ISSN 10214437, Russian Journal of Plant Physiology, 2010, Vol. 57, No. 4, pp. 469479. Pleiades Publishing, Ltd.

., 2010.
Published in Russian in Fiziologiya Rastenii, 2010, Vol. 57, No.4, pp. 503514.

RESEARCH
PAPERS

Computational Predicting Novel MicroRNAs in Tomato

and Validating with RTPCR1
Fulei Luan2, Yousheng Han2, Hongliang Zhu, Yi Shao, Anjun Chen, Huiqin Tian,
Yunbo Luo, and Benzhong Zhu
Laboratory of Fruit Biology, College of Food Science and Nutritional Engineering,
China Agricultural University, Beijing, 100083 China;
fax: +86 (10) 62737538; email: cauzbz@yahoo.com.cn
Received April 29, 2008

AbstractMicroRNAs (miRNAs) are a newly discovered class of nonproteincoding small RNAs with the
length of ~21 nucleotides that regulate gene posttranscriptional expression in animals and plants. By far, the
researches have indicated that miRNAs may play multiple roles in plant growth and development. It is diffi
cult to identify some miRNAs by experimental methods because of their low expressional levels and tissue
specificity, while bioinformatics is an effective strategy in the prediction of this kind of miRNAs. In this study,
we presented an approach of expressed sequence tag (EST) analysis for predicting novel miRNAs as well as
their targets in tomato (Lycopersicon esculentum). The database of tomato ESTs was compared with previously
known miRNA sequences of other plants using BLAST to search for potential miRNAs. Eight potential miRNAs
were found following a range of filtering criteria, including stemloop structure, mismatches, the content of
A + U, minimal folding free energy indices, and others with subsequent validated by touchdown RTPCR assay
in fruit tissue. Three unknown miRNAs, LemiR157a, LemiR172i, and LemiR399, which were not reported
in previous study, were found in tomato. Tomato mRNA database was further compared with the newly iden
tified miRNA sequences with BLAST, and 42 potential targets of miRNAs were identified. According to the
annotations of tomato mRNAs provided by the website (http://ted.bti.cornell.edu/digital/sRNA), miRNA
target genes were classified into four groups, in which transcription factors regulating growth and develop
ment, signal pathway transduction, and metabolism of tomato plants were in the majority.
Key words: Lycopersicon esculentum, fruit, EST, miRNA, targets
DOI: 10.1134/S1021443710040035
21

INTRODUCTION

1 This text was submitted by the authors in English.

2 These authors contributed equally to this work.

cated that miRNAs participate in broad regulating

gene expression on posttranscriptional level, and they
play a vital role in the network of gene expression and
regulation [1, 3]. By computational prediction and exper
imental validation, most of the targets of miRNAs are
transcription factors [4]. Thereby the genes targeted by
miRNAs control the metabolism during plant growth
and development, such as morphogenesis of the roots,
stems, leaves, and flowers, cellular differentiation, and
tissue growth [4, 5]. In addition, miRNAs may be reg
ulated by environment stresses, such as pathogens,
drought, salt, heat, and cold [5, 6].

Abbreviations:
ACC1aminocyclopropane1carboxylate;
ACS1aminocyclopropane1carboxylate synthase; AGO1
Argonaute protein; cDNAcomplementary DNA; DDBJ
DNA Data Bank of Japan; DMSOdimethyl sulfoxide; EST
expressed sequence tag; ERFethylene response elementbind
ing factor; HMWhigh molecular weight; LMWlow molecu
lar weight; MFEminimal folding free energy; MFEImini
mal folding free energy index; NCBINational Center for Bio
technology Information; ntnucleotide; PTGSpost
transcriptional gene silencing; RNAiRNA interference;
RISCRNAinduced silencing complex; RTPCRreverse
transcription polymerase chain reaction; SBPSquamosapro
moter binding proteins; TGStranscriptional gene silencing.

