Physiologia Plantarum 138: 226237.

2010 Copyright Physiologia Plantarum 2009, ISSN 0031-9317

Expression analysis suggests potential roles of microRNAs
for phosphate and arbuscular mycorrhizal signaling in
Solanum lycopersicum
Mian Gu
a
, Ke Xu
a
, Aiqun Chen
a
, Yiyong Zhu
a
, Guiliang Tang
b
and Guohua Xu
a,
a
State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural
University, Nanjing 210095, China
b
Gene Suppression Laboratory, Department of Plant and Soil Sciences and Kentucky Tobacco Research and Development Center, University of
Kentucky, Lexington, KY 40546,USA
Correspondence
*Corresponding author,
e-mail: ghxu@njau.edu.cn
Received 11 October 2009; revised 26
October 2009
doi:10.1111/j.1399-3054.2009.01320.x
MicroRNAs (miRNAs) have emerged as a class of gene expression regulators
that play crucial roles in many biological processes. Recently, several reports
have revealed that micoRNAs participate in regulationof symbiotic interaction
between plants and nitrogen-xing rhizobia bacteria. However, the role of
miRNAs in another type of plant microbe interaction, arbuscular mycorrhizal
(AM) symbiosis, has not been documented. We carried out a microarray
screen and poly(A)-tailed reverse transcriptase-polymerase chain reaction
(RT-PCR) validation for miRNA expression in tomato (Solanum lycopersicum)
under varying phosphate (Pi) availability and AM symbiosis conditions. In
roots, miRNA158, miRNA862-3p, miRNA319, miRNA394 and miR399 were
differentially regulated under three different treatments, Pi sufcient (+P), Pi
decient (P) and AM symbiosis (+M). In leaves, up to 14 miRNAs were up-
or down-regulated under either or both of the Pi treatments and AMsymbiosis,
of which miR158, miR319 and miR399 were responsive to the treatments
in both roots and leaves. We detected that miR395, miR779.1, miR840 and
miR867 in leaves were specically responsive to AM symbiosis, which is
independent of Pi availability, whereas miR398 in leaves and miR399 in both
roots and leaves were Pi starvation induced. Furthermore, miR158 in roots as
well as miR837-3p in leaves were responsive to both Pi deprivation and AM
colonization. In contrast, miR862-3p in roots was responsive to Pi nutrition,
but not to AM symbiosis. Moreover, the group of miRNA consisting miR319
and miR394 in roots and miR158, miR169g*, miR172, miR172b*, miR319,
miR771 and miR775 in leaves were up- and down-regulated by Pi starvation,
respectively. The data suggest that altered expression of distinct groups of
miRNA is an essential component of Pi starvation-induced responses and AM
symbiosis.
Introduction
Phosphorus (P) is the most immobile element among
the three main macronutrients required by plants.
Plants have evolved a series of strategies to cope
with P deciency, including establishment of symbiotic
Abbreviations AM, arbuscular mycorrhizal; AMF, arbuscular mycorrhizal fungi; miRNA, microRNA; nt, nucleotide; P,
phosphorus; PHR1, Phosphate Starvation Responses 1; Pi, phosphate; PIP, plasma membrane intrinsic protein; RA, relative
abundance; RT-PCR, reverse transcriptase-polymerase chain reaction; TIP, tonoplast intrinsic protein; WPI, weeks post-
inoculation.
associations with arbuscular mycorrhizal fungi (AMF).
More than 80% terrestrial vascular owering plants are
able to form symbiotic relationship with the AMF, and
there are fossil evidences suggesting the existence of
AM symbiosis for more than 460 million years (Harrison
2006). Moreover, evidence indicates that even some of
226 Physiol. Plant. 138, 2010
the non-mycorrhizal plants of today accounting for about
10% of the plant species were mycorrhizal 100 million
years ago (Brundrett 2002).
MicroRNAs (miRNAs) are a class of small endogenous
non-coding RNAs that have been found in both
animals and plants. The mature miRNAs are usually
approximately 21 nucleotides (nt) in length and are
conserved across plant species (Jones-Rhoades et al.
2006). They function as negative post-transcription
regulators by specic binding and cleavage of their
target mRNAs, or by repression of target mRNA
translation (Jones-Rhoades et al. 2006). In recent
years, an increasing number of miRNAs have been
isolated/identied and functionally characterized (Jones-
Rhoades et al. 2006). A signicant amount of work has
been focused on unraveling the pivotal role that miRNAs
played in plant growth and development, including
nutrient stresses (Chiou 2007).
In Arabidopsis, miR395 was up-regulated during
sulfate starvation, and its accumulation was regulated by
the transcription factor SLIM1 (Kawashima et al. 2009).
MiR395 targeted not only the mRNAs of three members
of the ATP sulfurylase (APS) gene family (Allen et al.
