Genome-wide expressional and functional analysis of
calcium transport elements during abiotic stress and
development in rice
Amarjeet Singh1,*, Poonam Kanwar1,*, Akhilesh K. Yadav1,*, Manali Mishra1,2,*, Saroj K. Jha1,
Vinay Baranwal1, Amita Pandey1, Sanjay Kapoor1, Akhilesh K. Tyagi1,3 and Girdhar K. Pandey1
1 Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India
2 Max F. Perutz Laboratories, Vienna, Austria
3 National Institute of Plant Genome Research, New Delhi-110067, India
Keywords
abiotic stress; Ca2+ transporter;
development; expression; signal
transduction
Correspondence
G. K. Pandey, Department of Plant
Molecular Biology, University of Delhi South
Campus, Benito Juarez Road, Dhaula Kuan,
New Delhi-110021, India
Fax: +91-11-24111208
Tel: +91-11-24116615
E-mail: [email protected]
*These authors contributed equally to this
work.
(Received 31 July 2013, revised 18 October
2013, accepted 21 November 2013)
doi:10.1111/febs.12656
Ca2+ homeostasis is required to maintain a delicate balance of cytosolic
Ca2+ during normal and adverse growth conditions. Various Ca2+ transporters actively participate to maintain this delicate balance especially during abiotic stresses and developmental events in plants. In this study, we
present a genome-wide account, detailing expression profiles, subcellular
localization and functional analysis of rice Ca2+ transport elements.
Exhaustive in silico data mining and analysis resulted in the identification
of 81 Ca2+ transport element genes, which belong to various groups such
as Ca2+-ATPases (pumps), exchangers, channels, glutamate receptor homologs and annexins. Phylogenetic analysis revealed that different Ca2+
transporters are evolutionarily conserved across different plant species.
Comprehensive expression analysis by gene chip microarray and quantitative RT-PCR revealed that a substantial proportion of Ca2+ transporter
genes were expressed differentially under abiotic stresses (salt, cold and
drought) and reproductive developmental stages (panicle and seed) in rice.
These findings suggest a possible role of rice Ca2+ transporters in abiotic
stress and development triggered signaling pathways. Subcellular localization of Ca2+ transporters from different groups in Nicotiana benthamiana
revealed their variable localization to different compartments, which could
be their possible sites of action. Complementation of Ca2+ transport activity of K616 yeast mutant by Ca2+-ATPase OsACA7 and involvement in
salt tolerance verified its functional behavior. This study will encourage
detailed characterization of potential candidate Ca2+ transporters for their
functional role in planta.
Introduction
Calcium is one of the crucial ions regulating various
cellular processes in eukaryotes. Change in the cytosolic Ca2+ level is one of the primary responses to
external stimuli such as biotic and abiotic stresses
[1,2]. In response to various stimuli, generation of specific ‘Ca2+ signatures’ takes place which encompass
differences in Ca2+ oscillation frequency, amplitude
and location [3–6]. The specific changes in the Ca2+
Abbreviations
ACA, auto-inhibited calcium ATPase; CAX, H+/cation exchanger; CCX, cation/Ca2+ exchanger; CNGC, cyclic nucleotide gated channels; ECA,
ER type ATPases; EFCAX, EF-hand containing H+/cation exchanger; ER, endoplasmic reticulum; GFP, green fluorescent protein; GLR,
glutamate receptor homologs; PM, plasma membrane; qPCR, quantitative reverse transcriptase polymerase chain reaction; TMD,
transmembrane domain; TPC, two-pore channel.
