Transcriptional profiling in cadmium-treated rice seedling roots using suppressive subtractive hybridization
Cadmium (Cd), a non-essential metal, is a kind of toxic heavy metal to life, which can accumulate in rice tissues including seeds, thus posing a risk to human health through food chain. To investigate the molecular mechanisms of rice response to Cd exposure, suppression subtractive hybridization and mirror orientation selection were used to compare gene expression profiles in seedling roots of Cd-exposed and control (unexposed) rice plants (Oryza sativa L., Nipponbare). Approximately 1700 positive clones, with insertions ranging from 250 to 1300 bp, were identified through reverse cDNA microarray analysis. Gene expression was further confirmed by real time RT-PCR. A number of differentially expressed genes were found in Cd-exposed rice roots, including 28 up-regulated genes and 19 down-regulated genes. They were found to be involved in diverse biological processes, such as metabolism, stress response, ion transport and binding, protein structure and synthesis, as well as signal transduction. Notably a number of known functional genes were identified encoding membrane proteins and stress-related proteins such as heat shock proteins, monosaccharide transporters, CBL-interacting serine/threonine-protein kinases and metal tolerance proteins. The cDNAs isolated in this study contribute to our understanding of genes and the biochemical pathways that may play a key role in the response of plants to metal exposure in the environment.
Transcriptional proﬁling in cadmium-treated rice seedling roots using
suppressive subtractive hybridization
, Xuncheng Liu
, Lianyu Yuan
, Keqiang Wu
, Jun Duan
, Xiaolan Wang
, Lixiang Yang
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
Graduate University of the Chinese Academy of Sciences, Beijing 100039, China
Life Science School, Guangzhou University, Guangzhou 510006, China
Received 26 May 2011
Accepted 27 July 2011
Available online 9 August 2011
Suppressive subtractive hybridization (SSH)
Cadmium (Cd), a non-essential metal, is a kind of toxic heavy metal to life, which can accumulate in rice
tissues including seeds, thus posing a risk to human health through food chain. To investigate the
molecular mechanisms of rice response to Cd exposure, suppression subtractive hybridization and mirror
orientation selection were used to compare gene expression proﬁles in seedling roots of Cd-exposed and
control (unexposed) rice plants (Oryza sativa L., Nipponbare). Approximately 1700 positive clones, with
insertions ranging from 250 to 1300 bp, were identiﬁed through reverse cDNA microarray analysis. Gene
expression was further conﬁrmed by real time RT-PCR. A number of differentially expressed genes were
found in Cd-exposed rice roots, including 28 up-regulated genes and 19 down-regulated genes. They
were found to be involved in diverse biological processes, such as metabolism, stress response, ion
transport and binding, protein structure and synthesis, as well as signal transduction. Notably a number
of known functional genes were identiﬁed encoding membrane proteins and stress-related proteins such
as heat shock proteins, monosa ccharide transporters, CBL-interacting serine/threonine-protein kinases
and metal tolerance proteins. The cDNAs isolated in this study contribute to our understanding of genes
and the biochemical pathways that may play a key role in the response of plants to metal exposure in the
Ó 2011 Elsevier Masson SAS. All rights reserved.
The heavy metal contamination that results from various activi-
ties, including manufacturing, mining, petroleum reﬁning and
chemical production, has been identiﬁed as a major environmental
and human health risk factor in many countries . Based on their
inﬂuence on plant growth, heavy metals can be classiﬁed into two
categories .Theﬁrst category includes essential mineral elements,
such as Zn and Cu, for which trace amounts may be required for
adequate metabolism, growth and development and an excess of
which is toxic as soonas the concentration exceeds the threshold that
plants can endure. The other category includes non-essential metals,
such as Cd and Pb; even trace amounts of these can lead to cellular
damage in plants . Cadmium (Cd) is a major environmental
pollutant, to which animals, including humans, can be exposed
through a food ingestion pathway. Cd pollution has dramatically
increased over the last several decades due to the production of many
commercial and industrial products, including batteries, pigments,
metal coatings and plastics [4,5]. Cd, which is absorbed by the roots
and transported through the xylem to the vegetative and reproduc-
tive organs, can negatively affect nutrient uptake and homeostasis,
resulting in inhibited root and shoot growth and, ultimately, reduced
yield . Cd adversely impacts various physiological processes :
even at low concentrations, Cd can affect photosynthesis, alter the
synthesis of proteins, inhibit enzyme activities, disrupt the transport
and movement of essential ions, inhibit the function of the stoma,
generate oxidative stress and, eventually, cause severe wilting [8,9].