2005, Jones-Rhoades and Bartel 2004), but also the
sulfate transporter SULTR2;1 (Kawashima et al. 2009).
The miR399 is a Phosphate Starvation Responses 1
(PHR1) dependent phosphate (Pi) starvation-enhanced
miRNA in plants (Bari et al. 2006, Rubio et al. 2001).
It targets and directs the cleavage at the 5

UTR of
the mRNA of an ubiquitin-conjugating E2 enzyme-
UBC24, and its overexpression resulted in enhanced
Pi uptake and translocation of Pi within the plant
(Chiou et al. 2006, Fujii et al. 2005, Sunkar and Zhu
2004). In addition, chimeric Arabidopsis or tobacco
plants that were generated by micrografting revealed the
translocation of miR399 fromthe miR399 overexpressed
scions to the wild-type rootstocks (Lin et al. 2008,
Pant et al. 2008). These results indicated that phloem
transported miRNA from shoot to root could act as a
long-distance signal for the regulation of P homeostasis
(Lin et al. 2008, Pant et al. 2008).
The miRNAs are also involved in regulation of
plant microbe symbiosis. Combier et al. (2006) reported
that in the model legume Medicago truncatula,
miR169 was responsible for the post-transcriptional
repression of MtHAP2-1, a gene encoding a transcription
factor belonging to the CCAAT-binding family. Either
overexpression of miR169 or RNA interference (RNAi)
mediated knock-down of MtHAP2-1 in M. truncatula
would lead to defective nodule cells differentiation
(Combier et al. 2006). More recently, miR166, which
has two copies in the MtMiR166a precursor, was
shown as the post-transcriptional repressor of a novel
family of transcription factors associated with nodule
development, the class-III homeodomain-leucine zipper
(HD-ZIP III) genes. Plants overexpressing miR166
showed a characteristic phenotype with reduced number
of nodules as well as lateral roots (Boualem et al. 2008).
Twenty conserved and thirty-ve novel miRNA families
have been identied in soybean roots inoculated with
B. japonicum; among them a group of miRNAs was
differentially regulated upon inoculation (Subramanian
et al. 2008). These results offered the rst insights into
the miRNAs involved symbiotic signaling pathway in
plants.
It has been suggested that AM signaling pathway
involves a series of factors such as kinases, ion channels
and transcription factors (Harrison 2006). However, the
direct evidences for the involvement of these factors
as well as miRNAs are lacking. In this work, we
surveyed the expression of miRNAs in the roots and
leaves of tomato plants that were highly infected with
AMF or of tomato plants that were supplied with
high and low Pi in an attempt to identify conserved
miRNAs that may function in the stable plant AMF
symbiotic system. A preliminary screening for miRNAs
using microarray technology was performed and the
candidate regulatory miRNAs were selected, followed
by validation of their expression patterns by poly(A)-
tailed reverse transcriptase-polymerase chain reaction
(RT-PCR). Our results revealed that 16 conserved
miRNAs are differentially regulated by the Pi and/or AM
symbiosis, thus paving the way for further functional
characterization of miRNAs involved in Pi and AM
symbiosis signaling pathways.
Materials and methods
Plant material and culture condition
Seeds of tomato (Solanum lycopersicum L. cv. Micro-
Tom) were surface sterilized with a solution of 10%
commercial bleach (0.525% sodium hypochlorite) for
5 min, washed three times with sterile water and placed
on a wet sterile lter paper in a petri dish. The dish
was placed in dark at 28

C for 23 days. After germi-

nation, seedlings were placed in sterilized quartz-sand
until cotyledons were fully emerged. The seedlings were
grown for 10 days with application of a 1/4 strength
nutrient solution described below. The nutrient solu-
tion used for sand culture contains 2 mM KNO
3
, 1 mM
NH
4
NO
3
, 0.5 mM Ca(NO
3
)
2
, 0.25 mM CaCl
2
, 0.5 mM
MgSO
4
, 20 M Fe-EDTA, 9 M MnCl
2
, 46 M H
3
BO
3
,
8 M ZnSO
4
, 3 M CuSO
4
and 0.03 M (NH
4
)
2
MoO
4
.
Nutrient solution for Pi-sufcient plants was supple-
mented with 1mM NaH
2
PO
4
, and for Pi-decient and
AMF colonized plants, 50 M NaH
2
PO
4
was provided.
Physiol. Plant. 138, 2010 227
A sand-based mycorrhizal inoculum containing
Glomus intraradices was used for colonization (courtesy
of Prof. Xiangui Lin from the Soil Science Institute of
Nanjing, CAS). Each plantlet was inoculated with 3 g
inoculum placed in the sterilized sand around the roots.
One set of the plants provided with 50 M Pi was
colonized with the AM fungi, whereas another set was
cultivated with the autoclaved inoculum that served as
control.
The solution pH was adjusted to 5.5. The plants were
grown in a chamber with a 14-h light period at 2527

C
and a 10-h dark period at 1820

C for 7 weeks.
