Available online at www.sciencedirect.

com

Gene 414 (2008) 60 66

www.elsevier.com/locate/gene

Identification of conserved microRNAs and their target genes

in tomato (Lycopersicon esculentum)
Zujun Yin, Chunhe Li, Xiulan Han, Fafu Shen
State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai'an, Shandong 271018, PR China
Received 28 November 2007; received in revised form 12 February 2008; accepted 14 February 2008
Available online 20 February 2008
Received by Takashi Gojobori

Abstract
MicroRNAs (miRNAs) are a class of non-coding RNAs that have important gene regulation roles in various organisms. To date, a total of 1279
plant miRNAs have been deposited in the miRNA miRBase database (Release 10.1). Many of them are conserved during the evolution of land
plants suggesting that the well-conserved miRNAs may also retain homologous target interactions. Recently, little is known about the
experimental or computational identification of conserved miRNAs and their target genes in tomato. Here, using a computational homology search
approach, 21 conserved miRNAs were detected in the Expressed Sequence Tags (EST) and Genomic Survey Sequence (GSS) databases.
Following this, 57 potential target genes were predicted by searching the mRNA database. Most of the target mRNAs appeared to be involved in
plant growth and development. Our findings verified that the well-conserved tomato miRNAs have retained homologous target interactions
amongst divergent plant species. Some miRNAs express diverse combinations in different cell types and have been shown to regulate cell-specific
target genes coordinately. We believe that the targeting propensity for genes in different biological processes can be explained largely by their
protein connectivity.
2008 Elsevier B.V. All rights reserved.
Keywords: Conserved microRNAs; Tomato; Target genes; MFEI; Homology

1. Introduction
MicroRNAs (miRNAs) are single-stranded non-coding
RNAs that ranging in length from 19 nucleotides (nt) to 25 nt
Abbreviations: 3 UTR, 3 untranslated region; 5RLM-RACE, 5 RNA
ligase mediated rapid amplification of cDNA ends; G, folding free energies;
AGO1, Argonaute-1; Ap2, APETALA2; ARF, auxin response transcription
factor; DCL1, Dicer-like protein; EREBPs, ethylene-responsive element binding
proteins; EST, expressed sequence tag; GH3, Grim helix 3; GSS, genomic
survey sequence; MFE, minimal folding free energy; MFEI, minimal folding
free energy index; miRNA, microRNA; miRNA, opposite miRNA sequence;
nt, nucleotide(s); pre-miRNA, microRNA precursor; pri-miRNAs, microRNA
primary; Pro, proline; RISC, RNA-induced silencing complex; SBP, Squamosa
promoter Binding Proteins; Ser, serine; SPL, SBP-like proteins; Thr, threonine.
Corresponding author. State Key Laboratory of Crop Biology, Shandong
Agricultural University, Shandong, 271018, PR China. Tel.: +86 538 8242903;
fax: +86 538 8242226.
E-mail address: ffshen@sdau.edu.cn (F. Shen).
0378-1119/$ - see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2008.02.007

that modulating gene expression in both plants and animals, and

a large number of them are evolutionarily conserved across
species boundaries (Carrington and Ambros, 2003). In plants,
miRNA genes originate mostly from independent transcriptional
units, which are transcribed by RNA polymerase II into long
primary transcripts (pri-miRNAs) (Chen, 2005; Zhang et al.,
2006b). Subsequently the pri-miRNA is cut into miRNA
precursors (pre-miRNAs) with stem-loop (hairpin) structure(s).
The loop region of the hairpin is removed by the ribonuclease
III-like enzyme Dicer (DCL1) (Kurihara and Watanabe, 2004),
and the remainder is exported to the cytoplasm by Hasty (Park
et al., 2005). The mature miRNA is incorporated into the RNAinduced silencing complex (RISC) and guides RISC to
complementary mRNA targets. Eventually, the RISC inhibits
translation elongation or triggers the degradation of target
mRNA (Lin et al., 2005). An increasing number of studies
support the idea that plant miRNAs repress the expression of