Plant defense mechanisms include chelation and sequestration
of metals by ligands such as small peptides, organic acids and
amino acids that can bind free metal ions and buffer cytosolic metal
ions, resulting in metal detoxiﬁcation . The primary patterns of
heavy metal detoxiﬁcation in plants are the synthesis of intracel-
lular, high-afﬁnity binding sites that remove potentially damaging
ions by sequestration or efﬂux. The best-known chelators are the
small, cysteine-rich metallothionein (MT) proteins, the cysteine-
containing peptide, glutathione (GSH), and phytochelatins (PCs),
which bind metal ions through an S-containing amino acid ligand
[10e12] and reduce cellular metal toxicity.
Corresponding author. Tel.: þ86 20 37083652; fax: þ86 20 37087810.
E-mail address: email@example.com (M. Zhang).
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage: www.elsevier.com/locate/plaphy
0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
Plant Physiology and Biochemistry 50 (2012) 79e86
Although the exact metabolism pathway and detoxiﬁcation of Cd
exposure is still unclear, some reports have suggested that Cd could
affect the transcriptome and proteome of plants [4,13e16]. The
exposure of alfalfa seedlings to excess Cd has been shown to activate
mitogen-activated protein kinases (MAPKs) in roots , and it has
been reported that exposure of Arabidopsis thaliana roots to Cd
resulted in proteome changes that were detected by 2-D electro-
phoresis . In liverwort (Lunularia cruciata), which has a high
metal-accumulating capacity, gene expression patterns were shown
to be affected when the plant was grown in the presence of Cd salts
; changes in transcriptional regulation have also been analyzed
in response to Cd treatment in a heavy metal-accumulating plant,
Brassica juncea [4,16]. We suggest that further investigation to
identify the key genes responsible for Cd accumulation and trans-
port is important for understanding the molecular mechanisms of
the Cd stress response.
Rice is one of the most important cereal crops and is used as
a model for monocot plants. Rice is also a major source of Cd intake
to human beings . In China, some of the areas of rice cultivation
have been polluted by heavy metals, especially Cd ; therefore,
the Cd accumulated in rice seeds enters the body through the food
chain and can be very detrimental to human health. So, investiga-
tion of the rice mechanisms of Cd absorption and transport is
required. Furthermore, identifying the key genes and molecular
mechanisms responsible for Cd absorption and transport in rice is
important for altering the accumulation of Cd in rice plants through
genetic improvements, an important issue for food-safety with
regard to cadmium pollution. Our research presented here focused
mainly on the change in the expression pattern in rice seedling
roots when exposed to Cd and the identiﬁcation of the key genes
responsible for Cd stress responses.
Suppression subtractive hybridization (SSH) was employed to
identify the genes in rice seedling roots with a modulated expres-
sion following Cd treatment. The SSH technique, which is based on
the selective ampliﬁcation of differentially expressed sequences,
avoids the technical limitations of traditional subtraction methods
[18,19]. Together with cDNA microarray assays, SSH has been widely
used in the study of cell differentiation in both plants and animals.
To ensure that the identiﬁed genes were actually differentially
expressed, we used mirror orientation selection (MOS), which is
efﬁcient for eliminating background molecules in the subtracted
library . We also adopted reverse cDNA microarray analysis
and real time RT-PCR to eliminate false positive clones and increase
the selection efﬁciency. A number of differentially expressed
genes in Cd-exposed rice roots were found to be involved in diverse
biological processes, such as metabolism, stress response, ion
transport and binding, protein structure and synthesis and signal
transduction. Notably, a number of known functional genes were
identiﬁed that encode membrane proteins and stress-related
proteins, including heat shock proteins, monosaccharide trans-
porters, CBL-interacting serine/threonine-protein kinases and
metal tolerance proteins.
2.1. SSH cDNA library construction and quality of the libraries
In this study, we analyzed the transcriptomic changes of rice
seedling roots because Cd is readily taken up by the roots. Two-
week-old rice seedling roots treated with or without Cd were
harvested to construct the SSH libraries. The efﬁciency of subtrac-
tion was evaluated by PCR ampliﬁcation of a housekeeping gene,
rice actin7. If the subtraction is efﬁ
cient, as determined by PCR
of actin7, the actin7 transcripts should be reduced in
the subtracted cDNA population, whereas the actin transcripts
should remain normal in the unsubtracted cDNA population. The
actin fragment was barely detectable even after 33 cycles of
ampliﬁcation in the subtracted sample, whereas it was clearly
detectable in the unsubtracted sample after 28 cycles (Fig. 1), thus,
indicating that the housekeeping gene (actin7) transcript was
efﬁciently subtracted and the SSH libraries were reliable.