The primary miRNAs, which are long, capped, and

polyadenylated RNAs, are transcribed from miRNAs
genes in the nucleolus [7]. Different to the animals,
there is no a Drosha homolog in plants, while a Dicer
known as DCL1 catalyzes the processing of primary
miRNA to premiRNA, which also requires a dsRBD
protein (HYL1) [1, 7, 8]. Then miRNAmiRNA*
duplexes are exported to the cytoplasm with the help
of a protein called HASTY [7, 8]. In plants miRNA
miRNA* duplexes are methylated by HEN1, a kind of
enzyme, which does not exist in animals [911]. The

MicroRNAs (miRNAs) are a class of ~21 nt non

proteincoding RNAs that have been discovered in
recent years [1]. MicroRNAs are chemically and func
tionally similar to small interfering RNAs, which can
mediate RNA interference (RNAi), posttranscrip
tional gene silencing (PTGS), and transcriptional
gene silencing (TGS) [2]. By far the studies have indi

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LUAN et al.

Helicase catalyzes miRNAmiRNA* duplex conver

sion to helix, and mature miRNAs combine with an
Argonaute protein to form RNAinduced silencing
complex (RISC) [1214]. The miRNAs complement
with their targets more perfectly in plants than in ani
mals and generally direct cleavage of complementary
mRNAs; so the functions of miRNA in plants more
like siRNA [1518].
In recent years, the researches of miRNAs in plants
have obtained substantial advancements [1]. Many
miRNAs have been discovered in model plants, such
as Arabidopsis, Populus, and Oryza, but few miRNAs
have been found in tomato, which is a model plant in
the researches of fruit ripening and development [19,
20]. There are two common ways to discover miRNAs:
one is the usage of a direct clone, and the other one is
a bioinformatics strategy. The first technique is avail
able, but it always needs a long experimental progress,
and it is difficult to clone low abundance and tissue
specific miRNAs. Thus, only a few miRNAs can be
detected though direct cloning and deep sequencing
[21, 22]. For example, Rachel et al. [23] cloned 4018
small RNAs from tomato mature green fruit tissue and
detected only nine known miRNAs and three novel
putative miRNAs. The main characteristic of bioin
formatics way is high timesaving and laborsaving
[24]. The expressed sequence tag (EST) analysis is
most wellknown in the bioinformatics strategy [25].
ESTs are partially expressed gene sequences that have
been converted into cDNAs, and the EST analysis has
been proven to be an economical, feasible, and fast
method for gene discovery in species lacking a draft
genome sequence [25, 26].
Up to now, the EST analysis strategy has been
proven a very effective way to find conserved miRNA
in plants. Because many miRNAs in plants are con
served, known miRNAs in some species can be used to
discover miRNAs in other species, which have not
been studied [24]. It is reported that 481 miRNAs
belonging to 37 miRNA families in 71 various plant
species have been discovered by the EST analysis [24].
Therefore, the EST analysis is a better strategy to be
used to detect many miRNAs, especially which are of
low abundance in plant tissues and hard to be found in
the direct cloning method [24].
Tomato is an economic crop that is cultured
broadly in the world and very popular in many coun
tries. More important, it is a model plant for the
researches of fruits and vegetables during development
and ripening. So far, many researches of miRNAs have
focused on model plants and economic crops [19]. Up
to date, few miRNAs have been found in tomato.
Thus, in this study, we were intended to predict and
discover more miRNAs with the EST strategy and to
analyze their target genes. All the miRNAs and the
targets obtained in this study will provide evidence for
further study of small RNA regulation of functional
genes in tomato physiological processes.