Seeds of rice (Oryza sativa ssp. Japonica cv. Nippon-
bare) were surface sterilized and cultured as previously
described with minor modication (Li et al. 2006). After
7 days of growth, the plants were transferred to nutrient
solution, either with 0.3 mM Pi (Pi sufcient, +P) or
with no Pi supplied (Pi decient, P). The solution pH
was adjusted to 5.5, and the solution was replaced every
2 days. The plants were harvested for RNA isolation after
14 days.
Detection and analysis of AMF colonization
The development of mycorrhizal fungal colonization in
the root cortical cells was monitored regularly at 10-
day intervals, until 7 weeks post-inoculation (WPI). Part
of the tomato roots 7 WPI was used for microarray
experiments and RNA isolation, and the rest were taken
for observation of infection.
Root segments were treated with 10% (w/v) KOH
solution at 85

C for 3 h and then the medium was acidi-

ed with 1% HCl solution for 10 min, followed by three
washes with distilled water. The roots were then stained
with trypan blue and examined for mycorrhizal fun-
gal colonization under a binocular microscope (Leica,
DMR, Germany) by the magnied line intersect method
(David-Schwartz et al. 2001, McGonigle et al. 1990).
Measurement of P concentration of plant tissues
The measurement of P concentration was conducted as
previously reported (Chen et al. 2007, Murphy and Riley
1962). Briey, the dried crushed plant tissue powder
(0.05 g) was digested with 5 ml of 98% H
2
SO
4
and
3 ml of 30% hydrogen peroxide. After cooling, the
digested sample was diluted to 100 ml with distilled
water. The P concentration in the solution was measured
using the molybdate blue method with absorbance read
at 700 nm on a 722 UVvisible spectrophotometer
(JingHua Technology Instrument, Jing Hua, China).
miRNA microarray experiments and preliminary
screening
Total RNAs were isolated fromroots and leaves of tomato
with TRIzol reagent (Invitrogen, Carlsbad, CA). RNA
labeling was performed with miRCURY

Array Power
Labeling kit (Cat #208032-A, Exiqon). The labeling
reaction was performed at 16

C for 1 h. Hybridization
was performed at 56

C for 16 h. After hybridization,

the slides were washed using miRCURY Array, wash
buffer kit (Cat #208021, Exiqon), and then dried by
centrifugation at 1000 rpm for 5 min.
Clustering arrays was scanned with a scanner
(Genepix 4000B). Data were extracted from the TIFF
images using GENEPIX PRO 6.0 software (CapitalBio). Low
intensity spots were removed, for which fewer than 30%
of the signal pixels exceeded the median background
plus two times its standard deviation (SD). Then, normal-
ization was performed based on the mean array intensity
for inter-array comparison. For each miRNA probe, four
replicate spots were set on a microarray. The mean inten-
sity value of each probe was used for cluster analysis. The
raw data were log 2 transformed and median centered
by arrays and genes using the Adjust Data function of
CLUSTER 3.0 software and then further analyzed with hier-
archical clustering with average linkage. To determine
the differentially expressed miRNAs, signicance analy-
sis of microarrays (SAM, version 2.1) was carried out using
two-class unpaired comparison in the SAM procedure. All
hybridizations were normalized by total intensity with a
criterion of normalized value more than 50.
The miRNA populations from +P and P +M plants
were compared with P plants. The miRNAs with a
fold change >1.5 and q value <0.001 were adopted
as candidates that were differentially expressed in
responses to P nutrition and/or AM symbiosis.
RNA extraction and detection of miRNA expression
by poly(A)-tailed RT-PCR
Total RNA was extracted from plant tissues with TRIzol
reagent (Invitrogen, Carlsbad, CA). The poly(A)-tailed
RT-PCR was done as previously reported with minor
modication (Fu et al. 2005). Considering the limited
sequence information of tomato miRNAs (half of the
candidates were not deposited in any tomato miRNA
database), the poly(A)-tailed RT-PCR may not reect
the exact sequences of tomato miRNAs. However, this
validation method may tolerate one or two mismatches
on the miRNA sequences depending on the location of
mismatches (Table S1) and still shows the expression
trend of the miRNAs.
Four micrograms of total RNA was used in a 25-l
system for adding poly(A) tails with poly(A) polymerase
228 Physiol. Plant. 138, 2010
(Ambion). Reverse transcription was performed using
the poly(A)-tailed small RNA of plant tissues and 1.5 g
of RT primer (5-CGA ACA TGT ACA GTC CAT GGA
TAG-d(T)
30
(A, G or C) (A, G, C or T)-3) with 500 U
of M-MuLV reverse transcriptase (Fermentas, Ontario,
Canada) in a 50-l reaction system.