2.2. Isolation of Cd-responsive cDNA clones by SSH
The libraries consisted of approximately 1700 positive clones,
which were all conﬁrmed by cDNA microarray (Fig. 2), with inser-
tions ranging from 250 to 1300 bp by PCR ampliﬁ cation
(Supplementary data 2). After deleting the vector sequence and
poor-quality sequences, the average readable coding sequence was
approximately 500 bp in length. Approximately 280 clones with
different signal ratios were selected for 2 additional screenings,
after which, we chose 187 clones for sequence analysis. Af ter
removing the repetitive sequences and unknown sequence results,
28 cDNA clones were identiﬁed as being up-regulated, and 19 were
determined to be down-regulated (Table 1), with their mRNA levels
changing signiﬁcantly 48 h after Cd treatment.
2.3. Differentially expressed genes involved in metabolism, ion
transport, stress response and signal perception/transduction
Of the 47 clones that corresponded to known cDNAs or ESTs, 36
corresponded to unigenes, whereas 11 were homologous to
unknown ESTs or hypothetical proteins. These unigenes and ESTs
were classiﬁed into different categories (Table 1 and Fig. 3), including
metabolism (10; 21%), stress response (8; 17%), ion transport and
binding (10; 21%), protein structure and synthesis (4; 9%), signal
transduction (4; 9%) and unknown proteins or novel ESTs (11; 23%).
As shown in Table 1, several cDNA clones derived from the Cd-
exposed rice seedlings encode membrane proteins involved in
metal ion transport, such as Nramp (natural resistance-associated
macrophage protein) (Cd-de 555), a carrier membrane protein
Fig. 1. SSH subtraction efﬁciency detection. Subtraction efﬁciency was determined by
analyzing the amount of product of Osactin7 present in both subtracted cDNA and
unsubtracted cDNA through the use of increasing numbers of PCR cycles (for 18, 23, 28
or 33 cycles). Lane M, DL 2000 marker, from the top down means separately 2 kb, 1 kb,
750 bp, 500 bp, 250 bp, 100 bp； Lane 1e4, lane 5e8, lane 9e12, lane 13e16 were
forward subtracted samples, forward unsubtracted samples, reverse subtracted,
reverse unsubtracted sample separately.
M. Zhang et al. / Plant Physiology and Biochemistry 50 (2012) 79e8680
(Cd-in 7) and membrane transporter proteins (Cd-in 32 and Cd-129).
Several clones encoding putative stress-response proteins were
also identiﬁed, including a heat shock protein (HSP, Cd-in 356),
a probable copper amine oxidase (CAO, Cd-in 105) and a plant
metallothionein-like protein (MT, Cd-in 385).
Several clones encoding putative proteins related to signal
transduction were also recovered in the SSH libraries, including
phosphoglycerate kinase (Cd-in 94), CBL-interacting serine/threo-
nine-protein kinase (CIPK, Cd-in 307), GTP-binding regulatory
protein beta chain (Cd-in 397) and a pyruvate phosphate dikinase
family protein (Cd-de 175). In addition, many metabolism and
protein synthesis-related genes were also affected by Cd stress,
including genes that encode asparagine synthetase, farnesyl-
pyrophosphate synthetase, a cathepsin B-like cysteine protease
and formate dehydrogenase.
2.4. Expression patterns of candidate response genes
To conﬁrm the results of the cDNA microarray, we randomly
selected 11 up-regulated cDNAs and 9 down-regulated cDNAs to
analyze their expression patterns by RT-PCR. The real time RT-PCR
results are shown in Fig. 4. For all of the genes, the RT-PCR results
were generally in agreement with the microarray data (Table 1),
a result that indicated that the application of SSH in this study was
Cd is a non-essential trace element that is highly toxic to plants
at low concentrations . It can bind readily to free sulfhydryl
residues, leading to protein inactivation or denaturation. Cd
competes with and displaces essential cofactors, most notably Zn,
from a variety of proteins, leading to the inactivation of many
transcription factors or enzymes . In plants, Cd affects root
growth and biomass production by inhibiting photosynthesis,
respiration, mineral uptake and disruption of the water status
[23e25]. In higher plants, Cd is readily taken up by roots and
translocated into aerial organs where it can accumulate to high
levels  . In humans, ingestion of Cd through food or water can
lead to the accumulation of the metal and subsequent illnesses,
including itaieitai, as a result of eating Cd-contaminated rice .