MATERIALS AND METHODS

Sequences of miRNAs, EST, and mRNA. To dis
cover potential tomato miRNAs, we downloaded a
total of previously known 838 miRNAs and their pre
cursors from miRNA Registry Database (Release 10.0:
August 2007; http://microrna.sanger.ac.uk). These
sequences were from the species of Arabidopsis
thaliana, Oryza sativa, Populus trichocarpa, Glycine
max, Medicago truncatula, Physcomitrella patens, Sac
charum officinarum, Sorghum bicolor, and Zea mays.
In order to search the reference set of miRNA
sequences of tomato, the duplicated miRNA
sequences of the different species had been elimi
nated. The reference of miRNA sequences got from
the above progress was used as query sequences for
BLAST search with tomato ESTs.
The tomato EST sequences were obtained from
DNA Data Bank of Japan (DDBJ) (version 2.2.15)
(http://blast.ddbj.nig.ac.jp), and the tomato mRNA
sequences were obtained from the website (http://
ted.bti.cornell.edu/digital/sRNA).
Applied software for sequences analysis. The online
BLAST (version 2.2.15) was performed on the DDBJ
(http://blast.ddbj.nig.ac.jp/tope.html), and the anal
ysis of secondary structure of RNA was operated
online (http://frontend.bioinfo.rpi.edu/applications/
mfold/cgibin/rnaform1.cgi), with the mFold pro
gram (version 3.2) compiled by Zuker and Turner. A
web tool provided from website (http://ted.bti.cor
nell.edu/digital/sRNA) was used for analysis of poten
tial targets of miRNAs.
Prediction of tomato miRNAs. The process of
tomato miRNA prediction according to the method of
Zhang [24] with small modification is briefly described
in Fig. 1. All plant miRNAs were obtained from miRBase,
and all repeated sequences were removed. The
remaining miRNAs were compared with tomato ESTs
from DDBJ using BLAST. For searching appropriate
tomato ESTs, ESTs with 0, 1, or 2 mismatches against
known miRNAs were compared with the protein data
base from NCBI using BLASTX to eliminate EST
sequences encoding proteins. Online software mFold
was used to analyze the secondary structure of the
remaining RNA, because premiRNA can automati
cally fold into a special hairpin structure. The ESTs
with the corresponding hairpin structure were selected
as potential genes encoding tomato miRNAs. How
ever, the result above was so rude that the sequences
with a hairpin structure could not be ensured as pre
cursors of miRNAs because other RNAs (such as
mRNA, tRNA, and sRNA) may also form a hairpin
structure [27]. In order to obtain miRNAs and get rid
of the disturb of other RNAs, strict rules were estab
lished as follows: (1) Only 0, 1, or 2 mismatches were
permitted between predicted miRNAs and the known
miRNAs; (2) the predicted miRNA precursor
sequences could fold into an appropriate hairpin sec
ondary structure that contains the ~21 nt mature

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All plant miRNAs

registered in the miRBase
Remove repeated sequences

The remaining plant miRNAs

Tomato ESTs

BLAST

Sequences with 0, 1or 2

mismatches against known miRNAs
Blast with itself to
repeated
sequences
Sequences with 0, 1, or 2
mismatches against known miRNAs

Protein database

BLASTX
Remove encoding protein sequences
0, 1or 2 mismatches and noncoding proteins sequences

Predict secondary RNA structure by mFold

PremiRNA hairpin candidates

Novel potential coding miRNAs

Fig. 1. The procedure of searching tomato miRNAs with the previously discovered plant miRNAs and tomato ESTs.

miRNA sequence within one arm of the hairpin;

(3) the secondary structures of miRNA precursors had
the higher negative minimal free energies (MFEs)
than other types of RNAs; because the folding free
energy in miRNAs is not always higher than in other
RNAs, MFEI was introduced, which is a useful crite
rion for distinguishing miRNAs from other types of
coding or noncoding RNAs; it is indicated that most
miRNA precursors identified have a MFEI greater
than 0.85, a commonly used value to distinguish miRNAs
from other noncoding and coding RNAs because the
MFEI of tRNAs is 0.64, rRNAs is 0.59, and mRNAs
is from 0.62 to 0.66; (4) 3070% of the total miRNA
bases were composed of A+U; (5) there were no more
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than six mismatches between miRNA and the oppo

site miRNA* sequence in the other arm; and (6) the
loops or breaks in complementary miRNA* sequences
were not allowed [27, 28].
Prediction of miRNA targets. The previous
researches implicated a fact that most known plant
miRNAs bind their mRNA targets perfectly or nearly
perfectly and degrade the target mRNAs in a way
somewhat similar to RNA interference [29]. The
number of allowed mismatches between miRNAs and
their complementary potential mRNA targets was less
than four, and no gaps were allowed at the comple
mentary sites [29].
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LUAN et al.
Novel identified
tomato miRNA sequences

mRNA database of tomato

BLAST

<4 mismatches between miRNA and mRNA

Remove repeated
sequences
Potential targets of miRNA
Analysis according to annotations
Functional prediction miRNA targets
Fig. 2. The progress of searching the targets of novel identified tomato miRNA.