Z. Yin et al. / Gene 414 (2008) 6066

target RNAs through direct target cleavage or, in a few cases, by

translational repression (Schwab et al., 2005; Axtell et al., 2007).
Plant miRNAs negatively regulate their target genes, which
function in a range of developmental processes (Guo et al., 2005;
Laufs et al., 2004; Mallory et al., 2004) as well as in response to
environmental stimuli (Jones-Rhoades and Bartel, 2004; Sunkar
and Zhu, 2004). Identifying miRNAs and their target genes is
therefore central to understanding their function and the
underlying control mechanisms. Recently, computational
approaches have been used wildly as rapid, accurate, and
affordable methods for the identification of miRNAs. Zhang
et al. (2006d) classified these computational approaches into five
major categories: homology search-based, gene search-based,
neighbor stem-loop search-based, comparative genomic algorithm-based, and phylogenetic shadowing-based. The homology
search-based approach, which is based on conserved sequences
and secondary structures, searches nucleotide databases using
the BLAST program, and has been used to identify hundreds of
new miRNAs in the genomes of model species, such as Arabidopsis thaliana (Adai et al., 2005) and Oryza sativa (Li et al.,
2005, Zhang et al., 2006a). At present, the genomic sequences
are available for only a few plant species. Zhang et al. (2005)
developed an efficient strategy for identifying plant miRNAs
using expressed sequenced tag (EST) analysis. Using this
approach, more than 700 miRNAs have been identified in plants
and viruses (Zhang et al., 2006a; Pan et al., 2007). In order to
expand the scope of the search, Zhang et al. (2007), using
previously known miRNA sequences to blast the cotton EST and
Genomic Survey Sequences (GSS) databases, detected 30
potential miRNAs. With the development of computational
methods, several computer software programs have been
developed to help identify plant potential miRNA target genes
in mRNA sequences. These programs include MIRcheck
(Jones-Rhoades and Bartel, 2004) and miRU (Zhang, 2005).
Because almost all miRNAs show perfect or near-perfect
complementarity with their targets in plants, it is much easier
to predict miRNA targets using a BLAST search of mRNA
database for plants than it is for in animals. More and more
studies have shown this is a powerful approach that has been
used to successfully select potential miRNA targets in mRNA
sequences for experimental validation (Zhang et al., 2006d,
Gleave et al., 2008).
To date, a total of 1279 plant miRNAs have been discovered
and are deposited in the current edition of the miRNA Registry
(http://microrna.sanger.ac.uk/sequences/index.shtml). However, the majority have been identified from A. rabidopsis,
O. sativa and Populus trichocarpa where genome sequences
are available. In addition, many of them are conserved during
the evolution of land plants. This finding raises the question of
whether the well-conserved miRNAs retain homologous target
interactions and perform analogous molecular functions
amongst divergent plant species.
Tomato (Lycopersicon esculentum L.) is an important
vegetable crop of significant economic importance in horticultural industries worldwide (Leonardi et al., 2000). In addition to
its economic value, the tomato plant is an excellent experimental
system. Little is known, however, about the experimental or

61

computational identification of miRNAs in tomato, and nothing is

known about their target genes. Only recently, Pilcher et al. (2007)
and Itaya et al. (2008) discovered some novel putative miRNAs in
sRNAs library of tomato fruit and leaf tissue through direct
cloning. In this study, using a combined computational approach,
a total of 21 conserved miRNAs and 57 potential target genes
have been detected in tomato. Our results verified that the highly
conserved tomato miRNAs have retained homologous target
interactions amongst divergent plant species. These new miRNAs
and their targets could help guide experimental design and
improve our understanding of the possible roles of miRNAs in
regulating the growth and development of tomato.
2. Materials and methods
2.1. Sequences of miRNAs, expressed sequence tags (EST),
genomic survey sequences (GSS) and mRNA
To search for potential conserved miRNAs, the sequences of
previously identified mature miRNAs and their pre-miRNAs
were downloaded from the miRNA Registry Database (Release
9.2, May 2007; http://microrna.sanger.ac.uk/sequences/index.
shtml). This set contained 1110 miRNAs from 9 species
[A. thaliana (186), Glycine max (22), Medicago truncatula
(30), O. sativa (243), Physcomitrella patens (220), Populus
trichocarpa (215), Saccharum officinarum (16), Sorghum
bicolor (72) and Zea mays (96)]. The tomato EST, GSS, and
mRNA databases were obtained from the NCBI (http://www.
ncbi.nlm.nih.gov/).
2.2. Prediction of potential miRNAs
The procedure used to search for potential miRNAs in tomato
is shown in Fig. 1. To avoid overlap, repeat miRNA sequences
within the above species were removed. The remaining
sequences were used as BLAST search queries against the
tomato EST and GSS databases. The BLAST search was carried
out using BLAST 2.2.14. Adjusted blast parameter settings were
as follows: an expect value cutoff of 10; a low-complexity
sequence filter; 1000 descriptions and alignments; and automatically adjusted parameters for short input sequences to
improve the veracity of outputs. If the matched sequences were
less than the previously known mature miRNA sequences, the
non-aligned parts were inspected and compared manually to
determine the number of matching nucleotides. All BLAST
results were saved. EST or GSS sequences which have only 0
3 nt mismatches compared with the query miRNA sequences
were chosen manually.
The secondary structures of the selected entire EST or GSS
sequences were predicted and generated using the web-based
software Mfold 3.2 (publicly available at http://frontend.bioinfo.
rpi.edu/applications/mfold/cgi-bin/rna-form1.cgi). The following
parameters were used in predicting the secondary structures: a
fixed folding temperature of 37 C; ionic conditions set at 1 M
NaCl with no divalent ions; and grid lines in the energy dot plots
turned on. Other parameters followed the default parameters.
Generally, the lowest-energy structure corresponds to helices in

