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Genome-wide identification of rice class i
metallothionein gene: Tissue expression
patterns and induction in respons....
Article in Functional & Integrative Genomics · October 2012
DOI: 10.1007/s10142-012-0297-9 · Source: PubMed
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Funct Integr Genomics (2012) 12:635–647
DOI 10.1007/s10142-012-0297-9
ORIGINAL PAPER
Genome-wide identification of rice class I metallothionein
gene: tissue expression patterns and induction in response
to heavy metal stress
Neelam Gautam & Pankaj Kumar Verma &
Shikha Verma & Rudra Deo Tripathi &
Prabodh Kumar Trivedi & Bijan Adhikari &
Debasis Chakrabarty
Received: 23 April 2012 / Revised: 3 September 2012 / Accepted: 24 September 2012 / Published online: 10 October 2012
# Springer-Verlag Berlin Heidelberg 2012
Abstract Metallothioneins (MTs) are members of a family
of cysteine-rich low molecular weight polypeptides which
play an important role in heavy metal detoxification and
homeostasis of intracellular metal ions in plant. Though MT
genes from some selected plants have been characterized
with respect to their protein sequences, kinetic properties
and tissue-specific localization, no detailed study has been
carried out in rice. Here, we present genome-wide identification, structural and expression analyses of rice MT gene
family. Our analysis suggests presence of 11 class I MT
genes in rice genome (Release 7 of the MSU Rice Genome
Annotation Project) which are differentially expressed during growth and development, in various tissues and during
biotic and abiotic stresses. Our analyses suggest that class I
MT proteins in rice differ in tissue localization as well as in
heavy metal coordination chemistry. We also suggest that
some MTs have a predominant role in detoxification of As (V)
in arsenic-tolerant rice cultivars. Our analysis suggests that
apart from transcriptional regulation, post-transcriptional alternative splicing in some members of this family takes place
during growth and development, in various tissues and during
biotic and abiotic stresses.
Electronic supplementary material The online version of this article
(doi:10.1007/s10142-012-0297-9) contains supplementary material,
which is available to authorized users.
N. Gautam : P. K. Verma : S. Verma : R. D. Tripathi :
P. K. Trivedi : D. Chakrabarty (*)
CSIR-National Botanical Research Institute,
Rana Pratap Marg,
Lucknow 226 001 UP, India
e-mail: debasis1972@rediffmail.com
B. Adhikari
Rice Research Station, Department of Agriculture,
Chinsurah,
Hooghly, Government of West Bengal 712 102, India
Keywords Alternative splicing . Arsenic . Heavy metal .
Rice . Stress . Metallothionein
Introduction
Heavy metal ions, such as cadmium (Cd), arsenic (As),
chromium (Cr), lead (Pb), zinc (Zn) and mercury (Hg), are
highly reactive and toxic to living cells (Hall 2002). Plants
have evolved mechanisms such as chelation and sequestration of heavy metals by particular ligands to counter this
problem (Hall 2002). The two best-characterized heavy
metal-binding ligands in plant cells are the phytochelatins
(PCs), a family of enzymatically synthesized cysteine-rich
peptides and metallothioneins (MTs). Metallothioneins
(MTs) are low-molecular-weight (7–8 kDa), cysteine-rich
(20–30 %), metal-binding proteins (Margoshes and Vallee
1957). These proteins are involved in essential-metal homeostasis and impart protection against heavy metal toxicity
by sequestration (Mir et al. 2004; Hassinen et al. 2009a;
Huang and Wang 2010), scavenging of reactive oxygen
species (Hussain et al. 1996), regulation of metalloenzymes and transcription factors (Andrews 2000; Bratić
et al. 2009), metabolism of metallo-drugs and response to
stress conditions. There are more than 50 plant MT-like
proteins included in various databases, about one third of
all known metallothioneins (Liu et al. 2000).
In plants, the first evidence for the role of MTs in Cu and
Cd tolerance was provided through functional complementation of MT-deficient yeast by two Arabidopsis MT genes
(Zhou and Goldsbrough 1994). Further evidence for the
function of various MTs in plants was elucidated using
knock-down and over-expression lines, tissue-specific and
metal-regulated expression, as well as from the characterisation of MT–metal complexes (Guo et al. 2003, 2008;
636
Domènech et al. 2007a, b; Freisinger 2007; Yuan et al.
2008; Bratić et al. 2009; Hassinen et al. 2009a, b). Studies
on the localisation of MTs in specific plant parts, tissues and
subcellular organelles have also provided the role of these
proteins in metal detoxification and plant development
(Nakajima et al. 1991).
Plant MTs have been classified into classes I, II and III
based on the arrangement of Cys residues (Cobbett and
Goldsbrough 2002). Most of the plant MTs are class I
proteins containing two smaller Cys-rich domains (CRD)
and a large spacer region devoid of this amino acid. In class
II MTs cysteine residues are grouped into three cysteine-rich
domains separated by 10 to 15 amino acid residues (Lane et
al. 1987; Zhou et al. 2005). Class III consists of phytochelatins, enzymatically synthesized peptides with a poly(γGlu-Cys)-glycine structure (Cobbett and Goldsbrough
2002).
Class I MTs can be further divided into different types
(type I–IV) and differ in the arrangement and distribution of
cysteine residues. Class I MTs have the capacity to bind
both physiological (such as zinc, copper and selenium) and
xenobiotic heavy metals (such as cadmium, mercury, silver
and arsenic) through the thiol group of its cysteine residues.
The organization/distribution of cysteine residues confers on
different MT isoforms the ability to bind and sequester
different metal ions for detoxification and homeostasis.
Further, the four types of MTs are known to express in
different plant organs (Cobbett and Goldsbrough 2002).
Expression of metallothionein genes is regulated by abiotic
stress including metals and plays an important role in metal
detoxification and homeostasis (Usha et al. 2007, 2009;
Huang and Wang 2009, 2010; Singh et al. 2011). In addition, some plant MT-2 genes have been expressed in E. coli
to determine its metal-binding properties (Tommey et al.
1991; Jin et al. 2006; Huang and Wang 2010). These functional capabilities of MTs allow them to play a role in
mobilization of metal ions from senescing leaves and the
sequestration of excess metal ions in trichomes.