In the progress of predicting miRNA targets, novel

predicted tomato miRNA sequences were compared
with tomato mRNA database using BLAST. The result
< 4 mismatches between miRNAs and mRNAs was
allowed; then repeated RNA sequences were removed.
Finally, functional miRNA targets could be predicted
by further analysis (Fig. 2).
Tomato fruits and treatments. Tomato fruits were
harvested at five ripening stages: immature, mature
green, breaker, pink, and red. Cold stress (4C for
48 h), freezing stress (2C for 10 h), heat stress (50C
for 10 h), ethylene treatment, and pathogen infiltra
tion were applied to corresponding mature green
fruits. For ethylene treatment experiment, mature
green fruits were treated at ethylene concentrations of
150 l/l for 10 h at room temperature. In the pathogen
infiltration, the epidermis of mature green fruits was
bored and each hole was inoculated with 10 l of
Rhizopus nigricans Ehrenb. at the concentration of 2
105 cfu/ml and then incubated for 48 h at room tem
perature. After these treatments, appropriate fruit tis
sues were immediately frozen in liquid nitrogen and
stored at 80C for further analysis.
miRNA isolation and purification. Total RNA was
isolated from fruit tissues of five different maturation
phases and five different treatments using Trizol
reagent with 0.8 M guanidine thiocyanate, 0.4 M
ammonium thiocyanate, 0.1 M Naacetate (pH 5.0),
5% glycerol (v/v), and 38% phenol water (v/v). The
high molecular weight (HMW) RNAs were precipi
tated with 40% isopropanol (v/v); then the low molec
ular weight (LMW) RNAs were precipitated with the
final concentration of 50% isopropanol (v/v). miRNA
isolation was performed with 15% denaturing PAGE.
The liquid mixture of the gel was commingled thor
oughly and poured immediately, then placed at 37C

for at least 2 h; the sample of 510 mg of total RNA

dissolved in 500 l of RNasefree water was prepared;
the gel was run at 4C and 350 V for 5 h, then at 200 V
for 2 h. The gel was stained by soaking in 100 ml of
0.5X TBE buffer containing 0.25 g/ml ethidium bromide
for 10 min; then the gel slice encompassing 18 to 28nt
small RNAs defined by the mobility of the 10nt DNA
oligo ladder markers was excised. Finally miRNAs
were eluted from the gel using 0.3 M NaCl at 4C over
night.
Validation of predicted miRNA expression by touch
down RTPCR. miRNA 3'terminal was linked with
3'adapter (5',PrUrUrUCTATCCATGGACTGidT
3', where A, C, G, TDNA residues; rA, rURNA
residues; ,P5'phosphate; idT3'inverted deox
ythymidine) at 37C for 90 min in 10 l reaction mixture
with 6 l microRNA solution prepared as described
above, 1 l 100 M 3'adapter, 1 l DMSO, 1 l 10X T4
RNA Ligase Buffer, 0.5 l RNase inhibitor (Takara,
Japan), and 0.5 l T4 RNA Ligase (New England
Biolabs, United States).
cDNA was synthesized with a specific reverse
transcriptase (RT) primer, and the miRNA linked with
3'adapter acted as a template, at 42C for 60 min. The
reaction mixture (10 l) included 5 l miRNA linked
with 3' adapter, 1 l 10 M 3'primer (5'TACAGTC
CATGGATAGAAA3'), 1 l 10 mM dNTP mixture,
2 l 5X firststrand buffer, and 1 l MMLV reverse
transcriptase (Promega, United States).
Touchdown PCR was operated with cDNA synthe
sized above as templates in 50 l reaction mixture with
5 l 10X PCR buffer, 1 l 10 mM dNTP mixture, 1 l
10 M reverse primer, 1 l 10 M forward primer (cor
responding to miRNA sequences respectively), 1 l
cDNA synthesized above, 1 l Taq DNA Polymerase
(2 units) (Takara, Japan), 40 l double distilled water.