62

Z. Yin et al. / Gene 414 (2008) 6066

Fig. 1. Schematic representation of the miRNA gene search procedure used to identify tomato homology to known miRNAs.

optimal foldings. Thus, in each case, only the lowest-energy

structure was selected for manual inspection. According to the
structures, the potential pre-miRNA sequences were selected to
predict their secondary structures and calculate the folding free
energies. The following empirical criteria were used for selecting
potential miRNAs and pre-miRNAs: (a) predicted mature
miRNAs have only 03 nt mismatches compared with the query
sequences; (b) the approximately 23 nt potential miRNA sequence
is not located on the terminal loop of the hairpin structure;
(c) potential precursors have a higher minimal folding free energy
(MFE) index (MFEI) than other types of RNAs. The MFEI was
calculated using the following equation:

(http://www.ncbi.nlm.nih.gov/BLAST/) was used for the analysis of potential targets. The criteria were as follows: (a) four or
fewer mismatched nucleotides at complementary sites between
miRNA sequences and potential mRNA targets; (b) one mismatch in the complementary region of the miRNA at nucleotide
positions 212, but not at positions 10 or 11, which is a predicted cleavage site; and (c) up to three additional mismatches
between 12 nt and 23 nt but with no more than two continuous
mismatches within this region.

MFEI MFE=length of the RNA sequence  100=GC k

where MFE denotes the negative folding free energies (G); (d) the
miRNA has less than six mismatches with the opposite miRNA
sequence (miRNA*) on the other arm; (e) potential miRNAs are
located on the same arm of the stem-loop structure as their known
homologs; and (f) there is no large loop or break in the miRNA
sequence. Predicted miRNAs and their related information were
recorded. Closely related EST and GSS sequences were blasted
against each other and analyzed. If a high degree of similarity
(N 98%) was observed, the sequences were deemed to have been
created from the same sequences and considered as one miRNA.
2.3. Prediction of potential target genes and their functions
Fig. 2 summarizes the major steps employed in predicting potential target genes and their functions. BLASTX

Fig. 2. Schematic representation of the potential target genes search procedure by blasting mRNA databases of tomato with newly identified miRNA
sequences.

Z. Yin et al. / Gene 414 (2008) 6066

3. Results and discussion

3.1. Identification of potential tomato miRNAs
The high degree of sequence conservation of many miRNAs
within the plant kingdom (Axtell and Bartel, 2005) provides a
means to identify conserved miRNAs from all plant species
(Fahlgren et al., 2007). In this research, a homology search
approach was adopted to identify conserved miRNAs in tomato.
Following a set of strict filtering criteria, a total of 21 conserved
miRNAs were detected, of which 7 were identified in the EST
database and 14 in the GSS database (Table 1). The lengths of
these newly identified miRNAs varied from 20 nt to 22 nt, and a
total of 15 began with a 5' uridine, a characteristic feature of
miRNAs (Supplementary Fig. 1).
During screening of the potential miRNAs, sequences of the
candidate pre-miRNAs were evaluated for their A + U content,
which ranged from 45.52% to 83.33% (Table 1), in agreement
with previous results (Zhang et al., 2006a). Compared to the
number of nucleotides in animal pre-miRNAs (typically 70
80 nt), the tomato pre-miRNAs were more diverse in structure
and size (Table 1, Supplementary Fig. 1). The predicted hairpin
structures of the pre-miRNAs required 54355 nt, with the
majority (71.43%) requiring 70160 nt, similar to what has
been observed in A. thaliana and O. sativa (Li et al., 2005). The
different sizes and structures of the identified miRNAs suggest
unique functions in the regulation of miRNA biogenesis or gene
expression. The location of mature miRNA sequences in the
identified miRNAs also showed diversity (Supplementary Fig.
1), with sequences of miR156/157, miR160, miR167, miR168,
miR169, miR437, miR869.1 and miR1030 being located at the 5
end of the pre-miRNAs and all others at the 3 end.
The MFEI is a useful criterion for distinguishing miRNAs
from other types of coding or non-coding RNAs. In our results,

63

the newly identified plant pre-miRNAs had a high MFEI (0.70

2.02) (Table 1), with an average of about 1.01; this is significantly
higher than that for tRNAs (0.64), rRNAs (0.59), and mRNAs
(0.620.66) (Zhang et al., 2006c). The 21 conserved tomato
miRNAs belong to 14 miRNA families. miR156/157 was
represented by five members; miR167, miR172 and miR399 by
two members; and all other miRNA families including miR159,
miR437 and miR1030 by only one member. miR156/157,
miR168, miR169, miR172, and miR399 had more than 14
homologs of miRNAs in other plant species, while in contrast,
others showed a low level of conservation or were non-conserved.
For example, miR830 and miR869.1 matched only their
counterparts in A. thaliana, while miR1030 was found only in
P. patens. Interesting, although mosses are one of the most
ancient land plants present among the Earth's flora (Liang et al.,
2004), previously identified miRNAs in P. patens showed few
homologs in dicots or monocots during our filtering process of
repeat sequences. This discrepancy may be due to two nonmutually exclusive factors. First, the low or non-conserved
miRNAs may play important roles in the development of more
species-specific characteristics. Second, many likely arose in the
recent evolutionary past, with some miRNA families predating
the divergence of vascular plants and mosses.
In a recent investigation, it was suggested that about 1% of
genes predicted using a computational strategy were miRNAs (Lai
et al., 2003). The numbers identified in our study and other studies,
on the other hand, were very small. One explanation for this
discrepancy is that the molecular characteristics of miRNAs and
pre-miRNAs resulted in a low content in the EST and GSS
databases. First, the majority of pre-miRNAs are usually very
short (approximately 100 nt), and thus, processed rapidly in the
cell, and second, some have a low level of expression. Many
miRNAs or pre-miRNAs may therefore have a low probability of
detection. As more sequences become publicly available in the

