Journal of Plant Physiology 167 (2010) 1289–1297

Contents lists available at ScienceDirect

Journal of Plant Physiology

journal homepage: www.elsevier.de/jplph

Analysis of phosphorus-deficient responsive miRNAs and cis-elements from

soybean (Glycine max L.)
Hou Qing Zeng a,1 , Yi Yong Zhu b,1 , Si Qi Huang a , Zhi Min Yang a,∗
a
Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Tong Wen Road 1, Nanjing 210095, China
b
Department of Plant Nutrition, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, China

a r t i c l e i n f o a b s t r a c t

Article history: Phosphorus is one of the major factors controlling plant growth and productivity. Although physiological
Received 22 November 2009 and molecular processes of P deficiency have been intensively investigated, our current understanding of
Received in revised form 23 April 2010 the coordinated regulation of phosphate-responsive genes and signal networks is limited. In the present
Accepted 23 April 2010
study, we performed a microarray-based genome-wide transcriptional analysis of miRNAs from soybean
(Glycine max L.) under phosphate deficiency. miRNAs extracted from P-deficient and P-sufficient soybean
Keywords:
were hybridized to an array containing 853 known plant miRNA sequences. An induction ratio signif-
microRNA
icant at p < 0.01 was observed for 57 miRNAs belonging to 27 families. Among these miRNA families,
Glycine max
Phosphate deficiency
which differentially expressed, 7 and 8 were found to be up-regulated, whereas 17 and 6 were down-
Microarray regulated in leaves and roots, respectively. Seven representative individual miRNAs were selected for
qRT-PCR validation, and most showed an expression pattern similar to that on microarray. We further
predicted P-responsive cis-elements from the promoters of miRNAs in response to and non-responding
to P deficiency. In total, 125 putative cis-elements were identified for 24 soybean P-deficient responsive
miRNAs. Interestingly, those miRNAs (54) not responding to P deficiency were also found to contain the
same P-responsive motifs. A comparative analysis revealed that the frequency of the motif occurrence
in the promoters of miRNA genes in response to P deficiency was higher than that of miRNA genes not
responding to P deficiency. Our study provides initial evidence in soybean that a set of miRNAs with a high
frequency of P-responsive cis-elements may coordinately regulate the plant response to P deficiency.
© 2010 Elsevier GmbH. All rights reserved.

Introduction some nutrient deficiency responsive miRNA families in plants has

Phosphate (P) is an essential element required for plant growth
and development. Although abundant in farmland, P is often
unavailable for plants due to its low availability (Raghothama,
1999). Application of large quantities of fertilizers to soils for
adjustment of P availability is not economically sustainable and
often leads to environmental problems. Therefore, application of
phosphate fertilizer must be minimized. Recent efforts have been
directed to the understanding of the molecular basis of plant adap-
tation to P deficiency and identification of P-responsive genes
whose expression can be manipulated to enable plant growth
under low P environments. More importantly, characterization of
Abbreviations: miRNA, microRNA; IPS1, induced by phosphate starvation 1;
PHO2, ubiquitin E2 conjugase; pri-miRNA, primary miRNA; TSS, transcription start
site; PHT1, high affinity phosphate transporter 1; PHR1, regulator of P deficiency
response.
∗ Corresponding author. Tel.: +86 25 84395057.
E-mail address: zmyang@njau.edu.cn (Z.M. Yang).
1
These authors contributed equally to this work.
advanced our understanding of the pathway leading to the alloca-
tion and homeostasis of nutrients under stress conditions (Chiou,
2007; Hsieh et al., 2009; Pant et al., 2009).
miRNAs are endogenous single strand non-coding RNAs with a
length of about 21 nt. They are known to negatively regulate post-
transcription of genes through interactions with 3 untranslated
regions or coding regions of their targets (Bartel, 2004). In plants,
miRNAs regulate the expression of a large number of genes control-
ling development (Glazinska et al., 2009; Kidner and Martienssen,
2005; Kwak et al., 2009; Palatnik et al., 2003) and response to
nutrient deficiency (Pant et al., 2009; Sunkar et al., 2007) or other
abiotic stresses (Huang et al., 2009; Jia et al., 2009; Jian et al., 2010;
Liu et al., 2008; Zhou et al., 2008). As a regulatory component of
post-transcriptional gene expression, the miR399 family has been
recently found to regulate P acquisition in P deficiency plants (Bari
et al., 2006; Chiou et al., 2006; Fujii et al., 2005; Pant et al., 2008).
The miR399 family can guide the cleavage of PHO2, which encodes a
ubiquitin E2 conjugase-related protein that negatively affects shoot
P content and remobilization. Additionally, IPS1 mRNA has been
recently reported to host a motif with imperfect sequence comple-
mentarity to miR399; however, IPS1 mRNA is not cleaved but can