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Table 1. Newly identified eight miRNAs analyzed from tomato ESTs

Novel
miRNA

Gene ACC

NM, nt

LM, nt

LP

Side

A+U (%)

MFE

MFEI

157a
157b
159
167
168
172a
172i
399

DB708800
BM536323
CN385843
DB700536
BE461111
DB700858
BG627220
DB684524

0
1
0
0
1
0
2
2

21
21
21
21
21
21
21
21

100
96
178
73
145
106
45
71

5'
5'
3'
5'
5'
3'
5'
3'

61.9
57.1
57.1
52.4
33.3
61.9
47.6
47.6

43.40
16.60
73.61
27.30
59.80
39.80
11.10
30.24

1.20
0.43
1.11
1.05
0.76
1.11
0.53
1.03

Note: NMnumber of mismatches; LMlength of mature miRNAs; LPlength of precursors; MFEminimal folding free energy;
MFEIminimal folding free energy index.

Touchdown PCR was operated as follows: denaturing

at 94C for 30 s, the annealing temperature was grad
ually decreased by one degree from 68 to 60C with
2 cycles at each temperature and from 59 to 46C with
1 cycle at each temperature, then 10 cycles at 45C,
and each cycle of annealing spent 30 s, elongating at
72C for 30 s, and a final elongation at 72C for
10 min.
RESULTS AND DISCUSSION
Discovering miRNAs in Tomato
Although some nonconserved miRNAs have been
found, most mature miRNAs are evolutionarily con
served from species to species within the plant king
dom. This fact provides a powerful approach to pre
dict new miRNAs in other plant species from known
miRNAs [30].
A total of 357 365 tomato EST sequences were
obtained from the EST database in DDBJ. The
BLAST search for potential miRNAs was performed,
with the setting of e value at 10. After the first step of
searching with all previously known plant miRNAs
against the EST database of Lycopersicon esculentum
and the second step of removing the repeated and pro
teincoding sequences, eight potential miRNAs were
obtained (Table 1). They were LemiR157a/b,
LemiR159, LemiR167, LemiR168, LemiR172a/i, and
LemiR399. For the confirmation of potential miRNAs,
candidate miRNAs were evaluated for A+U content,
except miRNA168, which had A+U contents of
33.3%, other sequences of mature miRNA had A+U
contents ranging from 47.6 to 61.9%, corresponding to
the criteria that miRNA precursors and mature miRNAs
contained more A+U nt than G+C [27] (Fig. 3). To
avoid designating other noncoding RNAs as new
miRNAs, several uniform systems for annotating new
miRNAs have been established. Among these criteria,
negative minimal fold energy (MFE) is very important
because MFEs of miRNAs have higher values than
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MFEs of other noncoding and coding RNAs. But the