In rice, class I MTs, especially OsMT1a (Os11g47809)
have been characterized and it has been suggested that it
plays the pivotal role in zinc homeostasis and drought
tolerance (Yang et al. 2009). Yuan et al. (2008) characterized
another MT from Oryza sativa (Indica variety), OsMT2b
(Os01g74300), and reported that its expression was downregulated by cytokinins and it was expressed in rice immature panicles, scutellum of germinating embryos and
primordium of lateral roots. Dong et al. (2010) characterized
the OsMT-I-4b (Os12g38051) gene promoter which
expresses in the roots and the buds with its activity highly
upregulated by abscisic acid (ABA), drought, dark and
heavy metals including Cu, Zn, Pb, Al, Co and Cd. In this
study, 11 class I MT genes identified in Release 7 of the
MSU Rice Genome Annotation Project were shown to be
Funct Integr Genomics (2012) 12:635–647
differentially expressed during plant growth, development
and during various stresses in order to establish a model for
the detoxification of heavy metals such as As (V).
Materials and methods
Database search and sequence analysis
Genes encoding MT proteins were identified by keyword,
domain name and BLASTP searches available at Rice
Genome Annotation Project (Release 7 of the MSU Rice
Genome Annotation Project). The model Metallothio_2 (accession PF01439.11) of the Pfam database was used as a
query to search the protein and nucleotide databases of
NCBI (The National Center for Biotechnology
Information) and the matching genes were confirmed by
previous searches in Release 7 of the MSU Rice Genome
Annotation Project. A self BLAST of these sequences followed by manual editing to remove redundancy finally
resulted in the identification of 11 class I MTs in rice
(Cobbett and Goldsbrough 2002).
Multiple sequence alignment analyses were performed
using ClustalX (version 1.83) programme (Larkin et al.
2007). The intron–exon structure was identified by aligning
cDNA sequences of rice MTs with their corresponding
genome sequences. All the sequenced contigs of japonica
cv Nipponbare have been physically constructed as pseudomolecules at TIGR (http://rice.plantbiology.msu.edu/), representing the 12 rice chromosomes. Each of the MT proteinencoding genes was positioned on these rice chromosome
pseudomolecules by the BLASTN search. Genes separated
by ≤5 genes were considered to be putative tandem duplicates (Jain et al. 2010; Kumar et al. 2011). Such putative
duplicates were analysed using online Vista Tools for
Comparative Genomics (Frazer et al. 2004) for further
analysis.
Plant Material and RNA isolation
The rice variety IR-64 was germinated and allowed to grow
for 5 day at 37 °C and then transferred to Hewitt solution for
growth. After 10 days of growth, seedlings of uniform size
and growth were treated with different concentrations of Cr
(K2Cr2O7), Cd (CdCl2), AsV (Na2HAsO4) and Pb [Pb
(NO3)2] (100 μM) under standard physiological conditions
of 16-h light (115 μmolm−2 s−1) and 8-h dark photoperiod at
25±2 °C for 24 h and 7 days. In another experiment, one
tolerant cultivar Triguna and one sensitive cultivar IET 4786
for As(V) were grown similarly as discussed above and
treated with 50 μM As (V). All the samples were ground
in liquid N2 and stored at −80 °C till further use. Total RNA
was extracted from the treated rice roots using the QIAGEN
Funct Integr Genomics (2012) 12:635–647
637
RNeasy Plant Maxi Kit (QIAGEN, MD). The yield and
RNA purity were determined spectrophotometrically
(NanoDrop, Wilmington, DE) and by formaldehyde–agarose gel electrophoresis. First strand cDNA was synthesised
using 5 μg purified total RNA and RevertAid First Strand
cDNA synthesis Kit (Fermantas, Life Sciences, USA).
at P<0.05, P<0.01 and P<0.001, respectively, according to
Duncan’s multiple range test to determine the significant
difference between treatments.
qRT-PCR analysis
In silico identification and analysis of rice metallothioneins
Total RNA and the first cDNA strand were prepared as described above. qRT-PCR was carried out using the primer pairs
listed in Supplementary Table S1 which were designed by
using Primique online software http://cgi-www.daimi.au.dk/
cgi-chili/primique/front.py. Specificity of each primer to its
corresponding gene was checked using the BLASTN program
of the NCBI. One microgram aliquots of cDNA were subjected
to each qRT-PCR reaction in a final volume of 20 μl containing
12.5 μl SYBR Green Master Mix Reagent (Takara, Japan) and
specific primers (Supplementary Table S1). qRT-PCR reactions
were carried out in a StepOne realtime PCR machine (Applied
Biosystems, USA) as described by Wu et al. (2011). Three
biological replicas were performed for each sample. To normalize the total amount of cDNA present in each reaction, the
actin was co-amplified as an endogenous control for calibration
of relative expression. The comparative Ct method (ΔΔCT
method) of relative gene quantification recommended by
Applied Biosystems (CA, USA) was used to calculate the
expression levels of different treatments.
Database search and sequence analysis resulted in the identification of 11 class I MTs in rice. The multiple sequence
alignments of full-length MT protein sequences showed that
N-terminal and C-terminal of these MTs are highly conserved having two CRD connected by a spacer characteristic
to all plant MTs (Fig. 1a). Out of all the MTs, one OsMT-IIIa (Os01g05585) is a duplicated form of OsMT-I-IIb
(Os01g05650), which does not contain one N-terminal
CRD. Rice genome may have lost this domain during evolution. Another MT located on chromosome 12, OsMT-I-If
(Os12g38300) also lacks C-terminal CRD. The localization
of MTs on the chromosomes depicted the clustering of five
genes on the 12 chromosome (Fig. 1b). It has been reported
that genes separated by ≤5 genes were considered to be
putative tandem duplicates (Jain et al. 2010; Kumar et al.
2011). Such putative duplicates were analysed using online
VISTA Tools for Comparative Genomics (Frazer et al. 2004)
which resulted in identifying two genes that are tandem
duplicates, OsMT-I-Ic (Os12g38010) and OsMT0I-Id
(Os12g38051) (Supplementary Fig. S1). Different MTs
identified with their gene name, gene length, open reading
frame length, protein length, chromosomal location and
other related information are provided in Table 1.