0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2010.04.017
1290 H.Q. Zeng et al. / Journal of Plant Physiology 167 (2010) 1289–1297
sequester miR399; transgenic plants over-expressing IPS1 accumu-
late more PHO2 mRNA and less P content in shoots (Franco-Zorrilla
et al., 2007). These results indicate that miR399, together with its
up- or down-stream genes, coordinately regulates plant response
to phosphate deficiency.
Soybean is one of most important economic crops. Because
they develop important symbioses with rhizobia and mycorrhizal
fungi, soybeans exhibit distinct features in secondary metabolism
and pod development that cannot be adequately modeled with
Arabidopsis and rice. As one of the legumes, soybean has diverse
mechanisms for adaptation to abiotic stresses (Sharifi et al., 2004;
van Heerden and Krüger, 2002). Under P starvation, soybean may
display its unique strategies to improve its acquisition and remo-
bilization of phosphate. In comparison with Arabidopsis and rice,
fewer miRNAs in soybean have been identified (Subramanian et
al., 2008; Valdés-López et al., 2008; Zhu et al., 2010). Also, the
physiological and molecular processes in soybean under Pi starva-
tion appear more complex. Hence, a global survey of miRNA gene
expression in response to P deficiency is necessary to understand
the networks of gene expression related to phosphate acquisition,
transport, recycling, root development and signal transduction.
Using microarrays, it is possible to perform high-throughput profil-
ing of all known miRNA expression to examine their development
process and response to environmental stresses (Yin et al., 2007). In
this study, we analyzed the spatial expression patterns of miRNAs
from soybean subjected to phosphate starvation. A set of miRNAs
was shown to express differentially in response to P deficiency.
Further analysis of P-responsive cis-elements in the promoters of
miRNA genes showed the functional complexity of transcriptional
regulation. Our results uncovered a group of miRNAs that may serve
as an important basis for systemic regulation of plant adaptation to
P deficiency.
Materials and methods
Plant cultivation
Soybean seeds (Glycine max cv. NY205) were soaked in aerated
1 mM CaSO4 for 4 h and germinated at 22 ◦ C in the dark between
two layers of filter paper moistened with 1 mM CaSO4 . After 3
days, seedlings were transferred to a 50 L nutrient solution contain-
ing 2.5 mM KNO3 , 2.5 mM Ca(NO3 )2 , 1 mM MgSO4 , 0.25 mM K2 SO4 ,
12.5 ␮M Fe–EDTA, 4.57 ␮M MnCl2 , 0.38 ␮M ZnSO4 ·7H2 O, 1.57 ␮M
CuSO4 ·5H2 O, 0.09 ␮M (NH4 )6 MoO24 ·4H2 O and 23 ␮M H3 BO3 . The
control plants grew in the same solution with an additional 0.5 mM
KH2 PO4 . Plants were grown in a growth chamber under con-
trolled conditions. Lamps gave a light intensity of approximate
400 ␮E m2 s−1 , with a day/night cycle of 14/10 h at 25/20 ◦ C. Rel-
ative humidity was 70%. The solution pH was constantly kept at
6.0 by continuous titration with 0.1 M NaOH or 0.05 M H2 SO4 .
The nutrient solution was changed every 3 days. After 7 days of
cultivation, plant roots and leaves were separately harvested and
immediately frozen in liquid nitrogen for analysis.
Construction of miRNA microarray
The known miRNA sequences in plant species were obtained
from miRBase (http://microrna.sanger.ac.uk/) (Griffiths-Jones et al.,
2008). Overlapping and repeat sequences were removed. All miRNA
probe sequences were designed to be complementary to the full-
length mature miRNAs. Each probe was repeated four times in one
chip. The in situ synthesis of miRNA microarray was performed by
LC Sciences (Houston, Texan, USA) and the protocols were strictly
followed (Gao et al., 2004).
Total RNA was isolated from leaves and roots with TRIZOL
reagent. The RNA sample was size-fractionated using a YM-100
Microcon centrifugal filter (from Millipore, Beijing, China) and the
small RNAs (<300 nt) isolated were 3 -extended with a poly(A) tail
using poly(A) polymerase. An oligonucleotide tag was then ligated
to the poly(A) tail for fluorescent dye staining; two different tags
(Cy3 and Cy5) were used for the two RNA samples (+P and −P) in
dual-sample experiments. Hybridization was performed overnight
on a ␮ParafloTM microfluidic chip using a micro-circulation pump
(Atactic Technologies, Houston, TX, USA) (Gao et al., 2004). On
the microfluidic chip, each detection probe consisted of a chemi-
cally modified nucleotide coding segment complementary to target
miRNAs (http://microrna.sanger.ac.uk/sequences/) and a spacer
segment of polyethylene glycol to extend the coding segment
away from the substrate. The detection probes were made by in
situ synthesis using PGR (photo-generated reagent) chemistry. The
hybridization melting temperatures were balanced by chemical
modifications of the detection probes. Hybridization was carried
out in 100 ␮L 6× SSPE buffer containing 0.90 M NaCl, 60 mM
Na2 HPO4 , 6 mM EDTA and 25% formamide at pH 6.8 and 34 ◦ C.
Following hybridization, the fluorescence was labeled using tag-
specific Cy3 and Cy5 dyes. Hybridization images were collected
using a laser scanner (GenePix 4000B, Molecular Device, Sunny-
vale, CA, USA) and digitized by Array-Pro image analysis software
(Media Cybernetics, Bethesda, MD, USA). Data were analyzed by
first subtracting the background and then normalizing the signals
using a LOWESS filter (locally weighted regression) (Bolstad et al.,
2003). For each sample, three hybridizations were carried out. Each
miRNA probe had four replicate spots on a microarray. miRNAs
that revealed significant changes in their expression in minus P(−)
plants were subjected to t-test analysis (p < 0.01). The microarrays
that changed significantly were adopted to discriminate expres-
sion of miRNAs that were differentially altered in response to P
deficiency.
Target prediction and functional classification
Previous studies have demonstrated that plant miRNAs reg-
ulate gene expression by targeting their mRNA sequences at a
perfect or near-perfect complementary site, indicating that plant
miRNA targets can be recognized using a simple homology search.
To predict the targets for the miRNAs in response to P defi-
ciency, we searched for soybean genome sequences using miRU2
(http://bioinfo3.noble.org/miRU2/) (Zhang, 2005) and miRNAassist
(Xie et al., 2007). Target sequences that have not been anno-
tated were blasted against the TAIR Arabidopsis protein database
(http://www.arabidopsis.org/). The functional annotations were
assigned on the basis of sequence similarity (E value <1e−5). The
target functions were grouped based on the MIPS functional cate-
gory classification at http://mips.gsf.de/projects/plants.
RT-PCR analysis of selective pri-miRNAs
The primers for quantitative RT-PCR (qRT-PCR) analysis
were designed according to their primary miRNA (pri-miRNA)
sequences. To ensure maximum specificity and efficiency for
PCR amplification of pri-miRNA cDNAs under standard condi-
tions, a stringent set of criteria was used for primer design,
including predicted melting temperatures of 58 ± 2 ◦ C, limited self-
complementarity, a primer length between 18 and 24 nt and PCR
amplicon lengths of 50–150 bp. Stem-loop sequences for which no
satisfactory primers could be found were elongated by 100 bp of
flanking genomic sequences on each side before primer design was
reinitiated.
Total RNA was extracted from tissues using Trizol (Invitro-
gen, Carlsbad, CA, USA) and digested with DNase I (Ambion) to
 H.Q. Zeng et al. / Journal of Plant Physiology 167 (2010) 1289–1297 1291