The amplication of miRNAs was performed for 35
cycles at an annealing temperature of 58

C using miRNA
specic primers and primer (5-CGA ACA TGT ACA
GTC CAT GG-3). The PCR products were analyzed
on 12% polyacrlyamide gel with Ethidium bromide
(EB) staining. Then the gel slices containing DNA
fragments of about 80 bp were excised and the DNA
was puried using polyacrlyamide gel electrophoresis
(PAGE) purication kit (Bioteke). The DNA fragments
were directly subcloned into pMD19-T vector (TaKaRa)
and sequenced. The relative abundance (RA) of each
gene was calculated semi-quantitatively using the
expression intensity of the Actin gene as their internal
standard by software IMAGE-PRO PLUS (version 5.0, Media
Cybernetics).
Prediction of miRNA targets
To predict potential targets of miRNA candidates, we uti-
lized miRU (Zhang 2005), an online tool for prediction
of plant miRNA potential target (http://bioinfo3.noble.
org/miRNA/miRU.htm). Tomato miRNA sequences were
used as query when their sequences are identical to
Arabidopsis, otherwise the probe sequences derived
from Arabidopsis miRNAs on the microarray were used.
The default parameters were used for prediction which
adapted the lowest stringency levels to capture more
potential targets. The default input options were: score
for each 20 nt = 3; GU wobble pairs = 6; indels = 1
and other mismatches = 3. The dataset is from the TIGR
Tomato Gene Index 10.
After the miRU prediction, a re-screening of the
predicted potential targets following a series of standard
criteria reported previously was performed (Qiu et al.
2007, Rhoades et al. 2002, Yin et al. 2008).
Results
AM infection rate and effect of AM colonization on
Pi acquisition
To study the signicance of miRNAs in tomato Pi and
AM symbiosis pathways, we rst established a tomato
AM infection and colonization system. Inoculation of
G. Intraradices with the lowPi (0.05 mM) roots of tomato
for 7 weeks resulted in formation of arbuscular mycor-
rhiza in about 7085% of the root segments. The three
main AMF structures, intraradical hyphae, vesicles and
arbuscules, were clearly observed in the infected roots
(Fig. 1A). The AMF colonization increased the total P
uptake by 20% (Fig. 1B), even though the increase of
P concentration in the plant was not signicant, pos-
sibly because of the dilution effect of P by enhanced
growth (Fig. 1B). As expected, supply of 1 mM Pi to the
plant resulted in the highest P concentration and total
P uptake.
Regulation of Pht1 transporters by Pi starvation
and AM colonization
It has been well-documented in tomato that Pi deciency
enhanced expression of two Pi transporter genes, LePT1
in both roots and leaves and LePT2 in roots, whereas AM
colonization induced expression of LePT4 in the roots
(Liu et al. 1998, Muchhal and Raghothama 1999, Nagy
et al. 2005, Xu et al. 2007). In this study, we used LePT1,
LePT2 and LePT4 as marker genes for Pi deciency and
AMF colonization in the tomato plants. We sampled the
highly colonized roots and leaves at 7 WPI and extracted
total RNA for transcripts analysis. Besides the expected
expression patterns of LePT1 and LePT2 under high P
(+P) and low P (P) supply conditions in the roots and
leaves (Fig. 2), we observed that colonization of AMF in
the low P roots slightly decreased expression of LePT1
and LePT2 in roots and suppressed expression of LePT1
greatly in the leaves (the very faint PCR product bands
of LePT1 in high P and AMF colonized leaves are under
the detection limit of the software for band intensity
evaluation) (Fig. 2).
Preliminary screening of Pi and AM responsive
miRNAs using miRNA microarray analysis
We screened the miRNA microarray (Product #208002-
A, Lot #20478.06, Version: 9.2, Exiqon) containing
probes against conserved miRNAs from plants, animals
and microbes, among which 203 probes are against
Arabidopsis miRNAs. A series of standards as described
in the section Materials and methods was performed.
A total of 23 miRNAs with plant only derived
sequences expressed either in the roots, or in the leaves
or both that might recognize P nutrition and or AM
colonization derived signals (Fig. 3) were identied for
further validations by poly(A)-tailed RT-PCR.
Five miRNAs were differentially regulated in roots
during Pi starvation and AM colonization
We performed poly(A)-tailed RT-PCR using the total
RNA extracted from either the roots or leaves of tomato
at 7 WPI. The data showed that transcripts abundance
Physiol. Plant. 138, 2010 229
Fig. 1. AMF colonization of tomato roots (A) and P concentration and content in roots and shoots of tomato (B). (A) All the main AMF structures,
intraradical hyphae (ih), vesicles (v) and arbuscules (ar) together with extraradical hyphae (eh) and germinated spores (gs) are indicated by red arrows.