894
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
signature provide vital clues about the nature and
intensity of the stimuli to the cell [5,6]. Specific Ca2+
signatures are generated due to spatial and temporal
Ca2+ fluxes in response to stimuli, which are mediated
by regulated activity of membrane localized calcium
channels and other calcium transporting elements. In
plants, there are three major classes of calcium transporters: channels, pumps (ATPases) and exchangers
[7,8]. Apart from these, some groups of proteins have
been identified in plants which non-specifically transport Ca2+ and include cyclic nucleotide gated channels
(CNGCs), glutamate receptor homologs (GLRs) and
annexins [5,9,10]. Channels can be broadly classified as
voltage dependent and voltage independent/ligand or
stretch activated based on their mode of activation
[11,12]. In animals, three types of Ca2+ channels have
been found which are voltage-dependent calcium channels (VDCC), receptor opened calcium channels and
mechanical stimulation gated channels [13] but molecular studies have suggested that very few homologous
Ca2+ channels are present in plants [12,14]. Two-pore
channel 1 (TPC1) is the only plant channel; it is partly
homologous to animal VDCC a-1 subunit and belongs
to animal L-type depolarization activated calcium
channel similar to yeast plasma membrane Ca2+ channel [15,16]. The Arabidopsis genome has a single TPC1
gene, which encodes for TPC protein and possesses
voltage activated channel activity [17]. Ca2+-ATPases/
pumps are structurally conserved in animals and plants
[13]. Two types of Ca2+-ATPases exist in animal,
namely plasma membrane (PM) type and endoplasmic
reticulum (ER) type, and similarly plant Ca2+-ATPases belong to P-type ATPases, which are further classified as P-IIA or ER type ATPases (ECAs) and P-IIB
or auto-inhibited calcium ATPases (ACAs), which are
analogous to animal PM type [18,19]. ACAs contain
an auto-inhibitory domain at the N-terminal, which is
known to bind the calmodulin that leads to activation
of the Ca2+ pump, whereas ECAs are devoid of this
N-terminal domain [18,20]. This family of plant Ca2+
pumps has been implicated in maintenance of ion
homeostasis by Ca2+ efflux from cytosol [21]. A total
of 14 members of type II Ca2+-ATPases have been
reported in the Arabidopsis genome, which includes
four ECA and 10 ACA members [19]. Exchangers are
another important group of Ca2+ transporters, utilizing energy generated from the flow of one ion down
its concentration gradient to mobilize Ca2+ ions
against their concentration gradient [8]. In yeast Ca2+/
H+ exchangers are found to be located at the vacuolar
membrane and regulate Ca2+/H+ transport. Plant
Ca2+/H+ exchangers are highly homologous to their
yeast counterparts except for some changes in N- and
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
Calcium transport elements in rice
C-terminal [22]. Therefore, Ca2+/H+ transport seems
to be conserved in yeast and plants. Na+/Ca2+
exchangers have been reported in different animal tissues and they are categorized into two main groups:
Na+/Ca2+ exchangers, which neither require nor
transport K+, and Na+/Ca2+- K+, which require and
transport K+; these two types are structurally very
similar [23]. In plants, exchangers are mainly divided
into the H+/cation exchanger (CAX) family (also
known as antiporters) and the cation/Ca2+ exchanger
(CCX) family [24]. The Arabidopsis genome encodes
six and four members of the CAX and CCX family,
respectively. Some specific EF-hand containing CAX
members known as EFCAX have also been identified
in Arabidopsis and these are unique to plants [24].
Apart from these major calcium transporters other elements such as annexins, CNGCs and GLRs also participate significantly in Ca2+ transport in plants.
Annexins are a multigene family in plants and they
bind to phospholipids in a Ca2+-dependent manner
[25,26]. Plant annexins harbor the sequence and motifs
important for ATPase/GTPase activity and calcium
channel activity. Moreover, annexins harbor various
post-translational modification sites, which might be
potential regulators for their Ca2+-dependent activity
[27,28]. CNGCs are a group of cation channels mediating Ca2+ influx into the cell after they get activated
by binding to ligands such as cAMP and cGMP [6].
This group of channels is composed of a large gene
family represented by 20 members in Arabidopsis
[29,30]. Similarly, GLRs homologous to animal ionotropic glutamate receptors function as a non-selective
cation channel. The Arabidopsis genome encodes for
20 GLRs, more than humans where only 11 members
are reported [31]. CNGC and GLRs are fundamentally
the same in structure in animals and plants [13].
In plants, various calcium transporters have been
implicated in a number of cellular processes such as
hormone responses, biotic and abiotic stress responses,
light signaling and development [32–37]. The majority
of these findings have been established in Arabidopsis
and other plant species but knowledge is minuscule
about the role of the Ca2+ transporting elements in
crop plants, especially rice. Moreover, there are very
few reports [8,37,38] which present expression analysis
of selected calcium transporters in rice and hardly any
that undertake a comprehensive identification, phylogenetic and expression analysis of the entire repertoire
(including ATPases/pumps, channels, exchangers,
CNGCs, GLRs and annexins) of Ca2+ transport elements at the whole genome level in rice. The possibility of connection between the expression profile at the
transcript level and the functional role in planta was
895
Calcium transport elements in rice
set as a rationale to undertake a comprehensive genome-wide study of Ca2+ transporters in rice.