Table 1
List of the newly identified miRNAs in tomato
New miRNAs

miRNA sequences

Gene ID

Gene source

Location

NM (nt)

LM (nt)

LP (nt)

A+U (%)

G (kal/mol)

MFEIs

156a
156b
157a
157b
157c
159
160
162
167a
167b
168
169
172a
172b
399a
399b
403
437
830
869.1
1030

ugaaagauagagcagugagcac
ugacagaagagagagagcac
uugacagaagauagagagcac
uugacagaagauagagagcac
uugacagaagauagagagcac
uuuggauugaagggagcucua
ugccuggcuccuuguaugcca
ucgauaaaccucugcauccag
ugaagcugccagcaugaucua
uaaagcugccagcaugaucugg
ucgcuuggugcaggucgggac
Uagccaaaaaugacuugccag
agaaucuugaugaugcugcau
agaaucuugaugaugcugcau
ugccaaaggagaguugcccua
ugccaagggagaauugcccua
cuagauucacgcacaagcucg
aaaguuagagaaguuugaaau
ugacuauuaugagaagaagug
auugguuuaauuuugguguug
Aucugcaugugcaccugcacc

84260136
72526205
117704089
84273338
84236627
116645971
72471353
72276781
117695324
72348884
9505413
84234106
117691535
72400226
117723706
72479804
72312989
84192580
84049570
117684944
84291882

GSS
GSS
EST
GSS
GSS
EST
GSS
GSS
EST
GSS
EST
GSS
EST
GSS
EST
GSS
GSS
GSS
GSS
EST
GSS

5
5
5
5
5
3
5
3
5
5
5
5
3
3
3
3
3
5
3
5
5

3
0
0
0
0
0
0
0
0
2
1
3
0
0
0
1
2
3
3
2
2

22
20
21
21
21
21
21
21
21
22
21
21
21
21
21
21
21
21
21
21
21

85
355
100
84
83
178
80
98
73
237
145
68
106
135
71
72
76
54
115
61
139

60.00
49.56
64.00
60.71
61.45
62.92
53.75
53.06
64.38
75.53
45.52
51.47
66.04
72.59
57.75
58.33
60.52
83.33
71.30
63.93
58.27

22.56
95.70
43.40
39.10
40.20
73.61
33.30
33.70
27.30
60.90
59.80
27.50
39.80
74.80
30.24
31.30
28.90
8.30
22.50
15.40
35.40

0.71
0.70
1.20
1.18
1.26
1.11
0.90
0.73
1.05
1.05
0.76
0.83
1.11
2.02
1.03
1.04
0.96
1.30
0.74
0.70
0.73

NM: number of mismatch; LM: length of mature miRNAs; LP: length of precursor; G: folding free energies; MFEIs: minimal folding free energy indexes.

64

Z. Yin et al. / Gene 414 (2008) 6066

repositories of the EST and GSS databases, and as more miRNAs

are identified in plants, we believe that the likelihood of successfully determining miRNAs in these databases will increase.

Table 2
Potential targets of the identified miRNAs in tomato
miRNA

Targeted protein

Target function

156/157

Squamosa promoter
binding protein
(SBP)/ SBPL
Tospovirus
resistance protein
Lateral suppressor
protein
AUX/IAA protein