(B) The plants were grown under 1 mM Pi (+P) and 50 M Pi in the absence (P) or presence (+M) of G. intraradices for 7 weeks, respectively. Values
are mean SD, n = 5.
of miR158 and miR862-3p in the roots was reduced
under both Pi deciency (P) and AM symbiosis (+M)
as compared with that under Pi sufcient condition (+P)
(Fig. 4A). AM colonization in low P roots (+M/P)
further decreased the expression of miR158 (Fig. 4A).
In contrast, miR319 and miR394 tended to be up-
regulated by Pi starvation in the roots (Fig. 4B), whereas
their levels decreased under AM symbiosis as well
(Fig. 1). Similarly, expression of the well-characterized
miR399 was also increased by P deciency in roots,
which was consistent with published reports (Chiou
et al. 2006, Fujii et al. 2005).
Pi and AM symbiosis positively impact miRNA
expression in leaves
We tested the abundance of the mature fragments
of miR172, miR771, miR169g*, miR158, miR319,
miR172b* and miR775 in tomato leaves (Fig. 5). They
were all down-regulated under Pi deciency compared
with that under Pi sufcient condition. The AMF inoc-
ulation enhanced their expression nearly to the levels
observed under high Pi supply condition (Fig. 5).
Some miRNAs in leaves are up-regulated only
under AM colonization
It would be interesting to identify the AM symbiosis-
specic induced genes, especially the ones with
upstream regulatory functions, such as transcription
factors and miRNAs. Here, we detected more mature
fragments of miR395, miR779.1, miR840 and miR867
in leaves, particularly under AM colonization (Fig. 6).
Therefore, we predict that these four miRNAs in leaves
might also be involved in AM symbiosis signaling and
independent of P signaling in tomato.
230 Physiol. Plant. 138, 2010
Fig. 2. Expression of LePT1, LePT2 and LePT4 in response to Pi availability
and AMfungal colonization. RT-PCR was performed with total RNA from
roots and leaves of tomato. Actin was used for cDNA normalization. The
number below the band indicates RA of each gene with respect to the
loading control actin. ND indicates not detected.
Fig. 3. miRNA candidates selected by preliminary screening of miRNA
microarray data. Preliminary screening was performed as described in
the section Materials and methods. Gray circle, miRNAs selected from
roots; white circle, miRNAs selected from leaves.
miRNAs in leaves up- or down-regulated by Pi
deprivation and AM symbiosis
In the leaves, expression of miR399 was up-regulated
under Pi decient conditions as that in the roots
(Fig. 4B), whereas its expression was not regulatedby AM
colonization as some other Pi-starvation-inducible genes
(Burleigh and Harrison 1999) (Fig. 7 and 2). Interestingly,
another well-characterized miRNA, miR398, showed a
very similar expression pattern in leaves as compared
with that of miR399 (Fig. 7). These results indicated that
miR398 is also a Pi starvation-up-regulated miRNA in
leaves, and both miR398 and miR399 are not strongly
responsive to AM symbiosis.
In contrast to miR399 and miR398, miR837-3p was
abundant in the leaves under high Pi and greatly down-
regulated by Pi deciency (Fig. 7). Its expression in the
AMF colonized plants was barely detectable (Fig. 7).
Fig. 4. Expression of miR158 and miR862-3p (A), as well as miR319,
miR394 and miR399 (B) in response to Pi availability and AM fungal
colonization. RT-PCR was performed with poly(A)-tailed total RNA from
tomato roots. Actin was used for cDNA normalization. The number
below the band indicates RA of each gene with respect to the loading
control actin. ND indicates not detected.
Fig. 5. Expression of miR172, miR771, miR169g*, miR158, miR319,
miR172b* and miR775 in response to Pi availability and AM fungal
colonization. RT-PCR was performed with poly(A)-tailed total RNA from
tomato leaves. Actin was used for cDNA normalization. The number
below the band indicates RA of each gene with respect to the loading
control actin. ND indicates not detected.
Physiol. Plant. 138, 2010 231
Fig. 6. Expression of miR395, miR779.1, miR840 and miR867 in
response to Pi availability and AM fungal colonization. RT-PCR was
performed with poly(A)-tailed total RNA from tomato leaves. Actin was
used for cDNA normalization. The number below the band indicates RA
of each gene with respect to the loading control actin.
Fig. 7. Expression of miR399, miR398 and miR837-3p in response to
Pi availability and AM fungal colonization. RT-PCR was performed with
poly(A)-tailed total RNA from tomato leaves. Actin was used for cDNA
normalization. The number below the band indicates RA of each gene
with respect to the loading control actin. ND indicates not detected.
Many miRNAs are constitutively expressed in
tomato
Among the 23 miRNA candidates selected from our
microarray data (Fig. 3), miR861-5p in roots, and
miR171, miR847, miR839 and miR862-5p in leaves
showed either constitutive expression or slight changes
under our treatments (data not shown). This might be
because of the technical difference between miRNA
microarray experiment and RT-PCR validation method.