In this study we found that several cDNA clones derived from
Cd-exposed rice seedlings encode membrane proteins involved in
metal ion transport. Nramps (natural resistance-associated macro-
phage proteins) (Cd-de 555) are a group of proteins functioning in
divalent metal transport across the phagosomal membrane of
macrophages. Studies have shown that Nramps play a major role in
metal ion homeostasis , functioning as general metal ion
transporters of Mn, Zn, Cu, Fe, Cd, Ni and Co . Among the
identiﬁed plant Nramps, AtNramp6 is likely involved in the distri-
bution and availability of Cd within plant cells . Another cDNA
clone (Cd-in 7), encoding a secretory carrier membrane protein,
was also isolated in the forward subtraction library and was up-
regulated in the subsequent RT-PCR analysis. In plant cells, carrier
membrane proteins also participate in the translocation of metal
ions into the vacuoles . The altered expression of Nramp and the
carrier membrane protein gene may be an adaptive reaction of the
rice root cells to Cd stress.
Two additional membrane transporter protein cDNAs (Cd-in 32
and Cd-in 129), designated as rice metal tolerance proteins
(OsMTPs), were up-regulated in the Cd-treated rice. The up-
regulation of two
ests a signiﬁcant role in metal
detoxiﬁcation in rice, which is supported by research on other plant
species [31e33]. MTP (metal tolerance protein) belongs to the
cation diffusion facilitator (CDF) family, also called the cation efﬂux
(CE) family. This is a diverse protein family, found in bacteria, fungi,
plants and animals . The ﬁrst plant CDF protein was designated
ZAT (zinc transporter in A. thaliana); however, because of its asso-
ciation with heavy metal tolerance, it was later renamed AtMTP1
(metal tolerance protein 1) . The ectopic over-expression of
Fig. 2. Typical results of cDNA array. Four identical membranes were hybridized with DIG labeled probes prepared from (A1, B2) forward subtracted cDNA, (A2, B1) reverse
subtracted cDNA, (C1, D2) forward unsubtracted cDNA and (C2, D1) reverse unsubtracted cDNA. The left four membranes (A1e D1) represent the results of Cd-induced clone
hybridization. The right four membranes (A2eD2) represent the results of Cd-decreased clone hybridization.
M. Zhang et al. / Plant Physiology and Biochemistry 50 (2012) 79e86 81
MTP1 in A. thaliana confers improved growth when supra-optimal
zinc concentrations are present in the medium. In addition, the
T-DNA insertional mutant line of AtMTP1 displays enhanced
sensitivity to high Zn concentrations , implicating a role of
AtMTP1 in Zn tolerance. MTP genes have been cloned from other
plants, in particular, from some metal hyperaccumulators, such as
Arabidopsis halleri (AhMTP1) , Thlaspi goesingense (TgMTP1) 
and Stylosanthes hamata L (ShMTP1) . MTP is a Zn ﬁnger protein,
Differentially expressed genes in the suppression subtractive hybridization libraries.
Genes are listed according to their possible function. The numbers in brackets
behind the clones’ numbers represent the numbers of replication in cDNA micro-
array screening assay.
Clone Accession No. Gene Location or Identity
Ion transport and binding
Cd-in 7 (2) Os05g0503000 Secretory carrier membrane protein
Cd-in 32 (2) Os05g0128400 Similar to Arabidopsis Metal
tolerance protein 1
Cd-in 129 Os08g0422200 Similar to Arabidopsis Metal
tolerance protein 12
Cd-in 178 Os02g0817500 KCNAB voltage-gated K
Cd-in 180 (5) Os08g0178200 Monosaccharide transporter 3
Cd-in 385 (4) Os01g0886900 Metallothionein-like protein
Cd-in 481 Os01g0661000 Integral membrane protein,
Cd-de 15 (10) Os11g0134900 Transporter-like protein
Cd-de 404 Os03g0107300 Transmembrane protein, similar
to P protein
Cd-de 555 (2) Os02g0131800 Metal transporter Nramp1
Cd-in 105 Os04g0476100 Probable copper amine oxidase
Cd-in 312 CI200599 Exoglucanase I precursor
Cd-in 319 Os08g0478000 Early nodulin 75 precursor-like protein
Cd-in 344 (3) Os01g0348900 SalT protein precursor
Cd-in 356 (3) Os01g0136200 16.9 kDa class I heat shock protein 3
Cd-de 36 (7) Os07g0539100 Glucan endo-1,3-beta-glucosidase
Cd-de 429 Os03g0320100 SSH00543 Osmotic stress SSH library