Fig. 1. The number of soybean miRNA members and their families regulated by P deficiency. Plants were subjected to P deficiency for 7 days. After that, total RNA was
extracted separately from roots and leaves. The miRNAs were expressed on microarray and only difference in miRNA abundance at p < 0.01 remained.

eliminate genomic DNA contamination, according to the manufac-
turer’s instruction. cDNA was synthesized from 1.0 ␮g total RNA by
Moloney Murine Leukemia Virus Reverse Transcriptase (Promega)
using oligo(dT) primers. qRT-PCR was performed on a MyiQ Sin-
gle Color Real-time PCR system (Bio-Rad). Briefly, 5 ␮L of a 1/50
dilution of cDNA in water was added to 10 ␮L of the 2× SYBR Pre-
mix Ex Taq (TaKaRa), 200 nM of each primer and water to 20 ␮L.
The reaction was amplified for 5 s at 95 ◦ C and 30 s at 60 ◦ C for 40
cycles. All reactions were run in triplicate and included no template
and no reverse transcription controls for each gene. PCR efficiency
was determined by a series of 2-fold dilutions of cDNAs. The cal-
culated efficiency of all primers was 0.9–1.0. Relative expression
levels were normalized to that of an internal control Tubulin A (Jian
et al., 2008) and presented as 2−CT × 104 to simplify the presenta-
tion of data (Livak and Schmittgen, 2001; Schmittgen et al., 2004).
The miRNA specific primers designed are listed in Supplemental
Table S1.
based on the screening criteria (Fig. 1). There were 25 and 13
miRNA populations that expressed in leaves and roots, respectively
(Fig. 2A). miR474 and miR482 were only expressed in roots. The
tissue-specific expression suggests that roots and leaves respond
differentially to P deficiency by activating a set of distinct miRNA
genes. In addition, several miRNA populations such as miR159,
miR319 and miR398 were expressed in both leaves and roots. The
miR399 family was probed on arrays, but was not significantly
expressed and therefore is not included in Fig. 2. However, suffi-
cient expression of miR399 could be detected using qRT-PCR (see
below).
Among the miRNAs, there were eight and seven miRNA families
whose expressions were up-regulated in leaves and roots, respec-
tively (Fig. 2B). Expression of miRNAs such as miR159, miR894,
miR1507 and miR1509 was coordinately stimulated in leaves and

Analysis of miRNA promoter and cis-acting elements

Soybean pre-miRNA sequences were obtained from the miR-

Base (http://microrna.sanger.ac.uk/seguence/index.html) (Release
14.0, September, 2009). The soybean pre-miRNA sequences
were mapped to the soybean genome using an online pro-
gram from Phytozome (http://www.phytozome.net/soybean). The
2 kb sequences upstream of the pre-miRNAs were downloaded
from Soybean Genome Browser (http://www.phytozome.net/cgi-
bin/gbrowse/soybean/?name=Gm09). These sequences were used
to predict the transcription start site (TSS) and TATA-box (Cui et
al., 2009) and analyze P-responsive cis-elements and other stress-
responsive cis-elements (Higo et al., 1999).

Results

Analysis of P deficiency responsive miRNAs from soybean

A microarray containing 853 plant miRNA sequences was con-

structed to analyze the gene expression of soybean (Glycine max)
under P deficiency. Plants were hydroponically grown under P
deficiency for 7 days. Total RNA was isolated from roots and
leaves, respectively. The induction ratio was calculated as a rel-
ative increase in expression under P deficiency compared to the
expression of miRNAs under P-sufficient conditions. Only changes
at p < 0.01 for miRNA abundance within treatments were accepted.
The change was the average value that was calculated from each Fig. 2. Diagrams of unique and overlapping soybean miRNAs under P deficiency:
(A) total miRNAs; (B) up-regulated miRNAs (↑); (C) down-regulated miRNAs (↓).
value of individual miRNA. Therefore, the data for expression abun-
The soybean plants were subjected to P deficiency for 7 days. After that, total RNA
dance of all miRNAs within a family were expressed as average was extracted separately from roots and leaves. The miRNAs were expressed on
values. At least 57 miRNAs (27 families) were significantly changed microarray with significant changes of p < 0.01.
1292 H.Q. Zeng et al. / Journal of Plant Physiology 167 (2010) 1289–1297
Fig. 3. P deficiency-stimulated (log2 (−P/+P) > 0) and depressed (log2 (−P/+P) < 0) soybean miRNA gene expression analyzed by microarray (p < 0.01). A: in root; B: in leaf.
roots. Conversely, expression of 17 and 6 miRNA families were
down-regulated in leaves and roots, respectively (Fig. 2C). Also,
several other miRNA families such as miR165/166 and miR398
were down-regulated in the tissues. Diversity of P-responsive miR-
NAs was also found in expression abundance. miR894, miR474
and miR482 dominated among the up-regulated miRNAs in roots
(Fig. 3A). Similarly, miRNAs such as miR1507, miR894 and miR157
were strongly up-regulated in leaves (Fig. 3B). However, some
miRNA families showed very low abundance of expression in tis-
sues. The varied abundance of the miRNA families suggests that
miRNA genes would be differentially regulated by P starvation and
may play different roles in regulating the plant response to P defi-
ciency.
Experimental validation of miRNA expression
To confirm the microarray results, total RNA was extracted
from tissues with P deficiency, and pri-miRNA transcripts were
analyzed by quantitative RT-PCR. Seven representative miRNAs
such as miR159a, miR166a, miR319a, miR396a, miR398b, miR399a
and miR1507a were selected for analysis because most of them
respond to abiotic or nutrition stress (Huang et al., 2009; Liu et al.,
2008; Sunkar et al., 2007). The primers were designed for RT-PCR
amplification based on the sequences of the corresponding miRNA
genes. By employing this approach, all miRNAs were recovered and
expression abundance could be determined. Expression of miR159a
and miR399 was up-regulated, whereas expression of miR166a,
miR319a, miR396a, miR398b and miR1507a was down-regulated
in roots with P deficiency (Fig. 4). However, the expression pat-
tern of miR396a and miR1507a in roots was not in agreement with
that from microarray. The significant variation may result from the
different methods used.
Identification of P deficiency responsive cis-elements in upstream
of miRNA genes
Comparative genomics has proven to be a powerful tool for
discovery of a large number of genetic elements that are usually
difficult to discover due to their small size or lack of experimen-
tally validated information (Haberer et al., 2006). It is known that
pri-miRNAs are typically transcribed by RNA polymerase II (Xie et
al., 2005) and class II promoters contain both the core promot-
ers and other regulatory elements. To specify sequences in the
promoter regions of miRNA genes, we first searched for miRNAs
with sufficiently long upstream sequences. At least 22 individual
miRNA precursor sequences were obtained from the 27 miRNA
 H.Q. Zeng et al. / Journal of Plant Physiology 167 (2010) 1289–1297 1293