However, these results conrmed their presence in
tomato. We detected no or very low amplication of
miR847, miR169, miR779.1 and miR860 in the roots
(data not shown). The reasons for this absence could
be that (1) their abundance is too low to be detected
in the tissues; (2) these miRNAs are not inducible under
our treatment conditions and (3) some of them could be
false positive of microarray experiment.
Predicted targets of the selected miRNAs
We used the plant miRNAtarget nder tool, miRU(http://
bioinfo3.noble.org/miRNA/miRU.htm) (Zhang 2005) as
well as a series of standard criteria for predicting
the potential targets of the miRNAs regulated either
by Pi nutrition or AMF colonization or both. In
brief, the predicted target genes complementary to the
miRNAs with four or fewer mismatches were selected.
Mismatches at the 11th nt of miRNAs and gaps were
not allowed, and the non-canonical G:U pair was
considered as a mismatch.
A variety of potential miRNA targets was predicted, of
which some targets had annotated functions, whereas
the others were unknown proteins (Table 1). The
different targets of the predicted tomato miRNAs include
a group of proteins associated with regulatory and
metabolic pathways (Table 1). In general, the predicted
mRNA targets can be separated into three groups. The
members of the rst group are all genes encoding
transcription factors. Transcription factors are vital for
signaling process of plant growth, development and
stress responses. The second group includes genes
encoding proteins with biological importance. Among
these annotated proteins, some are kinases (miR172 and
miR867), whereas another is responsible for nucleic acid
binding (miR867). These proteins may have a close link
with P, indicating their potential involvement in the plant
Pi starvation responses. The last group is consisting of
unknown proteins.
Discussion
Delicate molecular mechanisms are required for plants
to accomplish the physiological and developmental
processes, as well as responses to environmental stimuli.
There are increasing evidences suggesting that miRNAs
are one essential member of these mechanisms (Chiou
2007, Chuck et al. 2008, Jones-Rhoades et al. 2006).
It is of interest and importance to determine whether
and how miRNAs play a role in plant adaptation to the
environmental stimuli. A logical rst step toward this
aim is the identication of miRNAs that are differentially
expressed in response to these stimuli. In this study, we
identied a total of 16 miRNAs in tomato (2 in roots, 11
in leaves and 3 in both roots and leaves), and found that
they were differentially regulated by either P nutrition
or AM colonization or both, indicating that miRNAs
are probably a component for the P nutrition and AM
symbiosis signaling.
232 Physiol. Plant. 138, 2010
Table 1. List of the potential targets of the selected miRNAs in S. lycopersicum. The target genes were predicted by using of miRU, plant microRNA
potential target nder (http://bioinfo3.noble.org/miRNA/miRU.htm). The number of mismatches between each miRNA and their target mRNA is
indicated in parentheses.
miRNA Targeted protein Target function Targeted sequence
miR158
miR169g*
miR172 Unknown proteins Unknown TC141594(2), BI935148(3),
TC140095(3),
PHAP2A protein Transcription factor TC136965(3), TC142465(3)
TC142472(3)
Homolog to AHAP2 Transcription factor TC147477(3)
Similar to LIPLESS1 Cell cycle TC152549(3)
HD-ZIP protein Transcription factor TC148798(2)
Similar to S-receptor kinase Metabolism TC151000(3),TC152100(3)
miR172b*
miR319 Similar to Teosinte branched1like protein Transcription factor TC150868(4)
Similar to AT5g04420/T32M21 20 Unknown TC144625(4)
miR394
miR395 Unknown protein Unknown CK714778(4), BI930279(4),
TC152978(4)
Homolog to sulfate adenylyltransferase Metabolism TC137219(4), TC144228(4)
Similar to At1g26110/F28B23 21 Unknown TC146681(4)
miR398 Superoxide dismutase [CuZn] 2 Stress response TC142292(3)
Weakly similar to disulde isomerase Protein folding and
stabilization
TC137151(4)
miR399
miR771
miR775
miR779.1 Unknown protein Unknown BE463063(3)
Similar to At1g14670/T5E21.14 Unknown TC136791(4)
miR837-3p Similar to branched-chain-amino-acid
aminotransferase-like protein 1
Metabolism TC143374(3)
Unknown protein Unknown TC143375(3)
miR840 Water-stress induced tonoplast intrinsic
protein (WSI-TIP)
Cellular transport and stress
response
BG130224(4)
Probable aquaporin PIP-type Cellular transport and stress
response
TC142344(4), TC142345(4)
miR862-3p Similar to hydroxyproline-rich
glycoprotein-like protein
Unknown TC143571(3)
Similar to GP|6633816|gb| F1N19.14 Unknown AW979387(4)
miR867 Casein kinase 2 catalytic subunit Metabolism AW429181(3),
Homolog to protein kinase CK2 alpha
subunit
Metabolism TC142234(3), TC142237(3)
Weakly similar to elongation factor Tu family
protein-related
Nucleic acid binding TC150360(3)
Recently, miRNA395, miRNA398, and miRNA399
have been well characterized and linked with nutri-
ent deciency-induced stresses (Chiou et al. 2006,
Kawashima et al. 2009, Yamasaki et al. 2009), of which
miRNA399 is specically induced by Pi starvation. This
uncovered another branch of the complex Pi starva-
tion signaling pathway. However, plants have evolved a
set of strategies to adjust to environment with limited Pi,
which involves alterations of root architecture, enhanced
excretion of organic acid and acid phosphatase, and for-
mation of symbiotic associations with AMF (Raghothama
1999). The manifestation of these alterations caused by
Pi deprivation is ne controlled by diverse molecular
mechanisms, in which miRNAs besides miR399 might
also display a signicant regulatory role. On the other
hand, in a well-established plant AM fungi symbiosis,
plants supply the endo-symbiont with sugar to sustain
their growth and development. AM colonization in turn
can help plants with exploration of P far away from the
rhizosphere, transform the unaccessible forms of P into
Pi, and thus improve the P nutrition of plants (Harrison
2006). Therefore, plants might share a common signaling
Physiol. Plant. 138, 2010 233
pathway to recognize the AM signal and the Pi signal.