folding free energy of miRNAs is not always higher
than that of other RNAs. Therefore, we introduced
MFEI, which is considered the best criterion for pre
diction of miRNAs. It is indicated that most miRNA
precursors identified have a MFEI greater than 0.85, a
commonly used value to distinguish miRNAs from
other noncoding and coding RNAs because the MFEI
of tRNAs is 0.64, rRNAs is 0.59, and mRNAs is from
0.62 to 0.66. As to eight miRNAs isolated in our study,
the newly identified tomato premiRNAs had a high
MFEI (from 1.39 to 4.28), with an average of about
2.46, which is obviously higher than 0.85 [27, 28, 31].
Two groups of miRNA researchers have used the
direct clone method to discover miRNAs in tomato,
Rachel et al. [23] obtained 12 miRNAs from 4018
small RNAs in mature green fruit of tomato, and
Biao Ding et al. [32] reported 1210 small RNAs (con
taining miRNAs, and the most small RNAs they
obtained are nonconserved and specific in tomato) in
flower buds, young and ripe fruit, as well as leaves. As
to bioinformatics method, Zhang et al. [24] studied
new miRNAs in plant kingdom by EST strategy to dis
cover a total of 481 miRNAs belonging to 71 different
plant species, which embraced nine miRNAs in
tomato.
Diversity and Multiplicity of miRNAs in Tomato
Eight newly predicted miRNAs in tomato belong to
six families, of which miRNA157 and miRNA172 had
2 members; the others had only a single member each.
The tomato miRNA precursors exhibited diverse struc
ture and size. The length of eight miRNA precursors in
tomato varied from 45 to 178 nt, and the secondary struc
tures were quite different. The different structure of the
predicted miRNAs within different families and different
length of the precursors suggested that different miRNAs
might perform different functions in gene expression and
regulation. Newly identified miRNAs differed also in
their locations in precursor miRNA sequences.
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Fig. 3. Mature and precursor sequences and the predicted stemloop structures of newly identified miRNAs in tomato.
The sequences are from 5' to 3' and the mature miRNAs are marked with line. The actual size of the precursors may be slightly
shorter than the presented (miR159).
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Fig. 3. Contd.

miRNA159, miRNA172a, and miRNA399 were located

at the 3'end of the miRNA precursors, while the others
were located at the 5'terminal (Fig. 3). The miRNA pre
cursor ESTs had been annotated that they come from dif
ferent tissues. miR157b precursor EST BM536323 and
miR168 precursor EST BE461111 were cloned from fruit
tissue. miR157a, miR167, miR172a, and miR399 pre
cursor ESTs were all cloned from the leaf. The precursor
ESTs of miR159 and miR172i were cloned from the root
and flower, respectively (http://getentry.ddbj.nig.ac.jp).
The precursor ESTs coming from different tissues impli
cated that the mature miRNAs might have tissuespecific
expression patterns and functions in plant growth and
development.

bp

Experimental Validation of Predicted miRNAs

In this study, eight tomato miRNAs were predicted
by ESTs screen, of which miR157b and miR168 pre
cursor ESTs were annotated to be cloned from the
fruit, while the precursor ESTs of other miRNAs were
cloned from other tissues. In order to validate the
result of the prediction, the touchdown RTPCR
assay, which could improve the specificity of miRNAs,
was designed.
Total RNA was isolated from fruit tissues of five dif
ferent mature phases and five different treatments
using Trizol reagent, and miRNAs were obtained by
the means described in Materials and Methods sec
tion. Because miRNAs participate in the regulation of
plant growth and fruit ripening and also response to
the environment stresses [24], the miRNAs isolated
from different maturities and different treatments were
commixed together to improve the detected sensitivity
of miRNAs, which were predicted in this research.
cDNAs, which were synthesized by a specific
reverse transcriptase (RT) with above miRNAs linked
with 3' adapters, acted as the templates of touchdown
PCR amplification. As the 3'terminals of the miRNAs
were appended with 3'adapters, reverse primer were
the reverse complementary sequences of the 3'adapt
ers, while the forward primer sequences were strictly
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

corresponding to miRNA sequences, the products of

RTPCR amplification should be detected in about
40 bp to validate the existence of miRNAs in this assay.
The RTPCR method was performed to identify the
predicted miRNAs in Brassica napus by Zhimin Yang
[31] and was approved to be feasible to detect miRNAs
by experimental results. Compared to the RTPCR
method used above, the touchdown RTPCR in this
study was a meliorated way, which can improve the
specificity of miRNAs to be detected.
All the predicted miRNAs could be detected in
fruit tissue during different maturities and different
treatments by the result of touchdown RTPCR

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200
100
40

DL 157a NC 157b NC 159 NC 167 NC

200
100
40

DL 168 NC 172a NC 172i NC 399 NC

Fig. 4. Touchdown RTPCR amplification to validate the
newly predicted miRNA expression in tomato fruit tissue.
NC and DL are representative of negative control and
20 bp DNA ladder marker (Takara), respectively, and the
negative control reaction system is correspond with
miRNA touchdown RTPCR system, excluding the syn
thesized cDNA template.
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Table 2. Prediction of miR172i and target mRNAs interactions

mRNA

Alignment

Score

Mis
match

Wobble

Indel

mRNA
direction

mRNA (717 737)