Table 1
Putative transcription start sites (TSSs) and TATA-boxes upstream of the P deficiency
responsive miRNA genes in Glycine max.

miRNA Chromosome TSS position TATA position

miR156a 17 −174 −207
−735 −761
−1396 −1412

miR156d 8 −143 −176

−1893 −1928

miR156e 18 −201 −237

−718 −742
−1276 −1290

miR159a 9 −299 −329

−548 TATA-less
−1780 −1802
Fig. 4. Relative expression of some representative miRNA genes in soybean roots miR166a 16 −189 TATA-less
subjected to phosphate deficiency (−P) for 7 days by quantitative RT-PCR. The error −876 −891
bars represent SD from three biological replicates. Primers and amplification num- −1424 −1459
bers of target genes are listed in Supplemental Table 1. −1824 −1858

miR166b 8 −618 −656

−1621 −1655

families in response to P deficiency (Table 1). Next, we searched miR167a 19 −354 TATA-less
for the putative core promoters (TATA-boxes) and transcription −467 −503
−789 TATA-less
start sites (TSSs) located in the upstream regions. Because the vast
−937 −971
majority of plant miRNAs are transcribed from their own genes −1367 −1381
located in the intergenic genome (Lee and Ambros, 2001), and most
miR167b 2 −354 −391
of primary transcripts of the intergenic miRNAs are shorter than −903 −936
protein-coding transcripts (Cui et al., 2009; Xie et al., 2005), the
miR167d 10 −715 −748
2 kb region upstream of the G. max miRNAs in response to P defi- −1586 −1603
ciency was retrieved for analysis. The core promoters have two
miR168 9 −151 TATA-less
elements: a TATA-box beginning at the −60 position and an ini-
−177 −214
tiator centered on the TSS beginning at −46 (Table 1). In total, 64 −769 TATA-less
TSSs and 53 TATA-boxes were detected in the promoters of miRNA −864 −897
genes. To understand the feature of the core promoter location, miR319a 5 −221 −255
we analyzed TSS occurrence from the start positions of the pre- −635 −668
miRNAs. Most of TSSs were found to lie within 0.8 kb upstream of −1071 −1086
−1707 −1737
miRNA precursors (Fig. 5). The first peak for TSSs was found close
to the pre-miRNA within 0.2 kb upstream region and accounted miR319b 8 −1437 −1461
for 17% of total TSSs. The second largest region around 0.8 kb −633 −661
−229 −264
contained 15.2% of TSSs. Although TSSs were located in the dif-
ferent regions upstream of miRNA genes, the distribution pattern miR396a 13 −174 −207
−920 −954
was very similar to that of rice (Cui et al., 2009) and other plant
−1325 −1362
species (Zhou et al., 2007). Like TSS, the TATA-box has a similar −1727 −1759
pattern of location on the promoter region. Most miRNA genes
miR398a 1 −203 −236
host at least two TATA-boxes (or TSSs), except for miR1507a/b −1518 −1533
genes, which contain only one (Table 1). However, several miR-
miR398b 2 −426 −460
NAs have no promoter sequences. The missing core promoter (or
−1122 TATA-less
−1276 −1305
−1973 TATA-less