Microarray data analysis
The microarray data was collected for different rice tissues/
organs and developmental stages, including germinating
seedling (GS), seedling root (R), mature leaf (ML), leaf
(YL; leaf subtending the shoot apical meristem), shoot apical meristem (SAM) and various stages of panicle (P1–P6)
and seed (S1–S5) development (Jain et al. 2007; GSE6893).
GSE7951 (expression profiling of stigma), GSE6901 (expression data for stress treatment), GSE7256 (expression
data for virulent infection by Magnaporthe grisea) and
GSE10373 (expression data for interaction with the parasitic
plant Striga hermonthica) were selected for the analysis of
probe sets corresponding to rice MT gene families. The CEL
files were downloaded from the Gene Expression Omnibus
database at the National Center for Biotechnology
Information and analyzed using dChip software (Li and
Wang 2001).
Statistical analysis
All experiments were repeated three times with three replicates per treatment. Bars represent SD of means. *, ** and
*** indicate values that differ significantly from the control
Results
Expression of MTs in various tissues and developmental
stages
We studied the expression of different rice MTs using microarray data available at the GEO database under accession
numbers GSE6893 and GSE7951, respectively (Jain et al.
2007; Li et al. 2007). All the tissues/organs and developmental stages for which microarray data were analysed in
this study are shown in Fig. 2a. It is clear from our analysis
that MTs play differential role during different developmental stages. Out of 11 MTs, four are root specific (OsMT-I-If:
Os12g38300, OsMT-I-Ie:Os12g38290, OsMT-I-Id:
Os12g38051 and OsMT-I-Ic:Os12g38010). The validation
of differential gene expression of these selected MT genes in
root/shoot tissues by real-time PCR analysis showed very
good agreement with the microarray results (Fig. 2b).
OsMT-I-Ic gene showed highest expression in root tissue
followed by OsMT-I-If and OsMT-I-If. OsMT-I-Ic gene is
also root specific but its expression level is lower than the
other root-specific MTs (Fig. 3b). Seven MTs expressed
638
Funct Integr Genomics (2012) 12:635–647
a
CRD
CRD
b
Fig. 1 a, b Alignment of protein sequences of OsMT class I gene
family members by ClustalX software. Conserved cysteine-rich
domains (CRD) are shown in black line (a). Graphical representation
of the location of different MT class 1 genes (TIGR locus IDs of rice
MTs) on rice chromosomes (b). The chromosome number is indicated
at the bottom of each chromosome
during seed development (OsMT-1-IIa, OsMT-1-IIb, OsMT1-IIc, OsMT-1-Ia, OsMT-1-IIIa, OsMT-1-Ib and OsMT-1-If),
among them four MTs also expressed in leaves (OsMT-1IIa, OsMT-1-IIb, OsMT-1-IIIa and OsMT-1-Ib).
We also searched the probe IDs assigned for different MT
gene families. Interestingly, we observed that out of 11 MTs,
five MTs have multiple probe IDs for its single gene. There
are three different probe ID present in OsMT-I-IIb
(Os01g05650), OsMT-I-IIa (Os01g05585) and OsMT-I-IIc
(Os01g74300). In OsMT-I-IIb and OsMT-I-IIc, two probe
IDs represent CDS whereas the third one is present in the
intron region (Os.35005.1.S1_at and Os.12410.2.S1_at, respectively). Heat map of these genes under various developmental stages and tissues clearly suggests that transcript
containing intron is present in large amount in tissues during
panicle and seed development whereas the functional transcript (without intron) content decreases (Fig. 2a). Similarly,
OsMT-I-Id (Os12g38051) gene also has four different probe
set IDs and one of the probe set ID is from intronic region
(Os.54914.1.S1_at) showing differential expression pattern.
This indicates that promoter during S5 stage is very active
and transcribes pre-mRNA (with intron). Surprisingly, processing of intron in OsMT-I-IIb (Os01g05650) and OsMT-IIIa (Os01g05585) is completely reverted in OsMT-I-IIc
(Os01g74300) suggesting that mRNA processing machinery is tightly regulated suggesting functional redundancy for
these MTs (Fig. 2a).
Expression of MTs during different heavy metal stress
To study responsiveness of MT gene family to different
heavy metal treatments in rice, real-time PCR was performed with total RNA isolated from the roots and shoots
of IR-64 rice treated with Pb, As(V), Cr and Cd (Figs. 3 and
Funct Integr Genomics (2012) 12:635–647
639
Table 1 Class I metallothionine genes (OsMTs) in rice
Sl no. Locus ID
MT type
cDNA
No. of Protein Mol wt pIf
Genea
length (bp) lengthb (bp) intronsc lengthd (kDa)e
Chromosome NCBI Acch.
no.g
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
OsMT-I-IIa
OsMT-IIb
OsMT-I-IIc
OsMT-I-Ia
OsMT-I-IIIa
OsMT-I-Ib
OsMT-I-Ic
OsMT-I-Id
OsMT-I-Ie
OsMT-I-IId
OsMT-I-If
1,047
1,952
1,007
585
2,217
838
999
8,444
981
1,026
652
1
1
1
3
5
11
12
12
12
12
12
195
249
243
219
198
225
237
240
231
237
240
2
2
3
2
3
2
3
3
3
2
3
64
82
81
73
66
75
79
80
77
79
80
6.307
7.85
7.6
7.13
6.85
7.48
7.59
7.71
7.54
7.64
7.72
4.79
4.5
4.29
4.85
4.68
6.88
5.68
6.42
4.53
5.62
8.61
NP_001042028
NP_001042028/D15602
NP_001045552/AB002820
NP_001049782/AK059587
A3B0Y1/AF001396
NP_001068544/U43529
NP_001067063
Q2QNE8/BE039194
ABA99660
ABA99658/AK103445
ABG22056/BE039221
Length of genomic fragment TIGR release 7
d
Length (no. of amino acids) of the deduced polypeptide
e
Molecular weight of the deduced polypeptide in kilodalton
f
Isoelectric point of the deduced polypeptide
g
Chromosome number on which the gene is present
h
Locus ID from Release 7 of the MSU Rice Genome Annotation Project was BLASTN in NCBI
S5
P6
P3
P4
P5
P1
P2
Ovary
Stigma
R
Fig. 2 a, b Expression profiles
of OsMT class I gene family in
different tissues and
developmental stages using
Affymetrix Rice Genome
Array. The profiles in
germinating seedlings, seedling
roots (R), mature leaves (ML),
young leaves (YL; leaf
subtending the shoot apical
meristem), shoot apical
meristem (SAM), stigma, ovary
and various stages of panicle
(P1–P6) and seed (S1–S5)
development are presented by
cluster display. The colour scale
(representing log signal values)
is shown at the bottom (a).