Transcription factor BG630869 BI927983

BI931517 BI928213
BI927982 AW933950
Stress response
BG125997

3.2. The diversity and multiplicity of miRNA targets in tomato

Gaining insight into the miRNA targets will help us to
understand the range of miRNA expression regulation and to more
coherently describe the functional importance of miRNAs.
Evolutionarily conserved sites seem to be functional, and therefore
the conserved sequence can serve as a filter to define likely target
regions (Ioshikhes et al., 2007; Lall et al., 2006). Using the newly
identified miRNA sequences as BLAST search queries, 57 target
genes were predicted in the tomato mRNA database using a set of
strict criteria (Table 2). According to the information provided by
NCBI, the identified mRNA targets could be separated into
several groups. The largest group contained targets thought to
encode transcription factors, which are known to be involved
mainly in plant growth and developmental patterning. This is
probably a general characteristic of plant miRNAs that tends to be
complementary to their regulatory targets (Qiu et al., 2007). The
next group contained targets encoding a range of different proteins
implicated in various metabolic processes, while another group
was involved in functions such as hormone responses, stress
defense and signaling. Interestingly, a miRNA can be complementary to more than one regulatory target (Table 2); for example,
10 sequences were detected as targets of miR156/157, and of
these, 6 were found to be Squamosa promoter Binding Proteins
(SBP). Whether these different miRNAs have the same authentic
complementary sites to their targets depends on whether the
miRNA complementary sites are within the context of a domain
strongly conserved among family members (Rhoades et al., 2002).
The well-conserved tomato miRNAs seem to have retained
homologous target interactions and performed analogous molecular functions amongst divergent plant species (Table 2).
Representative examples are as follows. miR156/157 was
predicted as the target of SBP or SBP-like proteins (Table 2), a
plant-specific family of transcription factors involved in early
flower development and vegetative phase changes (Achard et al.,
2004). SPL3, SPL4 and SPL5 (SPL3/4/5) are closely related
members of the SBP-like family in A. thaliana (Cardon et al.,
1999). The expression of juvenile vegetative traits and delayed
flowering is regulated by an increase in the expression of SPL3/4/
5, which occurs as a result of a decrease in the level of miR156
(Wu and Poethig, 2006). Some studies have suggested that SPL3/
4/5 are all cleaved in the middle of the miR156 target site located
within their 3 UTR and 5 RLM-RACE (Schwab et al., 2005, Wu
and Poethig, 2006). However, Gandikota et al. (2007) demonstrated that SPL3 prevents early flowering by translational
inhibition in seedlings. This functional miRNA response element
emerges also in the mRNA of the SBP-box genes in moss (Riese
et al., 2007), suggesting that SBP-box gene family members are
dependently regulated by the ancient origin of miRNA.
The phytohormone auxin plays critical roles during plant
growth, many of which are mediated by members of the auxin
response transcription factor (ARF) family (Guilfoyle et al.,
1998). Recent studies have shown that miR160 is complementary

160

167

168

172

869.1

DNA (cytosine-5)methyltransferase
Auxin-responsive
factor
Serine/threonine
protein kinase
Auxin-responsive
factor

Putative phosphatase
Sucrose transporter
mRNA binding
protein precursor
Ethylene-responsive
element binding
protein
Putative protease/
hydrolase
Phospholipase family
Insulin degrading
enzyme
Polyphenol oxidase
Eukaryotic translation
initiation factor
Threonine deaminase

Target genes

Transcription factor BF097000

Auxin-responsive
factor
Cell growth

BE436147
BI925272

Auxin-responsive BP880486
factor
Transcription factor AW038480
Auxin-responsive
factor

BI925927 BP902363
BE434602 CD003107
DB715336 AW735838
AW223741
Transcription factor DB697961 DB706552
BF097936
Transcription factor BE461110
Transcription factor BE462736
Transcription factor BM413247 AI486618
BI208808 BM410833
BM408872
Transcription factor AI484737
Transcription factor BP893968 BI935838
Metabolism
BG627220
Metabolism
AI774952
Transcription factor BE436427 BE432648
Transcription factor BI935333 BI9353116
BI933914 BI933284
BI933270 BI932869
BI932857 BI935963
BG134948 BM413156
BG127250 BG129951
BG126331 BG126303
BG123821
Transcription factor BG626979

Ethylene-responsive
nuclear protein
P69C protein
Transcription factor BI932424
Subtilisin-like protease Metabolism
BG421482 BI935794
NBS-LRR resistance Transcription factor BG132455 BM410377
protein-like protein

to ARF10, ARF16 and ARF17 (Megraw et al., 2006; Rhoades

et al., 2002; Wang et al., 2005), while miR167 is complementary
to ARF6 and ARF8 (Wu et al., 2006). Our results suggested that
miR160 and miR167 and the target ARFs are conserved in tomato
(Table 2). Increased levels of auxin accelerate proteolysis of Aux/
IAA proteins, which allows ARF proteins to homodimerize and
impose their regulatory functions on early auxin-response gene
expression (Dharmasiri and Estelle, 2002; Liscum and Reed,
2002). Intriguingly, both miR160 and miR167 regulate ARFs, but
they have different complementary sites and unrelated sequences.
In A. thaliana, miR167 was found only to cause transcript