Cd-de 748 Os08g0398400 Hypersensitive-induced response
Cd-in 339 Os03g0291500 Asparagine synthetase, putative
Cd-in 376 Os04g0589401 Flavohemoglobin
Cd-de 13 (3) Os08g0189200 Probable germin protein type 2
Cd-de 53 Os08g0486200 Putative splicing factor, arginine/serine-rich
Cd-de 105 (2) Os01g0703400 Farnesyl-pyrophosphate synthetase
Cd-de 184 Os11g0677400 Plant invertase/pectin methylesterase
Cd-de 185 Os07g0686900 Probable alpha-arabinofuranosidase
Cd-de 212 (2) Os06g0486800 Formate dehydrogenase precursor
Cd-de 252 (3) Os03g0320100 Alpha-
Cd-de 879 Os01g0290000 Aspartic proteinase
Protein structure and synthesis
Cd-in 50 Os12g0617100 Translation initiation factor eIF-2B delta
Cd-in 152 Os12g0617100 Initiation factor 2 subunit family protein
Cd-de 188 Os02g0321900 60S ribosomal protein L10A
Cd-de 205 Os02g0534800 40S ribosomal protein S14
Cd-in 94 Os06g0668200 Phosphoglycerate kinase
Cd-in 307 Os03g0339900 CBL-interacting serine/threonine-protein
Cd-in 397 Os03g0669200 GTP-binding regulatory protein beta chain
Cd-de 175 Os05g0405000 Pyruvate phosphate dikinase family protein
Cd-in 17 CI188952 Novel EST
Cd-in 19 Os03g0129400 Unknown protein
Cd-in 42 CI424729 Novel EST
Cd-in 74 (4) CI436338 Novel EST
Cd-in 113 CA753770 Novel EST
Cd-in 182 CI446736 Novel EST
Cd-in 231 Os02g0584200 Unknown protein
Cd-in 334 Os03g0273800 Unknown protein
Cd-in 402 Os03g0129400 Unknown protein
Cd-de 43 (4) Os06g0170200 GOS9 protein
Cd-de 128 CI767456 Novel EST
Fig. 3. Gene’s classiﬁcation statistics among six functional categories. The percent (out of
the total number of genes) of genes belonging to a particular functional group is shown.
Fig. 4. Real time RT-PCR analyses of Cd-affected genes. (A) 11 Cd-induced genes; (B) 9 Cd-
decreasedgenes. Rice seedling rootswerecollected 0, 24 and 48hafter 0.5mM Cd exposure.
M. Zhang et al. / Plant Physiology and Biochemistry 50 (2012) 79e8682
which binds Zn, although the type of binding is not speciﬁc. When
expressed in yeast, TgMTP1t1 confers signiﬁcant tolerance to Cd, Co
and Zn, whereas TgMTP1t2 promotes signiﬁcant tolerance to Ni. Cd
can also competitively bind to AtMTP1 to suppress the binding of
Zn. In our research, two OsMTPs were notably up-regulated by the
Cd treatment, indicating that OsMTPs may be involved in the
translocation of Cd and metal detoxiﬁcation. The functional
signiﬁcance of OsMTPs responding to Cd and other heavy metal
remains to be investigated further.
Recently, a rice gene (OsHMA3) responsible for Cd accumulation
has been isolated [39,40] that encodes a heavy metal transporter
belonging to the P
-type ATPase gene family. The OsHMA3 trans-
porter has been shown to be highly speciﬁc for Cd in rice, a ﬁnding
that differs from other HMA genes [41,42]. In rice, OsHMA3 selec-
tively sequesters Cd into the root vacuoles and then limits the
translocation of Cd from the roots to the aerial parts, thereby
decreasing the Cd accumulation in the grain. Another metal
transporter gene, TcHMA3, identiﬁed in the Cd hyperaccumulator,
Thlaspi caerulescens, seems to have a similar biological function in
the speciﬁc absorption and sequestration of cadmium into the leaf
vacuoles . It has been reported that the expression level of
TcHMA3 in a higher Cd-accumulating ecotype (Ganges) showed
a sevenfold higher expression than in a lower Cd-accumulating
ecotype (Prayon), indicating that a higher expression of TcHMA3
was required for Cd hypertolerance in the Ganges ecotype. TcMTP1
encodes another metal transporter in T. caerulescens and is also
involved in metal tolerance and accumulation. The expression of
TcMTP1 has been demonstrated to be high in young leaves , and
the high level of expression of TcMTP1 has been closely correlated
with Cd hyperaccumulation in T. caerulescens plants. Clearly, the
identiﬁcation and characterization of transporter genes is key in
elucidating the mechanisms for heavy metal accumulation in
plants. Additional efforts should be made to determine the bio-
logical functions and the regulation mechanisms of rice heavy
metal transporter genes, with the long-term goal of decreasing the
Cd content in rice grains through breeding programs.