Quantitative real-time PCR
analysis to study the rootspecific expression pattern of
MT gene family members (b)
significantly increased expression in roots (OsMT-I-IIa after
24 h and OsMT-I-IIIa after 7 days) but others showed
YL
ML
SAM
4). In Pb treatment, only two genes (OsMT-I-IIa:
Os01g05585 and OsMT-I-IIIa:Os05g11320) showed
S4
No. of introns within cDNA
S3
Length of cDNA
c
S2
b
S1
a
Os01g05585
Os01g05650
Os01g74300
Os03g17870
Os05g11320
Os11g47809
Os12g38010
Os12g38051
Os12g38290
Os12g38270
Os12g38300
a
Os.35005.1.S1_x_at
Os.35005.1.S1_at
Os.46398.1.S1_s_at
OsMT-1-IIa
Os.46398.1.S1_s_at
Os.5523.1.S1_x_at
Os.35005.1.S1_at
OsMT-1-IIb
Os.12410.1.S1_x_at
Os.12410.2.S1_at
Os.12410.3.S1_x_at
OsMT-1-IIc
OsAffx.7826.2.S1 at
OsAffx.7826.1.S1_at
Os.54914.1.S1_at
Os.37783.2.S1_x_at
Os.37783.1.S1 a at
OsAffx.32080.1.S1_at
Os.17004.1.S1_at
OsMT-1-Ia
OsMT-1-IIIa
OsMT-1-Ib
OsMT-1-IId
OsMT-1-If
b
900
Relative transcript level
(Genes/Actin)
800
SHOOT
ROOT
700
600
500
400
300
200
100
0
OsMT-I-Ie OsMT-I-Id OsMT-I-If
OsMT-I-Ic
OsMT-1-Ic
OsMT-1-Id
OsMT-1-Ie
Root
15
10
5
Bc
0
300
250
200
150
100
50
0
140
120
100
80
60
40
20
0
c
B
A
B
B
a
a
b
c
A
a
b
b
B
B
b
2.5
2
1.5
a
300
200
b
c
100
c
0
Control
Pb
As
Cr
Cd
B
1
b
b
0.5
B
b
B
Cc
0
30
15
b
A
A
b
10
0
c
Cd
B
C
A
12
10
B
8
6
4
2
A
6
5
B
4
B
3
2
C
1
c
0
C
C
a
a
a
a
a
0
A
A
a
8
b
6
b
4
2
B
Cc
0
D d
25
A
20
a
15
b
10
5
B
c
C
0
Control
a
b
b
Cc
10
20
5
7
12
a
25
1000
400
A
Relative transcript level
(OsMT-I-IIIa/Actin)
b
a
Relative transcript level
(OsMT-I-IIb/Actin)
Shoot
20
3
Relative transcript level
(OsMT-I-IId/Actin)
a
a
Relative transcript level
(OsMT-I-Ib/Actin)
25
Relative transcript level Relative transcript level
(OsMT-I-IIa/Actin)
(OsMT-I-Ia/Actin)
Funct Integr Genomics (2012) 12:635–647
Relative transcript level
(OsMT-I-Ic/Actin)
Relative transcript level
(OsMT-I-If/Actin)
Relative transcript level
(OsMT-I-Ie/Actin)
Relative transcript level
(OsMT-I-Id/Actin)
Relative transcript level
(OsMT-I-IIc/Actin)
640
Pb
B
d
d
As
Cr
Cd
800
600
400
200
b
0
control
Pb
As
Cr
Cd
Fig. 3 Quantitative real-time PCR analysis to study the expression
pattern of MT gene family members exposed to various heavy metal
stresses after 24 h. After 10 days of growth, seedlings of uniform size
and growth were treated with different concentrations of Cr (K2Cr2O7),
Cd (CdCl2), AsV (Na2HAsO4) and Pb [Pb(NO3)2] (100 μM) under
standard physiological conditions of 16-h light (115 μmolm−2 s−1) and
8-h dark photoperiod at 25±2 °C for 24 h and 7 days. The grey bars
represent root and black bars represent shoot. Y-axis represents relative
mRNA level in stressed or treated samples as compared to control
samples and various treatments are given on X-axis, namely lead (Pb),
arsenate (AsV), chromium (Cr) and cadmium (Cd). Actin expression
was used as internal control each time. The error bars indicate standard
error of mean values for three biological replicates. Three technical
replicates have been employed for each biological replicate
decreased expression revealing that Pb responsive MT
genes have different induction kinetics. The transcript levels
of 11 genes continuously increased throughout the time
course for other heavy metal [As(V), Cr and Cd] treatments.