Z. Yin et al. / Gene 414 (2008) 6066

degradation of ARF8 but not ARF6 (Ru et al., 2006); this was
explained by the fact that the fewer base-pairs between ARF6 and
miR167 may result in inefficient cleavage of the ARF6 transcripts.
Mallory et al. (2005) suggested that miR160 and miR167
coordinately modulate GH3-like mRNA expression by regulating
expression of repressing and activating ARF proteins encoded by
ARF17 and ARF8. Thus, in the future, it will be fascinating to
experimentally determine the subtle correlations among miR160,
miR167and ARFs.
Ethylene-responsive element binding proteins (EREBPs) and
APETALA2 (AP2) are prototypic members of a plant-specific
family of AP2/EREBP transcription factors (Nole-Wilson and
Krizek, 2000; Riechmann and Meyerowitz, 1998; Theissen and
Saedler, 1999). Recent studies using A. thaliana show that
miR172 and its AP2-Like target genes regulate flowering time and
floral organ identity (Aukerman and Sakai, 2003; Chen, 2004). In
addition, Glossy15, an AP2-like gene from maize that regulates
leaf epidermal cell identity, is also detected as a target of miR172
(Lauter et al., 2005). In this research, five predicted targets of
miR172 were found to be members of the AP2 gene family (Table
2). Aukerman and Sakai (2003) suggested that the progressive
accumulation of miR172 results in the complete absence of AP2
and other AP2-like proteins; as a consequence, plants set flowers
early with disrupted specification of floral organ identity.
Recently, another study demonstrated that flower development
is regulated by at least four miRNAs: miR156, miR159, miR164
and miR172 (Gandikota et al., 2007). In fact, miR156 and miR172
are expressed in opposite temporal patterns, suggesting that the
miR156 module may regulate the expression of miR172 (Willmann and Poethig, 2007). In addition, Nilsson et al. (2007)
suggested that the AP2 gene in spruce has the capacity to
substitute an A class gene required in flower development.
Additional studies are now required to determine the basis for this
regulation and the complicated regulating network.
As the above data suggest, the molecular identities of the
miRNAs and their targets seem to have remained constant, providing additional evidence for the real existence of these miRNAs.
Presently, however, it is not possible to determine whether this
conservation is the result of shared ancestry or functional convergence from an independent origin during evolution.
Different combinations of miRNAs are expressed in different
cell types and may regulate cell-specific target genes coordinately (Cimmino et al., 2005). For example, Vaucheret et al.
(2004) confirmed that AGO1 is controlled by miR168 in
A. thaliana. However, in this study, the predicted targets of
miR168 did not encode AGO family proteins, but rather
putative phosphatase and sucrose transporter (Table 2). Maybe,
it is some small changes in the temporal, spatial or environmental regulation of these modules over long periods of time
that result in large different effects in developmental processes
of other species. Moreover, among the detected targets of
miR156/157, miR160, miR167, miR172 and miR869.1, there
were some atypical target genes that have yet to be validated by
an experimental approach. Interestingly, serine/threonine protein kinase was among the targeted proteins of miR160.
Combined with the fact that transcriptional repressor ARFs all
have Pro-Ser-Thr-rich middle regions (Tiwari et al., 2004), this

65

confirms that the targeting propensity for genes related to

different biological processes can be explained largely by their
protein interaction. Thus, we believe that an investigation of
protein connectivity would offer critical information on the
interactions between miRNAs and their target genes.
With some miRNAs, such as miR159, miR162, miR169,
miR399, miR403, miR437, miR830 and miR1030, we failed
to discover any target in tomato (Table 2). This could have
resulted from incomplete coverage of the mRNA in the databases. It is likely that a number of mRNAs could not be
identified because they are poorly expressed or highly unstable,
or because their expression is restricted to times and locations
such that isolation of sufficient amounts of RNA for cloning is
impractical. Further analysis of this therefore requires sequencing of miRNA complements, and testing of these hypotheses
will become possible as more is learned about target specificity.
In summary, in the present study, we presented global predictions of conserved tomato miRNAs and their targets. A total of
21 potential miRNAs, belonging to 14 miRNA families, and 57 of
their potential target genes were detected. Most of the pooled
targets were predicted, with functions in a variety of biological
processes, including growth and developmental patterning, metabolic processes, hormone responses, stress defense and signaling.
It should be noted, however, that this study used only a computational approach, which can never replace biological verification and can be used only to guide experimental design. The next
major steps, therefore, are to experimentally analyze the functional categories suggested by our computational approach, determine the analogous molecular functions amongst divergent
plant species and further elucidate any significant correlation
between the miRNAs and their target genes.
Acknowledgments
This research was supported by the China Key Development
Project for Basic Research (973) (grant no. 2007CB116208)
and the China Agricultural Commonweal Industry Project
(nyhyzx07-005).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.gene.2008.02.007.
References
Adai, A., et al., 2005. Computational prediction of miRNAs in Arabidopsis
thaliana. Genome Res. 15, 7891.
Achard, P., Herr, A., Baulcombe, D.C., Harberd, N.P., 2004. Modulation of
floral development by a gibberellin-regulated microRNA. Development
131, 33573365.
Aukerman, M.J., Sakai, H., 2003. Regulation of flowering time and floral organ
identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15,
27302741.
Axtell, M.J., Bartel, D.P., 2005. Antiquity of microRNAs and their targets in
land plants. Plant Cell 17, 16581673.
Axtell, M.J., Snyder, J.A., Bartel, D.P., 2007. Common functions for diverse
small RNAs of land plants. Plant Cell 19, 17501769.
Cardon, G., et al., 1999. Molecular characterisation of the Arabidopsis SBP-box
genes. Gene 237, 91104.