In this study, the entire set of Ca2+ transporting elements has been identified in the rice genome, including
Ca2+-ATPases (ACA and ECA), Ca2+ exchangers
(CAX and CCX), CNGCs, GLRs, annexins and TPC.
Phylogenetic analysis was carried out to understand
the evolutionary relationship between various Ca2+
transporters in monocot and dicot plant species. Global expression analysis was performed for three abiotic
stresses (salinity, cold and drought) and during some
critical stages of rice development (including vegetative
and reproductive stages) by microarray and quantitative RT-PCR (qPCR). Specific expression analysis was
done for duplicated genes, which revealed significant
functional diversification of duplicated partners and
their evolutionary significance. Detailed subcellular
localization was carried out for the representative
members from different groups to get a clue about
their possible site of action. Also, functional activity
was validated for one potential candidate OsACA7 by
complementation of a mutant yeast strain and assessment for its salinity stress tolerant behaviour. Based
on our analysis in rice, we propose a hypothetical
model for the activity of various calcium transporters
at different subcellular locations in the plant cell.
Results
Identification of calcium transport elements in
rice genome
Keyword searches using different phrases and words
culminated in the identification of 16 calcium exchangers, including eight CAX, one EFCAX, four CCX,
two magnesium/proton exchangers (MHX), which are
plant homologs of animal Na+/Ca2+ exchangers, and
a single Na+/Ca2+-K+ exchanger. Another group of
calcium transporters included 12 calcium ATPases (10
ACA and 2 ECA), 10 annexins, 17 CNGCs, 24 GLRs
and a single gene encoding a TPC. Sequence homology
searches using various approaches resulted in the identification of a new calcium exchanger member
(LOC_Os02g14980) with no additional member from
other categories. Sequence, domain and motif analysis
proved this additional exchanger to be EFCAX, which
makes a total of 17 calcium exchangers in the rice genome. Further verification of retrieved entries for the
presence of characteristic domains, conserved
sequences and motifs proved their authenticity and
integrity (Table 1). Topology prediction employing
SCAMPI software for different groups of calcium transport elements revealed that their structure module was
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A. Singh et al.
Table 1. List of calcium transport elements in rice genome.
RGAP locus ID
Gene name
Calcium ATPases
LOC_Os01g71240 OsACA1
LOC_Os02g08018 OsACA2
LOC_Os03g10640 OsACA3
LOC_Os03g42020 OsACA4
LOC_Os04g51610 OsACA5
LOC_Os05g41580 OsACA6
LOC_Os10g28240 OsACA7
LOC_Os11g04460 OsACA8
LOC_Os12g04220 OsACA9
LOC_Os12g39660 OsACA10
LOC_Os03g17310 OsECA1
LOC_Os03g52090 OsECA2
Calcium exchangers
LOC_Os01g37690 CAX1
LOC_Os02g04630 CAX2
LOC_Os05g51610 CAX3
LOC_Os02g21009 CAX4
LOC_Os03g27960 CAX5
LOC_Os04g55940 CAX6
LOC_Os11g01580 CAX7
LOC_Os11g05070 CAX8
LOC_Os03g08230 OsCCX1
LOC_Os03g45370 OsCCX2
LOC_Os10g30070 OsCCX3
LOC_Os12g42910 OsCCX4
LOC_Os01g11414 OsEFCAX1
LOC_Os02g14980 OsEFCAX2