miR482 2 −58 −86

−655 −689

miR1507a 13 −257 TATA-less

−897 −930

miR1507b 17 −544 −558

−788 TATA-less

miR1508a 16 −173 −206

−1349 −1383

miR1508b 16 −1350 −1384

−174 −207

miR1509 17 −46 −60

−582 −619
−1133 −1159
−1626 TATA-less

miR1511 18 −184 −219

Fig. 5. Genomic distribution of TSS and TATA-box from the 5 end of the Glycine Max −1323 −1358
pre-miRNAs sequences. 22 pre-miRNAs in response to P deficiency and containing −1968 −1990
2 kb regions upstream of the mature miRNAs were used for predicting promoters.
1294 H.Q. Zeng et al. / Journal of Plant Physiology 167 (2010) 1289–1297
Fig. 6. Occurrence of P-responsive cis-elements in the promoter regions of miR-
NAs in response to P deficiency (full square) and non-responding to P deficiency
(open square). The frequency of occurrence of P-responsive cis-elements in the Y-
axis was expressed as the number of each distinct cis-element per miRNA gene.
The soybean miRNAs analyzed were grouped into 24 P deficiency responsive and
54 non-responsive miRNA sequences. The eight types of P-responsive cis-elements
were analyzed including PHR1 (PHR1 element), PHO (PHO element), PHO-like,
P-responsive (P-responsive element), W-box, TATA-box (TATA-box like), TC (TC
element) and NIT2 (NIT2-like).
TATA-less promoter) is frequently found in housekeeping genes
from animals and plants (Lee et al., 2004; Smale, 2001), which may
be one of the special features for regulating miRNA gene expres-
sion.
In addition, we examined P-responsive cis-elements for miRNAs
using the approaches described previously. The experiment was
undertaken for the consideration that some P-responsive miRNAs
may share commonly regulatory cis-elements with genes for phos-
phate acquisition or metabolism. We used a set of well-defined P
deficiency responsive cis-elements as a reference (Supplemental
Table S2) (Devaiah et al., 2007; Hammond et al., 2003; Mukatira
et al., 2001; Oshima et al., 1996; Rubio et al., 2001; Schünmann
et al., 2004; Wu et al., 2003). In total 125 putative cis-elements
were found for the 24 G. max miRNAs in response to P deficiency
(Table 2). Among them, miR168 contained 12 motifs. miR166a and
miR159a contained 10 and 8 cis-elements, respectively. The other
miRNAs have relatively fewer cis-elements. In miR156a, only two
motifs were detected. Variation of the cis-regulatory elements was
also shown in their locations in the promoter regions and the types
of the elements the miRNAs contained (Supplemental Table S3). For
instance, miR166a and miR168 contained six types of cis-elements;
miR159a, miR1507b and miR1511 each contained five types, and
the other miRNAs contained fewer cis-elements. Interestingly, a
set of soybean miRNAs (54) that did not respond to P starvation
were also found to contain P regulatory motifs (Supplemental Table
S4). Therefore, we assumed that the presence of the motifs was
more likely to occur in promoters that are P-responsive than those
that are not P-responsive. To address this question, we compared
the occurrence of the P-responsive elements in miRNA promoter
sequences. Our analysis revealed that the frequency of most of
the motifs (except NIT 2-like) was higher in P-deficient respon-
sive miRNAs than in miRNAs that do not respond to P deficiency
(Fig. 6). These results suggest that the P-responsive elements found
in miRNA promoters are likely to be functional in providing P-
deficient responsiveness.
Prediction of targets of miRNAs with P deficiency responsive
cis-elements
To predict target genes of miRNAs, the P responsive miRNA
sequences were mapped to the G. max genome. The detected mRNA
sequences must be highly complementary to the mature miRNA
sequences on the basis of screening criteria (Xie et al., 2007).
We found that 40 targets were predicted for 19 individual miR-
NAs with P-responsive motifs (Table 3). A comparison was made
between the predicted miRNA targets and others (Subramanian
et al., 2008). Only five targets for miR156 and miR166 over-
lapped. The target genes of miRNAs represent a large number
of functional categories. We used the MIPS functional category
classification (http://mips.gsf.de/projects/plants) to group these
targets into seven functional categories (Supplemental Fig. S1).
Among these, 44.1% were assigned to the largest category of infor-
mation pathways, which are representative of DNA binding and
processing, DNA conformation and recombination, and transcrip-
tion activation. The second largest category (20.6%) is related to the
localization of subcellular components. The third (12.1%) relates to
developmental processes. The fourth category (13.2%) is involved in
perception and response to environmental stimuli. The last three
smallest categories are involved in metabolism (5.1%), transport
(1.1%) and unknown proteins (3.7%), respectively.
Discussion
The microarray-based survey identified 27 soybean conserved
and known miRNA families in response to P deficiency, and all
of them were differentially expressed under P deficiency. Some
of miRNA families were tissue-specifically expressed. These miR-
NAs may contribute to the establishment and/or maintenance of
cellular identity or local specific response to P deficiency. On the
other hand, a set of miRNAs was shared by both roots and leaves
and can be coordinately expressed under P deficiency, implying
that these miRNAs would be involved in the systemic regulation
of biochemical or physiological pathways (Lin et al., 2008). To val-
idate the results from microarray, seven individual miRNAs were
determined by RT-PCR. miR159a, miR166a, miR319a, miR398b and
miR399a showed a pattern similar to that of microarray. However,
expression of miR396a and miR1057a determined by RT-PCR was
not in agreement with the pattern found in the microarray. The dis-
crepancy can likely be explained by the different methods used for
analyzing the miRNA expression. The RT-PCR assay quantified the
abundance of an individual pri-miRNA, while microarray detects
the mature miRNAs derived from their diverse or even total pre-
cursors. Also, each miRNA member may have its unique pattern of
expression, which is apparently different from the expression of a
miRNA family.
Expression of miR399 is sufficiently induced in Arabidopsis
under P deficiency (Bari et al., 2006; Chiou et al., 2006). How-
ever, such strong induction of miR399 is not always observed
in other plant species (Zhu et al., 2010). In the present study,
only slight, nonsignificant expression was probed on microarray
(data not shown). Although it could be detected using RT-PCR, the
degree to which miR399 was induced by P deficiency was low
(Fig. 4). A similar pattern has been found in white lupin (Lupi-
nus albus), a legume highly tolerant to P deficiency (Zhu et al.,
2010). The low abundance of miR399 expression in the legume
plant may result from the unique mechanism for resistance to P
starvation.
Previous studies have demonstrated that miR399 sufficiently
regulates its target genes and causes drastic physiological changes
in phosphorus allocation (Aung et al., 2006). As an individual
component, the action of miR399 is also negatively regulated by
 H.Q. Zeng et al. / Journal of Plant Physiology 167 (2010) 1289–1297 1295