66

Z. Yin et al. / Gene 414 (2008) 6066

Carrington, J.C., Ambros, V., 2003. Role of microRNAs in plant and animal
development. Science 301, 336338.
Chen, X., 2004. A microRNA as a translational repressor of APETALA2 in
Arabidopsis flower development. Science 303, 20222025.
Chen, X., 2005. MicroRNA biogenesis and function in plants. FEBS Lett. 579,
59235931.
Cimmino, A., et al., 2005. miR-15 and miR-16 induce apoptosis by targeting
BCL2. Proc. Natl. Acad. Sci. U. S. A 102, 1394413949.
Dharmasiri, S., Estelle, M., 2002. The role of regulated protein degradation in
auxin response. Plant Mol. Biol 49, 401409.
Fahlgren, N., et al., 2007. High-throughput sequencing of Arabidopsis
microRNAs: evidence for frequent birth and death of MIRNA genes.
PLoS. ONE. 2, e219.
Gandikota, M., Birkenbihl, R.P., Hohmann, S., Cardon, G.H., Saedler, H.,
Huijser, P., 2007. The miRNA156/157 recognition element in the 3' UTR of
the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J. 49, 683693.
Gleave, A.P., et al., 2008. Identification and characterisation of primary
microRNAs from apple (Malus domestica cv. Royal Gala) expressed
sequence tags. Tree Genetics & Genomes 4, 343358.
Guilfoyle, T.J., Ulmasov, T., Hagen, G., 1998. The ARF family of transcription
factors and their role in plant hormone-responsive transcription. Cell. Mol.
Life Sci 54, 619627.
Guo, H.S., Xie, Q., Fei, J.F., Chua, N.H., 2005. MicroRNA directs mRNA
cleavage of the transcription factor NAC1 to downregulate auxin signals for
Arabidopsis lateral root development. Plant Cell 17, 13761386.
Ioshikhes, I., Roy, S., Sen, C.K., 2007. Algorithms for mapping of mRNA
targets for microRNA. DNA Cell Biol. 26, 265272.
Itaya, A., et al., 2008. Small RNAs in tomato fruit and leaf development.
Biochim. Biophys. Acta. 1779, 99107.
Jones-Rhoades, M.W., Bartel, D.P., 2004. Computational identification of plant
microRNAs and their targets, including a stress-induced miRNA. Mol. Cell
14, 787799.
Kurihara, Y., Watanabe, Y., 2004. Arabidopsis micro-RNA biogenesis through
Dicer-like 1 protein functions. Proc. Natl. Acad. Sci. U. S. A 101, 1275312758.
Lai, E.C., Tomancak, P., Williams, R.W., Rubin, G.M., 2003. Computational
identification of Drosophila microRNA genes. Genome Biol. 4, R42.
Lall, S., et al., 2006. A genome-wide map of conserved microRNA targets in
C. elegans. Curr. Biol. 16, 460471.
Laufs, P., Peaucelle, A., Morin, H., Traas, J., 2004. MicroRNA regulation of the
CUC genes is required for boundary size control in Arabidopsis meristems.
Development 131, 43114322.
Lauter, N., Kampani, A., Carlson, S., Goebel, M., Moose, S.P., 2005. microRNA172
down-regulates glossy15 to promote vegetative phase change in maize. Proc.
Natl. Acad. Sci. U. S. A 102, 94129417.
Leonardi, C., Ambrosino, P., Esposito, F., Fogliano, V., 2000. Antioxidative
activity and carotenoid and tomatine contents in different typologies of fresh
consumption tomatoes. J. Agric. Food Chem. 48, 47234727.
Li, Y., Li, W., Jin, Y.X., 2005. Computational identification of novel family
members of microRNA genes in Arabidopsis thaliana and Oryza sativa.
Acta Biochim. Biophys. Sin. (Shanghai) 37, 7587.
Liang, C.Y., et al., 2004. Construction of a BAC library of Physcomitrella
patens and isolation of a LEA gene. Plant Sci. 167, 491498.
Lin, S.L., Chang, D., Ying, S.Y., 2005. Asymmetry of intronic pre-miRNA
structures in functional RISC assembly. Gene 356, 3238.
Liscum, E., Reed, J.W., 2002. Genetics of Aux/IAA and ARF action in plant
growth and development. Plant Mol. Biol 49, 387400.
Mallory, A.C., Bartel, D.P., Bartel, B., 2005. MicroRNA-directed regulation of
Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell
17, 13601375.
Mallory, A.C., Dugas, D.V., Bartel, D.P., Bartel, B., 2004. MicroRNA regulation
of NAC-domain targets is required for proper formation and separation of
adjacent embryonic, vegetative, and floral organs. Curr. Biol. 14,
10351046.
Megraw, M., Baev, V., Rusinov, V., Jensen, S.T., Kalantidis, K., Hatzigeorgiou,
A.G., 2006. MicroRNA promoter element discovery in Arabidopsis. RNA.
12, 16121619.