Cd treatment can induce both toxicity and osmotic stress,
causing multiple potential physiological responses in plants. In our
research, several clones encoding putative proteins that are related
to signal transduction were identiﬁed in the SSH libraries. In
addition to the clones encoding CBL-interacting serine/threonine-
protein kinase (CIPK, Cd-in 307), we identiﬁed clones encoding
a GTP-binding regulatory protein beta chain (Cd-in 397), putative
arginine/serine-rich splicing factor (Cd-de 53) and pyruvate phos-
phate dikinase family protein (Cd-de 175). The altered expression
patterns of these genes suggest Cd-induced effects at both cellular
and molecular levels.
Several clones found in our study encode putative stress-
response proteins. In our analysis, Cd-in 356 was predicted to
encode a small heat shock protein (HSP), proteins that are usually
undetectable in plant cells under normal physiological conditions
but can be induced by certain stress conditions, including drought,
salinity, oxidized species and low temperatures . Thus, HSPs
can act as molecular chaperones, maintaining native protein
conformation and playing a crucial role in protecting plants against
abiotic stresses . Furthermore, HSP levels have been shown to
rise in cultured cells in response to metal stress, suggesting that
they may contribute to heavy metal tolerance in plants . Cd can
alter membranes via spatial distortion of their associated proteins
 and affect cellular processes by the formation of an active
oxygen redox status , and HSP can assist in protein folding and
reintegration into the membrane and may also alter signal trans-
duction pathways to eliminate reactive oxygen species (ROS) in
plant cells. In our research, the transcript of Cd-in 356 increased in
rice roots after Cd treatment, which may re
destruction of cell membranes and, therefore, trigger HSP
production for the protection of cells and the maintenance of
homeostasis and protein activity in general.
The protein encoded by the Cd-in 105 clone is a putative copper
amine oxidase (CAO), an enzyme that catalyzes the oxidative de-
amination of polyamines, which are ubiquitous compounds
essential for cell growth and proliferation. In plants, CAOs are
mainly responsible for degrading cellular polyamines, a reaction
that generates hydrogen peroxide (H
) . As a signaling
has been correlated with cell wall maturation and
ligniﬁcation during development and with wound healing and cell
wall reinforcement during pathogen invasion and abiotic stress,
thereby, mediating cell death, the hypersensitive response and the
expression of defense genes. Cd and other heavy metals can often
cause a transient depletion of GSH and an inhibition of anti-
oxidative enzymes, especially glutathione reductase. Indeed, the
exposure of plants to Cd has been found to result in oxidative stress,
as indicated by lipid peroxidation, H
accumulation and an
oxidative burst . In the present study, the expression of Cd-in
105 (putative copper amine oxidase) was up-regulated by Cd
stress, an indication that Cd may trigger the accumulation of ROS in
rice root cells, thus, altering the ROS status and causing a change in
the expression of ROS signaling molecules. The increased expres-
sion of copper amine oxidase would be helpful in eliminating the
ROS in rice root cells and may aid in the protection of plant cells
from Cd stress.
A cDNA clone (Cd-in 385) showing similarity to plant
metallothionein-like proteins was also isolated. The microarray and
RT-PCR analyses indicated that this gene is likely induced by Cd
stress. Metallothioneins (MTs) are a group of low molecular weight,
cysteine-rich, metal-binding proteins that are involved in the
response to heavy metal stress in many plant species . High
concentrations of metal ions disrupt the water balance of a cell and,
thus, decrease the activity of several important metabolic enzymes,
a situation that results in oxidative stress . MTs help to keep
metal ion concentrations in plant cells in a stable range by forming
complexes with the metal and reducing their bioavailability. MTs
may also scavenge oxygen free radicals and decrease injury in
oxidative tissues. The toxicity of Cd is primarily associated with its
binding to sulfhydryl groups in proteins or its preferential binding
to proteins in place of Zn, thereby, inhibiting enzyme activity or
protein function . The induced expression of MTs in our study
indicated that, under Cd stress conditions, rice roots might produce
high amounts of MT proteins.
Many metabolism- and protein synthesis-related genes were
also affected by Cd stress. In plants, Cd can cause various delete-
rious effects, such as the inhibition of photosynthesis, respiration
and nitrogen metabolism and a decrease of water and mineral
uptake . These Cd-induced metabolism-related changes in
plants lead to an inhibition of plant growth and, when the
concentrations are high enough, may result in death. Twelve
metabolism- and protein synthesis-related clones were isolated in
our experiment, including genes encoding asparagine synthetase,
farnesyl-pyrophosphate synthetase, cathepsin B-like cysteine
protease and formate dehydrogenase. The altered expression of
these enzymes emphasizes the multiple cellular and physiological
aspects disrupted by Cd exposure.