As a matter of fact, the Pi and AM signaling pathways
share a great deal of upstream regulatory factors and
downstream structural genes as revealed previously by
microarray analyses (Guimil et al. 2005).
In this work, our results support the hypothesis
that there are indeed common and specic signalings
of the P nutrition and AM symbiosis processes. In
roots, the expression of both miR158 and miR862-3p
decreased under Pi starvation, and the expression level
of miR158 was even lower under AM colonization. This
indicated that miR158 is likely to be involved in the
signaling of both normal P nutrition and AM mediated
Pi uptake processes, whereas miR862-3p is expected to
display its function under non-mycorrhizal conditions.
In addition, the expression of miR319 and miR394
was only detectable under Pi starved condition. One
may postulate that increased Pi derived from either the
direct Pi uptake pathway or from the mycorrhizal uptake
pathway suppressed the gene expression. However, it
should be noted that when mycorrhizal fungi dominate
Pi supply to plants, there is a functional loss of direct Pi
uptake pathway in roots colonized by AMF irrespective
of growth responses (Smith et al. 2003). In addition, the
improved P nutrition status was limited in comparison
to high P supply in the tomato (Fig. 1). Therefore, we
predict that miR319 and miR394 in roots (Fig. 4A) might
be involved in AM symbiosis signaling Pi nutrition in
an independent manner in tomato or respond to Pi in
a dosage-dependent manner. It might be possible that
an improvement of Pi nutrition as much as that offered
by mycorrhizal uptake pathway is sufcient to suppress
their expression.
In leaves, up to 14 miRNAs were identied and found
differentially regulated. They were grouped into three
subclasses based on the similarity of their responses to
our treatments. The rst group consisted of miR172,
miR771, miR169g*, miR158, miR319, miR172b* and
miR775. These miRNAs in leaves were up-regulated by
either sufcient level of Pi or AM symbiosis in general
(Fig. 5). As miR319 and miR394 were commonly down-
regulated by either sufcient level of Pi or AM symbiosis
in roots (Fig. 4), we suggest that the miRNAs of the rst
group in leaves might also participate in AMcolonization
signaling independent of the Pi level or respond to Pi in a
dosage-dependent manner. Interestingly, miR319 which
might target a transcription factor (Table 1) showed
opposite expression patterns in response to Pi nutrition
in leaves and in roots (Fig. 4B and 5).
The miRNAs deposited in the second group, miR395,
miR779.1, miR840 and miR867, had a comparable
expression level under Pi sufcient and decient
conditions, and were specically up-regulated by AM
symbiosis, suggesting that an AMF derived signal might
be responsible for their up-regulation. Of these miRNAs,
miR395 was responsible for sulfate assimilation and
translocation of plants (Kawashima et al. 2009). In the
past decades, increasing evidences have revealed that
AM fungi were able to help the host plants with
acquisition of other mineral nutrients besides P, such
as nitrogen (N) (Govindarajulu et al. 2005, Jin et al.
2005, Ortiz-Ceballos et al. 2007, Perner et al. 2008).
More recently, AM fungi were reported to play a role
in sulfur (S) transport to plants (Allen and Shachar-Hill
2009). Sulfate taken up by the fungus was transferred to
mycorrhizal roots, which increased the root S contents
under S supply at moderate levels (Allen and Shachar-
Hill 2009). Therefore, to explore the potential role of
miR395 for plant uptake of S in the mycorrhizal uptake
pathway is also of interest. MiR840 was predicted to
target water-stress induced tonoplast intrinsic protein
(TIP) and a plasma membrane intrinsic protein (PIP)-
type aquaporin (Table 1). As the AMF also supply their
host plants with water in addition to nutrients (Parniske
2008), one can speculate that miR840 may be involved
in the water transport between plants and AMF.
