzyme in miRNA biogenesis, and miRNA function
requires a RISC complex. Both Dicer and RISC are found in
both plants and animals, and they play similar roles in miRNA
biogenesis or miRNA-guide gene regulation. Whether or not
miRNAs originated before or after the divergence of these two
major kingdoms is still unclear.
New novel approach for genetic studies and plant genetic
engineering
miRNAs downregulate gene expression by cleaving mRNA
or by repressing mRNA translation. As such, it may be possible
to design artificial miRNAs to suppress target gene expression
in order to study gene function, similar to the use of antisense
mRNA and RNAi which are widely used as tools for studying
gene function. Another possibility is the use of miRNA
knowledge to improve plant yields, quality, or resistance to
various environmental stresses including insect and pathogen
infection. For example, crop resistance to drought could be
improved through the induction of more lateral roots by
downregulating miR164. Further study of miRNAs could
provide us with new tools for increasing crop yield and/or
improving crop quality.
Acknowledgments
We greatly appreciate the two anonymous reviewers for
their thorough comments and suggestions for improving the
manuscript. We would like to thank all colleagues who have
done work on miRNAs and related fields. We apologize to
colleagues whose work in this rapidly changing field was not
directly cited in this review due to space limitations and timing.
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