There have also been reports concerning transcriptional proﬁles
 and proteomic analyses  of rice roots in response to Cd
treatment. A time-course expression analysis has been performed
using DNA microarray analysis, and this research showed that some
transporter genes, P450 family proteins, heat shock proteins,
glutathione S-transferases and genes encoding proteins involved in
signal transduction were strongly induced, whereas genes involved
in photosynthesis were mainly down-regulated, under 10
M. Zhang et al. / Plant Physiology and Biochemistry 50 (2012) 79e86 83
stress for 3 h . Comparing the results of the above study with
our research revealed some common effects, such as the induction
of transporter genes, heat shock proteins, and proteins involved in
signal transduction, whereas some genes involved in metabolism
were down-regulated. A proteomic analysis has also identiﬁed
some proteins that were up- or down-regulated following 100 mM
treatment for 24 h, mainly proteins involved in oxidative
stress and related mechanisms .
As opposed to what may occur in different plant organs, the
roots respond directly to heavy metals (including Cd) that are
present in the rhizosphere. The heavy metals are absorbed through
symplastic or apoplastic pathways in the roots and then enter the
root xylem where they are translocated to the aerial parts .
Accordingly, the gene responses in roots were most relevant to our
research, and we therefore chose rice roots for our Cd exposure-
induced transcriptional analysis and identiﬁed several groups of
differentially expressed genes that appear to be related to the
response to Cd stress in rice. Some membrane proteins and stress-
related proteins, including HSP, a monosaccharide transporter, CIPK
and MTP, stand out as likely subjects for future research. Under-
standing the function of these genes and how they are regulated in
response to Cd toxicity and nutrient availability may have signiﬁ-
cant agricultural applications. Experiments are currently underway
to determine whether the over-expression or under-expression of
these genes in transgenic plants can yield instructive or useful
changes in Cd accumulation in the seeds.
4. Materials and methods
4.1. Plant material and Cd treatment
Rice (Oryza sativa L., sp. Japonica, var. Nipponbare) seeds were
germinated in trays containing ¼ ionic strength Murashige and
Skoog (MS) liquid medium. The trays were held at a constant
C) in the dark for two days and then were
transferred into an incubator (25
C, with a 16-h/8-h light/dark
photoperiod). Fourteen-day-old seedlings were carefully removed
from the culture solution and divided into two groups. One group of
seedlings was transferred to a separate container with the roots
submersed in a ¼ MS liquid medium containing 0.5 mM CdCl
second group of seedlings was transferred to ¼ MS liquid medium
without Cd. The roots of each group were harvested after 48 h for
SSH library construction. For the RT-PCR analysis, the 14-day-old
rice seedlings were taken from the liquid medium and evenly
distributed to ¼ MS liquid medium with or without (control)
0.5 mM CdCl
, and the roots were harvested at different time
intervals (0, 24 and 48 h) for RT-PCR. Total RNA for reverse tran-
scription was extracted three times separately for the RT-PCR.
4.2. RNA extraction and library construction
Total root RNA was isolated with the TRIzol reagent (Invitrogen).
Poly (A)þ RNA was isolated from the total RNA using the FastTrack
MAG mRNA Isolation Kit (Invitrogen). The mRNA yield and quality
were determined by spectrophotometry at 260 and 280 nm. Using
the SMARTÔ PCR cDNA Synthesis Kit (Clontech), total mRNA
(200 ng) was reverse transcribed (to cDNA) at 42
C for 1 h in the
presence of both cDNA synthesis (CDS) primers and SMART II
oligonucleotides. Tricine-EDTA buffer (90
l) was added to 10
the ﬁrst-strand reaction product and incubated at 72
C for 7 min.
Diluted cDNA (1
l) was then added to a 100
l reaction mixture for
long-distance (LD) PCR. The mixture was pre-heated at 95
1 min to denature the cDNA. The LD PCR was performed at 95
15 s, 65
C for 30 s and 68
C for 3 min using a TETRAD2 Peltier
Thermal Cycler (MJ Research) for 17 cycles. The resulting PCR
product was used for the SSH cDNA library construction and
unsubtracted virtual northern blotting.