Two MTs, OsMT-I-Ie and OsMT-I-IIa (Os12g38290 and
Os01g05585, respectively) were highly upregulated in roots
a f t e r 24 h A s ( V ) t r e a t m e n t w h e r e a s O s M T-I- Ib
(Os11g47809), OsMT-I-IId (Os12g38270), OsMT-I-IIb
(Os01g05650) and OsMT-I-IIIa (Os05g11320) showed
higher accumulation in shoots. Interestingly OsMT-I-Ia
(Os12g38051) showed no change in expression after 24 h
treatment, however was significantly upregulated after 7 day
of As(V) treatment. After 7 days, ten out of 11 MTs were
upregulated especially in roots except OsMT-I-Ib
(Os11g47809) which was upregulated both in shoots and
roots after As(V)-treatment. In case of Cr, OsMT-I-Id,
OsMT-I-If and OsMT-I-Ic (Os12g38051, Os12g38300 and
Os12g38010, respectively) were downregulated after 24 h
whereas all the MTs were upregulated after 7 days of treatment. However, after 7 days of Cr-treatment OsMT-I-Ic
(Os12g38010) showed sharp increase in their transcript
level indicating that this MT is highly responsive to Cr
and thus is Cr specific. Interestingly, OsMT-I-Ib, OsMT-IIIb, OsMT-I-IIIa and OsMT-I-IId (Os11g47809, Os01g5650,
Os05g11320 and Os12g38270, respectively) showed higher
level of expression in shoots whereas OsMT-I-Ia and OsMTI-IIa (Os03g17870 and Os01g05585, respectively) showed
higher expression level both in shoots and roots of Crtreated rice seedlings. This suggests that Cr strongly regulates the expression pattern of MTs as compared to other
heavy metals. In the Cd treatment, out of 11 MTs, seven
MTs, OsMT-I-IIc, OsMT-I-Ia, OsMT-I-IIIa, OsMT-I-IIa,
OsMT-I-IIb, OsMT-I-If and OsMT-I-IId (Os01g74300,
Os03g17870, Os05g11320, Os01g05585, Os01g05650,
40
20
0
b
A
Cc
B
C
a
800
a
600
400
b
200
c
A
B
0
1400
1200
1000
800
600
400
200
0
a
b
c
A
A
d
2500
a
2000
1500
b
1000
500
c
0
Control
d
Pb
A
B
As
Cr
c
Cd
Relative transcript level
(OsMT-I-IIIa/Actin)
b
a
40
30
20
10
0
b
A
B
Cc
Bc
250
Relative transcript level
(OsMT-I-IIb/Actin)
60
50
a
200
150
100
b
b
50
0
c
c
A
A
A
20
A
15
B
a
10
a
C
5
b
D
b
D c
0
Relative transcript level
(OsMT-I-IId/Actin)
Root
Relative transcript level Relative transcript level
(OsMT-I-IIa/Actin)
(OsMT-I-Ia/Actin)
80
a
Shoot
Relative transcript level
(OsMT-I-Ib/Actin)
120
100
641
Relative transcript level
(OsMT-I-Ic/Actin)
Relative transcript level
(OsMT-I-If/Actin)
Relative transcript level
(OsMT-I-Ie/Actin)
Relative transcript level
(OsMT-I-Id/Actin)
Relative transcript level
(OsMT-I-IIc/Actin)
Funct Integr Genomics (2012) 12:635–647
50
a
40
30
b
b
20
A
10
0
Bd
B
A
A
c
250
a
200
150
100
50
0
400
350
300
250
200
150
100
50
0
b
c
A
Bc
d
a
b
b
B
c
Control
Pb
A
C
As
Cr
Cd
500
a
400
300
200
100
b
0
control
Pb
As
Cr
Cd
Fig. 4 Quantitative real-time PCR analysis to study the expression
pattern of MT gene family members exposed to various heavy metal
stresses after 7 days. After 10 days of growth, seedlings of uniform size
and growth were treated with different concentrations of Cr (K2Cr2O7),
Cd (CdCl2), AsV (Na2HAsO4) and Pb [Pb(NO3)2] (100 μM) under
standard physiological conditions of 16-h light (115 μmolm−2 s−1) and
8-h dark photoperiod at 25±2 °C for 24 h and 7 days. The grey bars
represent root and black bars represent shoot. Y-axis represents relative
mRNA level in stressed or treated samples as compared to control
samples and various treatments are given on X-axis, namely lead (Pb),
arsenate (AsV), chromium (Cr) and cadmium (Cd). Actin expression
was used as internal control each time. The error bars indicate standard
error of mean values for three biological replicates. Three technical
replicates have been employed for each biological replicate
Os12g38300 and Os12g38270, respectively) were highly
upregulated in roots. Only OsMT-I-Ie (Os12g38290)
showed shoot-specific expression pattern and OsMT-I-IIIa
(Os05g11320) was highly expressed both in shoot and root
after 24 h Cd treatment. After 7 days of Cd treatment,
similar expression pattern was noticed except for OsMT-IIIb (Os01g05650) and OsMT-Id (Os12g38051).
cultivar as compared to Azucena indicating their role in
AsV tolerance (Fig. 5a). In all the cases studied above, it
is clear that alternative splicing plays an important role in
regulating some of the MTs expression during different
stresses.