Table 2
Analysis of P deficiency responsive cis-elements upstream of the soybean miRNAs in response to P starvation.

miRNA Total PHR1 element PHO element PHO-like P-responsive W-box TATA-box like TC element NIT 2-like

miR156a 2 2
miR156c 7 3 4
miR156d 4 1 1 1 1
miR156e 5 1 2 1 1
miR159a 8 2 1 1 3 1
miR166a 10 1 1 1 2 2 3
miR166b 5 1 2 2
miR167a 5 1 2 2
miR167b 3 1 1 1
miR167d 3 1 1 1
miR168 12 1 3 1 2 2 3
miR319a 4 2 1 1
miR319b 4 1 1 2
miR396a 4 1 1 1 1
miR398a 5 1 1 1 1 1
miR398b 4 2 1 1
miR399 4 1 1 1 1
miR482 4 1 1 2
miR1507a 7 1 1 1 4
miR1507b 7 1 1 1 1 3
miR1508a 4 1 1 1 1
miR1508b 4 1 1 1 1
miR1509 4 3 1
miR1511 6 1 2 1 1 1

Table 3
Potential targets of Glycine max miRNAs in response to P deficiency. Functionally unknown proteins were not listed in.

miRNA Target protein Target function Target

miR156a/c/d/e Conserved protein with cbs domain Metabolism TC210466

Olfactory receptor Signaling TC214342
Pollen-specific protein-like Development TC234878
Conserved protein with cbs domain Metabolism TC210466
GCN5-related N-acetyltransferase Metabolism TC219621
S-locus protein kinase Metabolism TC214166
Squamosa promoter binding protein-like 9 Transcription factor TC209333
Squamosa promoter binding protein-like 6 Transcription factor TC232420
Squamosa promoter binding protein 1 Transcription factor BE058432

miR159a Boron transporter Transport TC218466

Ubiquitin thiolesterase Metabolism TC224244

miR166a/b Class III HD-Zip protein Transcription factor TC230399

Homeodomain transcription factor Transcription factor BM309730
PHAVOLUTA-like HD-ZIPIII protein Transcription factor TC221756

miR167a/b/d DEAD box RNA helicase Metabolism TC222871

Ribulose-1,5-bisphosphate carboxylase Metabolism TC210116

miR168 GRF1-interacting factor 3 Transcription activator TC226538

Ent-kaurene synthase Metabolism TC234580
Protein serine/threonine kinase-like Metabolism BG882680

miR396a Growth-regulating factor Transcription activator TC231066; TC218495 TC216688

Cysteine proteinase precursor Metabolism TC206710
ATP binding /ATP-dependent helicase Metabolism TC220021
Cytochrome B6-F complex iron–sulfur subunit, chloroplast precursor Metabolism TC214790
Heat shock protein Stress response TC219546
Calmodulin-binding family protein Signaling transduction BI700778

miR398a/b Cu–Zn superoxide dismutase Stress response TC204404

miR482 Disease resistance-like protein Stress response NP213090; TC210836 TC224117; TC233092
Resistance-like protein KNBS2 Stress response TC231944

miR1507a/b Cold acclimation protein WCOR413-like protein Stress response BU549097

NBS-LRR resistance-like protein J78 Stress response BG507325
Antigen 85-B precursor Stress response TC219096
Nitrate transporter Transport TC218389