The SSH was performed with the PCR Select cDNA Subtraction
Kit (Clontech). Mirror orientation selection (MOS) was completed
as described by Rebrikov . After MOS hybridization using the
SmaI-digested secondary PCR products, MOS PCR was conducted
using the NP2Rs primer (shorter than the NP2R primer used in the
secondary PCR of the SSH, 5
). The sizes of
the MOS PCR products ranged from 200 to 1000 bp, with the
majority approximately 500 bp long.
The cDNA fragments obtained by MOS were ligated into
a pGEM-T vector (Promega) and transformed into JM109 (Escher-
ichia. coli) competent cells for differential screening. Two different
subtractive cDNA libraries were constructed: the control (no Cd)
and treated plants (0.5 mM Cd) were used alternately as the driver
and tester. The cDNA from Cd-treated roots was used as the “tester”
cDNA pool, and the cDNA from untreated (control) roots was used
as the “driver” pool in the forward SSH experiment to isolate the
Cd-induced genes; the cDNA roles were switched in the reverse
SSH experiment to isolate the suppressed genes.
To ensure the accuracy of the SSH assay, we analyzed the efﬁ-
ciency of ligation to verify that at least 25% of the tester cDNA
fragments had adaptors on both ends. We chose a housekeeping
gene, rice actin7 (accession number: X15863) as a negative control
to estimate the efﬁciency of subtraction. The forward and reverse
actin7 primers used in this experiment were 5
4.3. Microarray as a form of reverse northern blotting
Additional analysis using a microarray dot blot was conducted
on 1700 randomly selected clones, which were cultured in 1 ml of
LB medium containing ampicillin overnight on a shaker table
(200 rpm, 37
C, 16e20 h). An aliquot of the culture (1
l) was used
for the PCR with the NP2Rs primer in a 20
l of reaction mixture. A
small volume (0.4
l) of each of the PCR products was spotted onto
Immobilon-Nyþ transfer membranes (Millipore) in a 0.25 cm
The membrane was then placed on wet ﬁlter paper, rinsed with
0.4 M NaOH for 5 min to denature the DNA and immediately rinsed
with 2x SSC buffer to remove any residual NaOH. The membrane
was heated at 80
C for 2 h for crosslinking, and the membrane was
then hybridized with speciﬁc probes or stored at room temperature
for future use.
Digoxigenin (DIG)-labeled DNA probes were produced using
a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche)
according to the manufacturer’s instructions. To eliminate any false
positive clones that had been found in previous studies ,we
chose four kinds of cDNA as the templates to prepare the probes for
hybridization. Screening was conducted as described previously
. Putative differential clones were selected for DNA sequencing.
4.4. Sequence analysis and Blast search
The nucleotide sequences of the selected cDNA clones were
determined using an ABI PRISM 3730 Genetic Analyzer (Perkin
Elmer), and sequence homologies were analyzed by sequence
comparison with entries in the GenBank database (http://www.
4.5. RT-PCR analyses
To conﬁrm the results of the cDNA microarray, we randomly
selected 11 up-regulated cDNAs and 9 down-regulated cDNAs to
analyze their expression patterns by RT-PCR. The primer sequences
used in the RT-PCR are listed in Supplementary data 1. The relative
M. Zhang et al. / Plant Physiology and Biochemistry 50 (2012) 79e8684
abundances were determined utilizing the housekeeping gene, rice
actin7, as an internal control. Total root RNA from three growth
stages (0, 24 and 48 h after initiation of the experiment) were used,
and ﬁrst-strand cDNAs were synthesized from DNaseI-treated
total RNA using Superscript II reverse transcriptase (Invitrogen)
according to the manufacturer’s instructions. RT-PCR was per-
formed using an optical 96-well plate with an ABI PRISM 7500 real
time PCR system (Applied Biosystems). Rice actin7 was used as an
internal control for the RT-PCR analysis. Each reaction contained
l of 2x SYBR Green Master Mix reagent (Applied Biosystems),
l of the cDNA sample, and 200 nM each of the gene-speciﬁc
primers in a ﬁnal volume of 25
l. The thermal cycle used was as
C for 3 min; 45 cycles of 95
C for 30 s, 60
C for 30 s,
C for 40 s. All of the primers for the candidate genes were
designed by Primer 3 (http://frodo.wi.mit.edu/). The speciﬁcity of
the reactions was checked by melting curve analysis, and three
replicates of each cDNA sample were used for the analysis.
This project was ﬁnancially supported by grants from the China
National Natural Sciences Foundation (Grant no. 30900892),
International Foundation for Science (C/4787-1), Knowledge Inno-
vation Project for Young Scientist of South China Botanical Garden,
CAS (200724) to Mei Zhang and the CAS ‘100 Talents’ Program to
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