Our previous study on large-scale screening of rice germplasm for grain arsenic level and sensitivity to arsenic (Tuli
et al. 2010; Tripathi et al. 2012) suggests that two cultivars
(Triguna and IET 4786) are contrasting in their arsenic
sensitivity (Tripathi et al. 2012; Rai et al. 2011). To study
the expression pattern of rice MT genes that are highly
expressed during As (V) stress (OsMT-I-IIc, OsMT-I-IIa,
OsMT-I-Ic, OsMT-I-Ie and OsMT-I-IId), these two cultivars,
Triguna (tolerant) and IET 4786 (sensitive), were grown in
50 μM As (V) for 7 days and studied expression through
qRT-PCR. Results of expression in roots showed the similar
results as of Bala and Azucena. OsMT-I-IIa, OsMT-I-IIc and
OsMT-I-IId showed higher expression pattern in tolerant
Expression of MTs in contrasting lines of As-tolerant rice
cultivars
Expression analysis from rice varieties Azucena (arsenate
sensitive) and Bala (arsenate tolerant) grown in the presence
or absence of 13.3 μm sodium arsenate for 7 days was
studied by Norton et al. (2008). We also analyzed the
expression pattern of the 11 MTs in the same database and
observed that OsMT-I-IIc, OsMT-I-Ic, OsMT-I-Id, OsMT-I-Ie
and OsMT-I-IId genes are highly upregulated in tolerant
642
Funct Integr Genomics (2012) 12:635–647
Relative transcript level
(OsMT-I-IIa/Actin)
Bala (AsV)
Os.46398.1.S1_s_at
Os.5523.1.S1_x_at
Os.35005.1.S1 at
OsMT-1-IIb
Os.12410.1.S1_x_at
Os.12410.2.S1_at
Os.12410.3.S1 x at
OsMT-1-IIc
OsAffx.7826.2.S1 at
OsMT-1-Ic
OsAffx.7826.1.S1_at
Os.54914.1.S1_at
Os.37783.2.S1_x_at
Os.37783.1.S1_a_at
OsAffx.32080.1.S1_at
Os.17004.1.S1 at
2.5
*
2
1.5
*
2
1.5
1
1
0.5
0.5
0
2.5
0
OsMT-1-Id
**
3
2.5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
**
**
Relative transcript level
(OsMT-I-Id/Actin)
OsMT-1-IIa
**
3
*
*
2
1.5
1
0.5
IET4786 C
IET4786 AS(V)
0
TRIGUNA C TRIGUNA AS(V)
Relative transcript level
(OsMT-I-IId/Actin)
Os.35005.1.S1_x_at
Os.35005.1.S1_at
Os.46398.1.S1 s at
Relative transcript level
(OsMT-I-IIc/Actin)
3.5
3.5
Relative transcript level
(OsMT-I-Ie/Actin)
Azucena (AsV)
b
Bala (C)
Azucena (C)
a
OsMT-1-Ie
OsMT-1-Ia
OsMT-1-IIIa
OsMT-1-Ib
OsMT-1-IId
OsMT-1-If
9
7
*
6
5
4
3
2
1
0
IET4786 C
Fig. 5 a, b Expression profile of OsMT class I gene family in rice
varieties Azucena (arsenate-sensitive) and Bala (arsenate-tolerant)
grown in the presence or absence of 13.3 μm sodium arsenate for
7 days. The colour scale (representing log signal values) is shown at
cultivar Triguna as compare to sensitive cultivar IET 4786
(Fig. 5b).
Expression of MTs during cold, drought and salinity
The role of MTs is well documented in the protection of
plants from abiotic stresses (Yang et al. 2009). Considering
the role of MTs in abiotic stresses, microarray data available
under series accession no. GSE6901 (Jain et al. 2007) for
abiotic stresses (desiccation, salt and cold) were analysed.
This analysis showed that most of the MTs are differentially
expressed in different abiotic stresses. OsMT-I-IIa and
OsMT-I-IIb are highly upregulated in both desiccation and
salt stress. However, except these two MTs others are highly
downregulated in desiccation stress. Similarly, OsMT-I-IId
is highly upregulated in salt stress. It is also interesting to
observe that alternative splicing plays an important role for
regulating the expression pattern of OsMT-I-Id as processed
transcript is not accumulating in drought stress (higher accumulation of pre-mRNA in drought stress) whereas the
final processed transcript is accumulating in higher amount
in cold stress (Fig. 6).
**
8
IET4786 AS(V)
TRIUNA C
TRIGUNA AS(V)
the bottom (a). Quantitative real-time PCR analysis to study the rootspecific expression pattern of selected MT gene family members in
Triguna and IET 4786 rice varieties treated with 50 μM of arsenate
(AsV) for 7 days (b)
C CS DS SS
Os.35005.1.S1_x_at
Os.35005.1.S1_at
Os.46398.1.S1_s_at
OsMT-1-IIa
Os.46398.1.S1_s_at
Os.5523.1.S1_x_at
Os.35005.1.S1_at
OsMT-1-IIb
Os.12410.1.S1_x_at
Os.12410.2.S1_at
Os.12410.3.S1_x_at
OsMT-1-IIc
OsAffx.7826.2.S1_at
OsMT-1-Ic
OsAffx.7826.1.S1_at
Os.54914.1.S1_at
OsMT-1-Id
Os.37783.2.S1_x_at
Os.37783.1.S1_a_at
OsAffx.32080.1.S1_at OsMT-1-Ie
Os.17004.1.S1_at
OsMT-1-Ia
OsMT-1-IIIa
OsMT-1-Ib
OsMT-1-IId
OsMT-1-If
Expression of MTs during biotic stresses
Ascomycete fungus M. grisea causes serious and widespread diseases of rice blast. Recently, a transcriptome
Fig. 6 Expression profile analysis of OsMT class I gene family during
cold (CS), drought (DS), and salinity (SS) stresses. The colour scale
(representing log signal values) is shown at the bottom
Funct Integr Genomics (2012) 12:635–647
643
analysis of a fully susceptible infection of rice (cultivar
Nipponbare) by a compatible M. grisea isolate (FR13) was
performed to understand the molecular mechanism involved
in their interaction (Ribot et al. 2008). We analysed these
data to establish role of the rice MT gene family in this host–
pathogen interaction (Fig. 7). The data included microarray
analysis of 2-week-old rice seedlings (cultivar Nipponbare)
treated with M. grisea (virulent isolate FR13) spore suspension on gelatine or gelatine alone after 3 days [3 days postinoculation (dpi), without disease symptoms) and 4 days
(4 dpi, with disease symptoms] post inoculation. Our analysis suggests that OsMT-I-IId, OsMT-I-IIa and OsMT-I-IIb
genes are significantly upregulated after 4 dpi indicating
MTs role in biotic stress tolerance (Fig. 7).
Similarly, we also studied the expression profiles of rice
MT genes in roots of susceptible (IAC165) and highly resistant (Nipponbare) cultivars in response to infection with S.
hermonthica after 2, 4 and 11 dpi (Swarbrick et al. 2008).
Expression analysis of 11 MTs shows that OsMT-I-IIa, OsMTI-IIb, OsMT-I-Ia and OsMT-I-Ib genes are highly upregulated
in susceptible IAC165 in response to infection with rice roots
and hemiparasite (Fig. 7).