miR1508a Syringolide-induced protein 19-1-5 Metabolism AW306720

Histone H1 Metabolism TC204652

miR1509 Mitochondrial processing peptidase alpha subunit, mitochondrial precursor Development TC205568
1296 H.Q. Zeng et al. / Journal of Plant Physiology 167 (2010) 1289–1297
IPS1/At4 (Franco-Zorrilla et al., 2007; Lin et al., 2008) and pos-
itively regulated by PHR1 (Bari et al., 2006). In this case, PHR1,
miR399s, PHO2, PHT1 and IPS1/At4 constitute a pathway respon-
sible for the phosphate acquisition in plants. miR399 may play a
central role in mediating P-signaling by interacting its multiple
up- or down-stream genes. However, from the viewpoint of sys-
tematic biology, miR399 may only represent the local pathway
for P homeostasis. In fact, molecular mechanisms that maintain
P homeostasis are most likely to be associated with the global
architecture of miRNA regulatory networks, and miRNAs may
work in combinations. Coordinated expression of genes during
P deficiency initiates a series of physiological changes such as
phosphate acquisition and translocation, root branching, and phos-
phate metabolism and recycling (Fang et al., 2009). In each case,
a subset of genes including miRNAs is involved in the regula-
tion of a branch of P-signaling pathways. A set of miRNAs may
act in a combinatorial fashion to regulate coordinated expression
of genes related to plant resistance to P starvation. Such a net-
work may have evolutionary advantage and beneficial functions.
The present study provides a comprehensive analysis of a collec-
tion of miRNAs responding to P deficiency. The genes targeted by
these miRNAs cover a wide range of categories, including signal-
ing, metabolism, transport and stress, and may regulate a series
of physiological changes to improve the survival of plants under P
deficiency.
In plants, P deficiency induces expression of diverse genes for
multiple functions (Misson et al., 2005; Morcuende et al., 2007).
Some genes encode multiple transcription factors (TFs), and each
of these further regulates a group of down-stream genes for tol-
erance to P starvation (Hammond et al., 2004). The TF-targeted
down-stream genes may share common regulatory sequences in
their promoters. The cis-elements act as binding sites for TF and
respond to the early signaling of P deficiency. The present study
demonstrates that several P-responsive cis-elements do exist in
the promoter regions of the miRNA genes, suggesting that their
expression might be coordinately activated. For instance, Pho4, a
transcript factor, has an amphipathic helix-loop-helix-type DNA
binding domain that can interact with specific promoter sequences
of genes such as P transporters and phosphatases (Oshima et
al., 1996). Similarly, protein factors such as Pho4 may also bind
the promoter of miRNA genes with the same cis-elements. How-
ever, the existence of target sites in promoters does not mean
the sufficient activation of a miRNA gene. Similarly, expression
of a group of miRNAs that contain specific elements may not be
induced by P deficiency. These results suggest that the occurrence
of a P-responsive element may be necessary, but not sufficient,
for miRNA gene expression. To date, little is known about how
miRNAs are regulated through their promoter activation by P defi-
ciency.
It is possible that the miRNAs, in response to P deficiency,
would be regulated by other stresses because most of the
miRNA genes contain several other stress-responsive cis-elements
(Supplemental Tables S5 and S6). This finding is attributed to the
fact that plants have redundancy with other stress response path-
ways in genetic response to P deficiency (Hammond et al., 2004).
miRNA genes with functional redundancy are most likely to contain
multiple cis-regulatory elements that respond to various stresses.
For example, the miR1509 gene contains at least 6 cis-regulatory
elements, including EIRE (ethylene response), LTRE (cold, drought
and ABA), MYB (ABA and dehydration) and MYC (cold and dehy-
dration) sites in its promoter regions. The occurrence of multiple
cis-regulatory elements in miRNA promoters suggests that they
would be involved in multiple pathways. Hence, the protein-coding
genes and miRNAs carrying the same or similar cis-regulatory ele-
ments in their promoters may be co-expressed or co-regulated
under the same stress conditions.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation (30700488), the Science and Technology Foun-
dation of the National Education Ministry (107060), the National
Basic Research Program of China (863 Project) (2007AA10Z143) in
China, and the Innovation Project for College Graduate Students
from Jiang Su (Si Qi Huang). We thank the two anonymous review-
ers for their constructive suggestions.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.jplph.2010.04.017.
References
Aung K, Lin SI, Wu CC, Huang YT, Su CI, Chiou TJ. pho2, a phosphate overaccumulator,
is caused by a nonsense mutation in a microRNA399 target gene. Plant Physiol
2006;141:1000–11.
Bari R, Pant BD, Stitt M, Scheible WR. PHO2, microRNA399, and PHR1 define a
phosphate-signaling pathway in plants. Plant Physiol 2006;141:988–99.
Bartel D. MicroRNAs: genomics, biogenesis, mechanism and function. Cell
2004;116:281–97.
Bolstad BM, Irizarry RA, Astrandand M, Speed TP. A comparison of normalization
methods for high density oligonucleotide array data based on variance and bias.
Bioinformatics 2003;19:85–193.
Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Su CI. Regulation of phosphate homeostasis
by microRNA in Arabidopsis. Plant Cell 2006;8:412–21.
Chiou TJ. The role of microRNAs in sensing nutrient stress. Plant Cell Environ
2007;30:323–32.
Cui X, Xu SM, Mu DS, Yang ZM. Genomic analysis of rice microRNA promoters and
clusters. Gene 2009;431:61–6.
Devaiah BN, Nagarajan VK, Raghothama KG. WRKY75 transcription factor is a mod-
ulator of phosphate acquisition and root development in Arabidopsis. Plant
Physiol 2007;143:1789–801.
Fang Z, Shao C, Meng Y, Wu P, Chen M. Phosphate signaling in Arabidopsis and Oryza
sativa. Plant Sci 2009;176:170–80.
Franco-Zorrilla JM, Valli A, Todsco M, Mateos I, Puga MI, Rubio-Somoza I, et al. Target
mimicry provides a new mechanism for regulation of microRNA activity. Nat
Genet 2007;39:1033–7.
Fujii H, Chiou TJ, Lin SI, Aung K, Zhu JK. A miRNA involved in phosphate-starvation
response in Arabidopsis. Curr Biol 2005;15:2038–43.
Gao X, Gulari E, Zhou X. In situ synthesis of oligonucleotide microarrays. Biopolymers
2004;73:579–96.
Glazinska P, Zienkiewicz A, Wojciechowski W, Kopcewicz J. The putative miR172
target gene InAPETALA2-like is involved in the photoperiodic flower induction