SGN mRNA 5' CGGCAGCAUCAUCAGGAUUCU3'
U325104
172i

3' GCCGUCGUAGUAGUCCUAAGA5'

mRNA (157 177)

SGN
U325725

mRNA 5' CUGCAGCAUCAUCAGGAUUCU3'

172i

3' GCCGUCGUAGUAGUCCUAAGA5'

mRNA (547 567)

SGN mRNA 5' CUGCAGCAUCAUCAGGAUUCC3'
U314861
172i

3' GCCGUCGUAGUAGUCCUAAGA5'

mRNA (98 118)

SGN mRNA 5' CUGCAGCAUCAUCAGGAUUCC3'
U314856
172i

3' GCCGUCGUAGUAGUCCUAAGA5'

mRNA (742 762)

SGN mRNA 5' CUGCAGCAUCAUCAGGAUUCC3'
U314859
172i

3' GCCGUCGUAGUAGUCCUAAGA5'

mRNA (1589 1609)

SGN mRNA 5' CUGCAGCAUCAUCAGGAUUCC3'
U314858
172i

3' GCCGUCGUAGUAGUCCUAAGA5'

amplification shown in Fig. 4. The result of touch

down RTPCR also revealed that miR172a could
rarely be detected in fruit tissue during different matu
rities and different treatments; this implicated that
miR172a might be less important than other predicted
miRNAs in the process of fruit development or envi
ronment stress. Among the eight miRNAs predicted in
this study, only miR157b and miR168 precursor ESTs
were annotated to be cloned from fruit tissue
(http://getentry.ddbj.nig.ac.jp). The existence of other
miRNAs (miR157a, miR159, miR167, miR172a,
miR172i, and miR399), whose precursor ESTs were
cloned from other tissues except fruit (http://geten
try.ddbj.nig.ac.jp), were proven by the touchdown RT
PCR. This phenomenon indicated that the precursor
ESTs of these miRNAs might also express in normal
fruit or treated fruit, but they were not cloned from
fruit tissue in previous researches, or these miRNAs

had other precursors, which expressed in fruit tissue in

addition to the precursor ESTs predicted in this study.
Prediction of miRNA Targets and Their Complementary
Sites with mRNAs
A website tool of tomato small RNAs (http://
ted.bti.cornell.edu/digital/sRNA) under Tomato
Expression Database has been established by Biao
Ding et al. [32] recently. Using this tool and the newly
identified miRNA sequences, the targets of tomato
miRNAs could be mined by informatics analysis.
Three or fewer mismatches between the detected
mRNA sequences and the eight newly acquired miRNAs
were allowed [17]. Gaps and noncanonical pairs
except G:U were considered as mismatches according
to the screen criteria and were discarded [17] (the
newly identified miR172i as an example in Table 2).
After cutoff of the candidates beyond the criteria and

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Table 3. The potential targets of newly identified miRNAs in tomato