MPSS-based expression analysis of MT gene family
We also used the data from rice MPSS database (http://
mpss.udel.edu/in9311/) to quantify the expression of individual members of the MT gene family. MPSS technology
provides a quantitative measure of transcript accumulation
of virtually all the genes in a tissue sample in terms of the
Discussion
Plants have evolved multiple strategies to tolerate and response various stresses. Some of these strategies include
immobilization, exclusion, chelation and compartmentalization especially for heavy metal stress (Gasic and Korban
2007). It has been demonstrated that MTs and PCs are
important molecules for metal detoxification in plants
(Huang and Wang 2010; Shukla et al. 2012). Though information about these small proteins and their involvement in
Nipponbare
IAC165
Mg-Mock-3dpi
Mg-3dpi
Mg-Mock-4dpi
Mg-4dpi
Sh-Mock-2dpi
Sh-2dpi
Sh-Mock-4dpi
Sh-4dpi
Sh-Mock-11dpi
Sh-11dpi
Sh-Mock-2dpi
Sh-2dpi
Sh-Mock-4dpi
Sh-4dpi
Sh-Mock-11dpi
Sh-11dpi
Fig. 7 Differential expression
of OsMT class I gene family in
response to various biotic stress
conditions. The colour scale for
fold change values is shown at
the bottom. Numbers on each
panel represent days postinoculation (dpi)
number of small signature sequences corresponding to each
gene (Brenner et al. 2000). A survey of 46 rice MPSS
libraries representing different tissue samples as well as
biotic and abiotic stress conditions showed that all the MT
genes have corresponding 17-base signatures in these, suggesting that expression of different members of MTs vary in
different tissues and stresses (Fig. 8). OsMT-I-Ic, OsMT-I-Id,
OsMT-I-Ie and OsMT-I-If are root specific and they
expressed during different stressed conditions. This result
supports our microarray and q-RT-PCR data. Our MPSS
analysis also suggests OsMT-I-IIc is specifically expressed
in panicle and seed development which is well correlated
with the microarray analysis. However, OsMT-I-IIc is also
highly expressed in meristematic tissue. Otherwise, our
analysis of the transcript abundance of the MT gene family
from rice in different tissues and stress-specific libraries
revealed that they are differentially expressed which are well
correlated with our microarray and qRT-PCR data.
Os.35005.1.S1_x_at
Os.35005.1.S1_at
Os.46398.1.S1 s at
OsMT-1-IIa
Os.46398.1.S1_s_at
Os.5523.1.S1_x_at
Os.35005.1.S1 at
OsMT-1-IIb
Os.12410.1.S1_x_at
Os.12410.2.S1_at
Os.12410.3.S1 x at
OsMT-1-IIc
OsAffx.7826.2.S1_at
OsMT-1-Ic
OsAffx.7826.1.S1_at
Os.54914.1.S1_at
Os.37783.2.S1_x_at
Os.37783.1.S1_a_at
OsMT-1-Id
OsAffx.32080.1.S1_at
Os.17004.1.S1 at
OsMT-1-Ie
OsMT-1-Ia
OsMT-1-IIIa
OsMT-1-Ib
OsMT-1-IId
OsMT-1-If
644
Funct Integr Genomics (2012) 12:635–647
14000
90000
NYR
NPO
NSL
XC06
XR48
MR03
MS06
MC24
12000
12000
NRA
NOS
NDR
XC24
XS03
MR06
MS12
PLA
NST
NIP
NDL
XR03
XS06
MR12
MS24
PLW
NYL
NGS
NCR
XR06
XS12
MR24
MS48
PLC
NLA
NCA
NCL
XR12
XS24
MR48
MS96
NME
NSR
XC00
XR24
XS48
MS03
MC00
10000
10000
8000
8000
6000
6000
4000
4000
2000
2000
00
OsMT-I-IIb OsMT-I-IIc
OsMT-I-Ia OsMT-I-IIIa OsMT-I-Ib
OsMT-I-Ic
OsMT-I-Id
OsMT-I-Ie
OsMT-I-IId
OsMT-I-If
Fig. 8 Transcript abundance of OsMT gene family members in plant
growth and development and salinity, drought and cold libraries from
the MPSS database. Different libraries in the MPSS database were
analysed for the expression level of OsMT genes. NYR 14 days—
young roots, NRA 60 days—mature roots A, NST 60 days—stem,
NYL 14 days—young leaves, NYL 60 days—mature leaves A, NLA
60 days—mature leaves A, NME 60 days—crown vegetative meristemetic tissue, NPO mature pollen, NOS ovary and mature stigma, NIP
90 days—immature panicle, NGS 3 days—germinating seed, NCA
35 days—callus, NSR 14 days—young leaves stressed in 250 mM
NaCl for 24 h, NSL 14 days—young roots stressed in 250 mM NaCl
for 24 h, NDR 14 days—young roots stressed in drought for 5 days,
NDL 14 days—young leaves stressed in drought for 5 days, NCR
14 days—young roots stressed in 4C cold for 24 h, NCL 14 days—
young leaves stressed in 4C cold for 24 h, XC00 unwounded control—
Nipponbare Xa21-0h, XC06 mock treatment—6 h, XC24 mock treatment—24 h, XR03 X. oryzae-R—3 h, XR06 X. oryzae-R—6 h, XR12 X.
oryzae-R—12 h, XR24 X. oryzae-R—24 h, XR48 X. oryzae-R—48 h,
XS03 X. oryzae-S—3 h, XS06 X. oryzae-S—6 h, XS12 X. oryzae-S—
12 h, XS24 X. oryzae-S—24 h, XS48 X. oryzae-S—48 h, MR03
M.grisea-R—3 h, MR06 M. grisea-R—6 h, MR12 M. grisea-R—
12 h, MR24 M. grisea-R—24 h, MR48 M. grisea-R—48 h, MS03 M.
grisea-S—3 h, MS06 M. grisea-S—6 h, MS12 M. grisea-S—12 h,
MS24 M. grisea-S—24 h, MS48 M. grisea-S—48 h, MS96 M. grisea-S
—96 h, MC00 mock treatment—0 h, MC24 mock treatment—24 h,
PLA rice leaf, beet armyworm damaged, 24 h, PLW rice leaf, water
weevil damaged, 24 h, PLC rice leaf, mechanical damaged, 24 h
stress response is known in some plants, no detailed study
has been carried out in rice. The objectives of this study
were to determine the expression patterns of members of
class I OsMT gene family during plant growth and development and under different stresses especially during heavy
metal stresses with aim to select the most appropriate candidate genes which can be exploited for improving stress
tolerance in future.