The expression of the members in the last group
was either strongly increased (miR399 and miR398)
or strongly decreased (miR837-3p) upon Pi starvation.
MiR399 have been well characterized as reported
previously (Aung et al. 2006, Bari et al. 2006, Chiou
et al. 2006, Lin et al. 2008), and we have conrmed its
conserved expression pattern in tomato (Fig. 4B and 7).
Moreover, we observed that miR837-3p was completely
suppressed by AMF inoculation in the leaves (Fig. 7),
which prompts us to explore the potential role of
miR837-3p played in the AM signaling.
MiR398 is another well-characterizedmiRNAinduced
by copper deciency (Yamasaki et al. 2007, 2009).
It has been reported to target two Cu/Zn superoxide
dismutases, CSD1 (localized to cytoplasm) and CSD2
(localized to chloroplast), and be involved in a broad
range of plant responses to diverse abiotic and biotic
stresses, such as oxidative stress, copper deciency and
addition of sucrose, paraquat, ozone or plant pathogen
(Dugas and Bartel 2008, Jagadeeswaran et al. 2009,
Sunkar et al. 2006, Yamasaki et al. 2007, 2009).
Very recently, one of the three members of the
Arabidopsis miR398 family, ath-miR398a, was found to
be strongly down-regulated in response to Pi starvation
(Pant et al. 2009), which is contrary to our ndings
in tomato. However, Jia et al. reported that miR398
responded completely opposite or differentially to ABA
and NaCl stress in poplars (Populus tremula) and
Arabidopsis, respectively (Jia et al. 2009). This highlights
the possibility that evolutionarily conserved miRNAs
234 Physiol. Plant. 138, 2010
might respond differently to a certain abiotic stress
and thus play distinct roles in diverse species. In
order to further conrm this possibility, we tested
the abundance of the two members of miR398
family in rice (O. sativa), Osa-miR398a and Osa-
miR398b. Osa-miR398a was dramatically induced by
Pi starvation (Figure S1), which is consistent with that
we found in tomato, whereas Osa-miR398b tended to
be constitutively expressed (Figure S1). Furthermore,
a genomic fragment of 2000 bp upstream in relation
to each of the OsmiRNA398s precursor sequence
together with that of AtmiRNA398s was retrieved from
National Center for Biotechnology Information (NCBI,
http://www.ncbi.nlm.nih.gov/), respectively, and then
set to PLACE (a database of Plant cis-acting Regulatory
DNA Elements, http://www.dna.affrc.go.jp/PLACE/) for
the Pi starvation related cis-regulatory element analysis.
W Box (Devaiah et al. 2007) motif is commonly
distributed in the promoter regions surveyed (in
OsmiR398a, OsmiR398b and AtmiR398a) (Table S2).
Whereas most of these miRNAs are not up-regulated by
Pi starvation, indicating W Box might not be functional
in these genes. The PHR1 binding site, P1BS, (Rubio
et al. 2001) is found in the 5 upstream region of both
OsmiRNA398a and AtmiR398c. We suggest that the
up-regulation of OsmiR398a in this study might be
mediated by the P1BS motif, although whether P1BS
leads to the up-regulation of AtmiR398c is unknown.
Moreover, another potential Pi starvation-responsive cis-
element identied before, PHO, (Hammond et al. 2003,
Mukatira et al. 2001) is only presented in the 5 upstream
of AtmiR398c. Whether AtmiR398c responding to Pi
stress through PHO also needs to be investigated.
In summary, in the present study, we used the
miRNA candidates acquired by preliminary screening
of the microarray data for further expression analysis. A
subset of miRNAs in tomato was identied and found
differentially regulated by either P nutrition or AMF
colonization or both, conrming the hypothesis that
miRNAs may play important roles in the complex signal
transduction networks of P nutrition and/or AM derived
signaling. In addition, it should be noted that there is
a possibility that some early responding and/or novel
miRNAs expression are not detected by the microarray
analysis. The future work, therefore, will be focused on
the identication of some early responding and/or novel
miRNAs, if any, under our treatments, as well as the
elucidation of the correlations between the miRNAs and
their target genes and the functional characterization of
the targets.
Acknowledgements This work was supported by the 863
project (2006AA10Z134), 973 project (2005CB120903)
and the National Natural Science Foundation of China
(30571108, 30971855). We thank Prof. Shubin Sun and
Dr Yibing Hu for technical support and Prof. K. G.
Raghothama and Dr Wangxia Wang for critical reading
of this manuscript.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1: The PCR primers used in this study.
Table S2: Sequences related to the Pi starvation-
responsive motifs found at the upstream region of
AtmiR398s and OsmiR398s.
Figure S1: miR398s in response to Pi starvation in
O. sativa.
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Edited by J. K. Schjrring
Physiol. Plant. 138, 2010 237