of Ipomoea nil. J Plant Physiol 2009;166:1801–13.
Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ. miRBase: tools for microRNA
genomics (Database Issue). Nucleic Acids Res 2008;36:D154–8.
Haberer G, Mader MT, Kosarev P, Spannagl M, Yang L, Mayer KFX. Large-scale
cis-element detection by analysis of correlated expression and sequence
conservation between Arabidopsis and Brassica oleracea. Plant Physiol
2006;142:1589–602.
Hammond JP, Bennett MJ, Bowen HC, Broadley MR, Eastwood DC, May ST, et al.
Changes in gene expression in Arabidopsis shoots during phosphate starvation
and the potential for developing smart plants. Plant Physiol 2003;132:578–96.
Hammond JP, Broadley MR, White PJ. Genetic responses to phosphorus deficiency.
Ann Bot 2004;94:323–32.
Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements
(PLACE) database. Nucleic Acids Res 1999;27:297–300.
Hsieh LC, Lin SI, Shih AC, Chen JW, Lin WY, Tseng CY, et al. Uncovering small RNA-
mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.
Plant Physiol 2009;151:2120–32.
Huang SQ, Peng J, Qiu CX, Yang ZM. Heavy metal-regulated new microRNAs from
rice. J Inorg Biochem 2009;103:282–7.
Jia X, Ren L, Chen QJ, Li R, Tang G. UV-B-responsive microRNAs in Populus tremula.
J Plant Physiol 2009;166:2046–57.
Jian B, Liu B, Bi Y, Hou W, Wu C, Han T. Validation of internal control for gene expres-
sion study in soybean by quantitative real-time PCR. BMC Mol Biol 2008;9:59.
Jian X, Zhang L, Li G, Zhang L, Wang X, Cao X, et al. Identification of novel stress-
regulated microRNAs from Oryza sativa L. Genomics 2010;95:47–55.
Kidner CA, Martienssen RA. The developmental role of microRNA in plants. Curr
Opin Plant Biol 2005;8:38–44.
Kwak PB, Wang QQ, Chen XS, Qiu CX, Yang ZM. Enrichment of a set of microRNAs
during the cotton fiber development. BMC Genomics 2009;10:457.
Lee R, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science
2001;294:862–4.
Lee Y, Kim M, Han J, Yeom K, Lee S, Baek SH, et al. MicroRNA genes are transcribed
by RNA polymerase II. EMBO J 2004;23:4051–60.
 H.Q. Zeng et al. / Journal of Plant Physiology 167 (2010) 1289–1297
Lin SI, Chiang SU, Lin WY, Chen JW, Tseng CY, Wu PC, et al. Regulatory network of
microRNA399 and PHO2 by systemic signaling. Plant Physiol 2008;147:732–46.
Liu HH, Tian X, Li YJ, Wu CA, Zheng CC. Microarray-based analysis of stress-regulated
microRNAs in Arabidopsis thaliana. RNA 2008;14:1–8.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-
time quantitative PCR and the 2-[delta][delta]CT method. Methods Mol Biol
2001;25:402–8.
Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, Bligny R, et al. A genome-
wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips
determined plant responses to phosphate deprivation. Proc Natl Acad Sci USA
2005;102:11934–9.
Morcuende R, Bari R, Gibon Y, Zheng WM, Pant BD, Blasing O, et al. Genome-
wide reprogramming of metabolism and regulatory networks of Arabidopsis
in response to phosphorus. Plant Cell Environ 2007;30:85–112.
Mukatira UT, Liu C, Varadarajan DK, Raghothama KG. Negative regulation of phos-
phate starvation-inducible genes. Plant Physiol 2001;127:1854–62.
Oshima Y, Ogawa N, Harashima S. Regulation of phosphatase synthesis in Sacchar-
momyces cerevisae. Gene 1996;179:171–7.
Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, et al. Control of
leaf morphogenesis by microRNAs. Nature 2003;425:257–63.
Pant BD, Buhtz A, Kehr J, Scheible WR. MicroRNA399 is a long-distance signal for the
regulation of plant phosphate homeostasis. Plant J 2008;53:731–8.
Pant BD, Musialak-Lange M, Nuc P, May P, Buhtz A, Kehr J, et al. Identification of
nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive
real-time polymerase chain reaction profiling and small RNA sequencing. Plant
Physiol 2009;150:1541–55.
Raghothama K. Phosphate acquisition. Annu Rev Plant Physiol Plant Mol Biol
1999;50:665–93.
Rubio V, Linhares F, Solano R, Martín AC, Iglesias J, Leyva A, et al. A conserved MYB
transcription factor involved in phosphate starvation signaling both in vascular
plants and in unicellular algae. Genes Dev 2001;15:2122–33.
Schmittgen TD, Jiang J, Liu Q, Yang L. A high-throughput method to monitor the
expression of microRNA precursors. Nucleic Acids Res 2004;32:e43.
Schünmann PHD, Richardson AE, Smith FW, Delhaize E. Characterization of pro-
moter expression patterns derived from the Pht1 phosphate transporter genes
of barley (Hordeum vulgare L.). J Exp Bot 2004;55:855–65.
1297
Sharifi M, Ghorbanli M, Ebrahimzadeh H. Improved growth of salinity-stressed soy-
bean after inoculation with salt pre-treated mycorrhizal fungi. J Plant Physiol
2004;164:1144–151.
Smale ST. Core promoters: active contributors to combinatorial gene regulation.
Gene Dev 2001;15:2503–8.
Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O. Novel and nodulation-
regulated microRNAs in soybean roots. BMC Genomics 2008;9:160.
Sunkar R, Chinnusamy V, Zhu J, Zhu JK. Small RNAs as big players in plant abiotic
stress responses and nutrient deprivation. Trend Plant Sci 2007;12:301–9.
Valdés-López O, Arenas-Huertero C, Ramírez M, Girard L, Sánchez F, Vance CP, et
al. Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399
in phosphorus-deficiency signalling in common bean roots. Plant Cell Environ
2008;31:1834–43.
van Heerden PDR, Krüger GHJ. Separately and simultaneously induced dark chilling
and drought stress effects on photosynthesis, proline accumulation and antiox-
idant metabolism in soybean. J Plant Physiol 2002;159:1077–86.
Wu P, Ma LG, Hou XL, Wang MY, Wu YR, Liu FY, et al. Phosphate starvation triggers
distinct alterations of genome expression in Arabidopsis roots and leaves. Plant
Physiol 2003;132:1260–71.
Xie FL, Huang SQ, Guo K, Zhu YY, Nie L, Yang ZM. Computational identification
of novel microRNAs and targets in Brassica napus. FEBS Lett 2007;581:1464–
73.
Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC. Expression of Ara-
bidopsis miRNA genes. Plant Physiol 2005;138:2145–54.
Yin JQ, Zhao RC, Morris KV. Profiling microRNA expression with microarrays. Trends
Biotechnol 2007;26:71–6.
Zhang Y. miRU: an automated plant miRNA target prediction server. Nucleic Acids
Res 2005;33:W701–4.
Zhou X, Ruan J, Wang G, Zhang W. Characterization and identification of miRNA core
promoters in four model species. Plos Comput Biol 2007;3:412–23.
Zhou ZS, Huang SQ, Yang ZM. Bioinformatic identification and expression analysis
of new microRNAs from Medicago truncatula. Biochem Biophy Res Commun
2008;374:538–42.
Zhu YY, Zeng HQ, Dong CX, Yin XM, Shen QR, Yang ZM. microRNA expression profiles
associated with phosphorus deficiency in white lupin (Lupinus albus L.). Plant
Sci 2010;178:23–9.