In this study, 11 class I OsMT genes were identified in
Release 7 of the MSU Rice Genome Annotation Project
which is in agreement with earlier reports for presence of
multiple copies of rice MTs (Zhou et al. 2006). From our
study, it is clear that the number of class I OsMT genes
increased rapidly during the course of evolution and whole
genome duplication and/or tandem/segmental duplication
played an important role in the expansion of MT genes in
rice (Kumar et al. 2011).
Differential expression of class I OsMT genes in various
organs/developmental stages
The gene expression patterns can provide important clues
for the gene function. We studied expression of all the class I
OsMT genes in MPSS databases as well as microarray data
from 51 arrays representing 17 stages of development
throughout the life cycle of rice. The hierarchical cluster
analysis based on average log signal values as well as MPSS
signatures for 11 class I OsMT indicated that this gene
Funct Integr Genomics (2012) 12:635–647
family display diverse expression patterns. In our study, the
preferential expression of class I OsMT was found especially in roots, leaves and developing seeds indicating its specific role in developmental stages which was not reported
earlier (Zhou et al. 2006).
Role of class OsMT under different stresses
In the present study, we also established that class I MT
genes were differentially regulated by different heavy metal
stresses and might play a crucial role in different heavy
metal metabolism. Our result revealed that some of rice
MTs was regulated by a specific heavy metal only; however,
most of the class I MTs were regulated by more than one
heavy metal stress condition. Our study also indicates that
MTs (OsMT-I-IIa, OsMT-I-IIc and OsMT-I-IId) might play
very crucial role for detoxification of As (V) in tolerant rice
cultivars. There were plenty of evidence that MT plays a
role in heavy metal tolerance in fungi and animals (Hamer
1986). Although, it has been reported that expression of
some MT is strongly induced by Cu, Zn, Pb, Al, Co and
Cd in Arabidopsis and rice, the role of MTs in heavy metal
detoxification in plants remains to be established. Grispen et
al. (2009) overexpressed AtMT2b in Nicotiana tabacum and
found that the highest AtMT2b expressing line exhibited a
significantly decreased arsenic accumulation in roots with increased accumulation in shoots, while the total amount of
arsenic taken up remained unchanged indicating that AtMT2b
expression enhanced the arsenic root to shoot transport. In our
previous study, we also reported that three (Os12g38300;
Os12g38290 and Os12g38051) and two class I (Os12g38290
and Os12g38051) MTs were upregulated in As (V) and As (III)
stresses, respectively (Chakrabarty et al. 2009).
To understand the role of class I OsMT genes during
various abiotic stress conditions, their expression patterns
were investigated in rice seedlings subjected to desiccation,
salt and cold. Our results revealed that OsMT-I-IId gene
showed response to salt stress only, while others were responsive to two or more stress treatments. The role of MTs
is not so well documented in the protection of plants from
biotic (Shim et al. 2004; Choi et al. 1996) and abiotic
stresses (Yang et al. 2009). Xue et al. (2009) reported a
cotton MT (GhMT3a) was upregulated not only by high
salinity, drought and low temperature stresses, but also by
heavy metal ions, ABA, ethylene and reactive oxygen species (ROS) in cotton seedlings. They concluded that
GhMT3a could function as an effective ROS scavenger
and its expression could be regulated by abiotic stresses
through ROS signalling. Similarly, Yang et al. (2009) also
reported that a rice class I MT, OsMT1a (Os11g47809) plays
an important role in drought tolerance in rice. Microarray
data from two earlier studies on transcriptome analysis of
rice (Nipponbare) after infection with M. grisea and gene
645
expression profiling in roots of susceptible (IAC165) and
highly resistant (Nipponbare) cultivars after infection with
S. hermonthica revealed that OsMT-I-IIa, OsMT-I-IIb,
OsMT-I-IId, OsMT-I-Ia and OsMT-I-Ib genes showed response to biotic stress conditions. It has already been
reported that metallothionein was induced during M. grisea
infection in wild rice (Oryza minuta) (Shim et al. 2004).
Choi et al. (1996) reported a wound and pathogen inducible
metallothionein-like cDNA from Nicotiana glutinosa while
cloning plant disease resistance–response genes by subtractive hybridization. Induced expression MTs with other
stress-related genes which evoke stress responses have also
been observed during banana fruit ripening (Kesari et al.
2007) suggesting importance of MTs in defence response.
Our findings may lead in a way to develop counter defences
against biotic and abiotic stress factors.
Regulation of expression of class I OsMT genes:
post-transcriptional alternative splicing
Apart from transcriptional regulation, post-transcriptional alternative splicing in some members of this family takes place
during growth and development, in various tissues and various
abiotic and biotic stresses. Similar observations were also made
by Kumar et al. (2011) with alternative splicing of members of
sulphate transporter gene family in different tissues indicating
that alternative splicing plays an important role to modulate the
gene expression during different developmental stages in regulating the level of functional transcripts via a mechanism
termed regulated unproductive splicing and translation (Lewis
et al. 2003; Lareau et al. 2007). It has already been reported that
intron retention is involved in important plant processes, such
as floral development, where alternative splicing of
Arabidopsis FCA pre-mRNA regulates the switch from the
vegetative to the reproductive phase (Quesada et al. 2003;
Razem et al. 2006; Reddy 2007). Although an earlier report
indicated tissue-specific expression of rice MTs (EST database
of all MTs from NCBI including japonica and indica subspecies, Zhou et al. 2006) no information was given about alternative splicing which we argue is an important step in gene
regulation. Several reports demonstrate that alternative splicing
can be influenced by abiotic and biotic stresses (Kumar et al.
2011). It was suggested that genes required to ameliorate various stress responses respond to changing environmental conditions by evolving rapidly, and the acquisition of alternative
splice isoforms (in our case accumulation of pre-mRNA) may
provide an additional mechanism to facilitate such behaviour.
Acknowledgements The authors are thankful to Director, National
Botanical Research Institute, Lucknow for the facilities and for the
financial support from CSIR Network Project, New Delhi, India. NG
acknowledges the financial support from ICMR. PKV acknowledge the
financial support from CSIR. The authors declare that they have no
conflict of interest associated with this work.
646
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