Nilsson, L., Carlsbecker, A., Sundas-Larsson, A., Vahala, T., 2007. APETALA2
like genes from Picea abies show functional similarities to their Arabidopsis
homologues. Planta 225, 589602.
Nole-Wilson, S., Krizek, B.A., 2000. DNA binding properties of the Arabidopsis floral development protein AINTEGUMENTA. Nucleic Acids Res.
28, 40764082.
Pan, X., Zhang, B., San, F.M., Cobb, G.P., 2007. Characterizing viral
microRNAs and its application on identifying new microRNAs in viruses.
J. Cell Physiol. 211, 1018.
Park, M.Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H., Poethig, R.S., 2005.
Nuclear processing and export of microRNAs in Arabidopsis. Proc. Natl.
Acad. Sci. U. S. A 102, 36913696.
Pilcher, R.L., et al., 2007. Identification of novel small RNAs in tomato
(Solanum lycopersicum). Planta 226, 709717.
Qiu, C.X., et al., 2007. Computational identification of microRNAs and their
targets in Gossypium hirsutum expressed sequence tags. Gene 395, 4961.
Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B., Bartel, D.P.,
2002. Prediction of plant microRNA targets. Cell 110, 513520.
Riechmann, J.L., Meyerowitz, E.M., 1998. The AP2/EREBP family of plant
transcription factors. Biol. Chem. 379, 633646.
Riese, M., Hohmann, S., Saedler, H., Munster, T., Huijser, P., 2007. Comparative analysis of the SBP-box gene families in P. patens and seed plants.
Gene 401, 2837.
Ru, P., Xu, L., Ma, H., Huang, H., 2006. Plant fertility defects induced by the
enhanced expression of microRNA167. Cell Res. 16, 457465.
Schwab, R., Palatnik, J.F., Riester, M., Schommer, C., Schmid, M., Weigel, D.,
2005. Specific effects of microRNAs on the plant transcriptome. Dev. Cell 8,
517527.
Sunkar, R., Zhu, J.K., 2004. Novel and stress-regulated microRNAs and other
small RNAs from Arabidopsis. Plant Cell 16, 20012019.
Theissen, G., Saedler, H., 1999. The golden decade of molecular floral development (19901999): A cheerful obituary. Dev Genet. 25, 181193.
Tiwari, S.B., Hagen, G., Guilfoyle, T.J., 2004. Aux/IAA proteins contain a
potent transcriptional repression domain. Plant Cell 16, 533543.
Vaucheret, H., Vazquez, F., Crete, P., Bartel, D.P., 2004. The action of
ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA
pathway are crucial for plant development. Genes Dev. 18, 11871197.
Wang, J.W., Wang, L.J., Mao, Y.B., Cai, W.J., Xue, H.W., Chen, X.Y., 2005.
Control of root cap formation by MicroRNA-targeted auxin response factors
in Arabidopsis. Plant Cell 17, 22042216.
Willmann, M.R., Poethig, R.S., 2007. Conservation and evolution of miRNA
regulatory programs in plant development. Curr. Opin. Plant Biol. 10, 503511.
Wu, G., Poethig, R.S., 2006. Temporal regulation of shoot development in
Arabidopsis thaliana by miR156 and its target SPL3. Development 133,
35393547.
Wu, M.F., Tian, Q., Reed, J.W., 2006. Arabidopsis microRNA167 controls
patterns of ARF6 and ARF8 expression, and regulates both female and male
reproduction. Development 133, 42114218.
Zhang, B.H., Pan, X.P., Wang, Q.L., Cobb, G.P., Anderson, T.A., 2005.
Identification and characterization of new plant microRNAs using EST
analysis. Cell Res. 15, 336360.
Zhang, B.H., Pan, X.P., Cannon, C.H., Cobb, G.P., Anderson, T.A., 2006a.
Conservation and divergence of plant microRNA genes. Plant J. 46, 243259.
Zhang, B.H., Pan, X.P., Cobb, G.P., Anderson, T.A., 2006b. Plant microRNA: a
small regulatory molecule with big impact. Dev. Biol. 289, 316.
Zhang, B.H., Pan, X.P., Cox, S.B., Cobb, G.P., Anderson, T.A., 2006c. Evidence
that miRNAs are different from other RNAs. Cell Mol. Life Sci. 63,
246254.
Zhang, B.H., Pan, X.P., Wang, Q.L., Cobb, G.P., Anderson, T.A., 2006d.
Computational identification of microRNAs and their targets. Comput. Biol.
Chem. 30, 395407.
Zhang, B.H., et al., 2007. Identification of cotton microRNAs and their targets.
Gene 397, 2637.
Zhang, Y., 2005. miRU: an automated plant miRNA target prediction server.
Nucleic Acids Res. 33, W701W704.