miRNA
157a

Targeted protein

Target function

Targeted gene

Squamosa promoter binding

proteinhomologue

transcription factor

SGNU317177, SGNU324312,
SGNU317176, SGNU342074,
SGNU325281, SGNU323360

Ribosomeassociated membrane RAMP4

metabolism

SGNU339626

33 kD secretory proteinrelated

metabolism

SGNU319877

157b

Squamosa promoter binding

proteinhomologue

transcription factor

SGNU317177, SGNU317176,
SGNU324312, SGNU342074,
SGNU323360, SGNU325281,
SGNU319736, SGNU319735

159

Aminotransferase, class I and II; ACS

signal transduction

SGNU332362

168

Argonaute

miRNA metabolism

SGNU338285, SGNU338286

Homeoboxleucine zipper protein 3

(HAT3)/HDZIP protein 3

transcription factor

SGNU328990

ERF

transcription factor

SGNU314861

AP2A

transcription factor

SGNU314859,
SGNU314858

AP2 domaincontaining transcription

factor RAP2.7

transcription factor

SGNU325104

PHAP2A

transcription factor

SGNU314856

SLocus protein kinase

signal transduction

SGNU321990

Expressed protein

metabolism

SGNU324615

AP2 domaincontaining transcription

factor RAP2.7

transcription factor

SGNU325104

Zinc finger binding DNA

transcription factor

SGNU314859

ERF

transcription factor

SGNU314861, SGNU314856,
SGNU314858

172a

172i

the repeated ones, 42 targets were gained, which were

classified into four groups by their functions (Table 3).
The first and largest group contains targets predicted
to encode transcription factors. The second group is a
series of enzymes, which may play crucial roles in
plant metabolism. The third group of miRNA targets
involves protein kinase, representative macromole
cules of signal transduction. However, some targets are
with no annotations, and their functions have been
unknown up to now; thus, these mRNAs comprised
the fourth group.
It is reported that most of the predicted targets were
transcription factors, which may play crucial roles in
cell differentiation, tissue development, and plant
growth [1, 2]. Plant miRNAs tend to complement to
their target mRNAs perfectly or nearly perfectly; thus,
it is easier to find plant miRNA targets than animal
ones [17]. Generally, a miRNA can be complementary
with more than one regulatory target [17]. In some
cases, the targets can be grouped into several gene
families. For example, the targets of miR172a contain
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

Vol. 57

three groups, which are transcription factor, protein

kinase, and unknown functional protein. In other
cases, one miRNAs family may have the same or sim
ilar functional targets with another family [17]. These
results indicate that miRNAs participate in the expres
sion and regulation of many functional genes in plant.
miRNA157a/b have been predicted to target Squa
mosapromoter Binding Proteins (SBP) or SBPlike
protein, which are reported to regulate the Antirrhi
num floral meristem identity gene Squamosa. This
implicates that Squamosa may also be regulated by
miRNA157a/b in tomato [33].
miRNA172a/i have been predicted to target a
homologue gene coding HDZIP protein 3. There are
five HDZIP III genes in Arabidopsis genome, includ
ing REVOLUTA (REV), PHABULOSA (PHB),
PHAVOLUTA (PHV), ATHB8, and ATHB15 [34].
miR172a/i have also been predicted to target ethyl
eneresponse element binding factor (ERF). ERFs are
validated as key regulators in the pathway of ethylene
signal transduction and also regarded as important
No. 4

2010

478

LUAN et al.

elements of plant defense responses signal system [35].

It is reported that several members of the ERF family
bind specifically to the GCCbox (AGCCGCC) of
ethyleneregulated genes through the conserved ERF
domain, initiating a transcriptional response in ethyl
ene signal transduction pathway [36].
miR168 has been predicted to target Argonaute
protein (AGO1). Argonaute protein family is a central
component of RNAinduced silencing complex
(RISC). Among 10 Argonaute proteins in Arabidopsis,
AGO1 is the only Argonaute gene known to be
required for miRNA function in Arabidopsis. Because
Arabidopsis AGO1 binds miRNAs and catalyzes target
mRNAs cleavage, the cleavage of target mRNAs are
repressed in ago1 mutants, and the levels of miRNA
targets in this kind of mutants elevate in vivo [37]. This
evidence suggested that miR168 might also participate
in the regulation of other miRNAs in tomato.
Tomato 1aminocyclopropane1carboxylate syn
thase gene 8 (LeACS8), encoding a key enzyme in the
ethylene biosynthesis has been predicted as one of the
targets of miR159. ACC synthase is a cytosolic enzyme
that catalyzes the ratelimiting step in ethylene bio
synthesis in higher plants [38]. The precursor EST of
miR159 was CN385843 which was annotated to
express in the tomato root tissue, which indicates that
miR159 might regulate root growth and development
via controlling LeACS8 in tomato.
ACKNOWLEDGMENTS
This study was funded by National Science Foun
dation of China (grant nos. 30430490 and 30600421)
and National Key Technologies R&D program in the
11th FiveYear Plan (2006BAD22B01).
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