LOC_Os02g43110 OsMHX1
LOC_Os11g43860 OsMHX2
LOC_Os03g01330 NCKX1
Annexins
LOC_Os01g31270 OsANN1
LOC_Os02g51750 OsANN2
LOC_Os05g31750 OsANN3
LOC_Os05g31760 OsANN4
LOC_Os06g11800 OsANN5
LOC_Os07g46550 OsANN6
LOC_Os08g32970 OsANN7
LOC_Os09g20330 OsANN8
LOC_Os09g23160 OsANN9
LOC_Os09g27990 OsANN10
Channel
LOC_Os01g48680 OsTPC1
RGAP locus ID
Gene name
Cyclic nucleotide gated channel
LOC_Os01g57370 OsCNGC1
LOC_Os02g15580 OsCNGC2
LOC_Os02g41710 OsCNGC3
LOC_Os02g53340 OsCNGC4
LOC_Os02g54760 OsCNGC5
LOC_Os03g44440 OsCNGC6
LOC_Os03g55100 OsCNGC7
LOC_Os04g55080 OsCNGC8
LOC_Os05g42250 OsCNGC9
LOC_Os06g08850 OsCNGC10
LOC_Os06g10580 OsCNGC11
LOC_Os06g33570 OsCNGC12
LOC_Os06g33600 OsCNGC13
LOC_Os06g33610 OsCNGC14
LOC_Os09g38580 OsCNGC15
LOC_Os12g06570 OsCNGC16
LOC_Os12g28260 OsCNGC17
Glutamate receptor homologs
LOC_Os09g26160 OsGLR1.1
LOC_Os02g54640 OsGLR1.2
LOC_Os09g26144 OsGLR1.3
LOC_Os09g25960 OsGLR2.1
LOC_Os09g25980 OsGLR2.2
LOC_Os09g25990 OsGLR2.3
LOC_Os09g26000 OsGLR2.4
LOC_Os02g02540 OsGLR3.1
LOC_Os04g49570 OsGLR3.2
LOC_Os06g06130 OsGLR3.3
LOC_Os06g13730 OsGLR3.4
LOC_Os06g46670 OsGLR3.5
LOC_Os07g01310 OsGLR3.6
LOC_Os07g33790 OsGLR3.7
LOC_Os09g31160 OsGLR3.8
LOC_Os06g08880 OsGLR4.1
LOC_Os06g08890 OsGLR4.2
LOC_Os06g08900 OsGLR4.3
LOC_Os06g08910 OsGLR4.4
LOC_Os06g08930 OsGLR4.5
LOC_Os06g09050 OsGLR4.6
LOC_Os06g09090 OsGLR4.7
LOC_Os06g09120 OsGLR4.8
LOC_Os06g09130 OsGLR4.9
comparable with the respective group of calcium transporting proteins in other organisms (Fig. 1). A majority
of
calcium
exchangers
harbored
11–13
transmembrane domains (TMDs), cytoplasmic Nterminal and non-cytoplasmic C-terminal. All rice calcium ATPases except OsACA2 were predicted to have
8–10 TMDs, and OsACA2 was predicted to have
only three TMDs and a reverse orientation of the terminals compared with other calcium ATPases. Most
of the CNGCs were predicted to have six TMDs. The
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
Calcium transport elements in rice
Autoinhibited Calcium ATPase
Cation/Calcium transporter
COOH
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S1
S13
NH2
S3
S2
S4
S6
S5
S9 S10
S7 S8
M
Ca
NH2
COOH
Cyclic nucleotide gated channels
ER-type calcium ATPases
P
S1
S2
S3
S5
S4
S6
S7
S8
S2
S1
S9 S10
NH2
S4
S3
S5
S6
NH2
cNMP
COOH
COOH
Glutamate Receptors
Two pore channel
NH2
1
Gln
Gln2
S1
S2
S3
S1
S4
COOH
S2
NH2
S3
S4
S5
S6
S7
EF
hand
EF
hand
S8
S9
S10
S11
S12
COOH
Fig. 1. Structural topology of different rice calcium transporter families. SCAMPI software was used to predict the topology of different
calcium transporters. The figure depicts the generalized structural topology predicted for most of the members belonging to a particular
calcium transporter group.
majority of the GLRs were predicted to have five
TMDs, a single TPC member was made up of 12
TMDs and as expected none of the annexins was predicted to bear any TMDs.
Evolutionary analysis of calcium transport
elements in rice and Arabidopsis
Phylogenetic analysis was performed using protein
sequences from both rice and Arabidopsis calcium
transport elements to comprehend their evolutionary
relatedness or divergence. Based on statistical analysis
and a bootstrap support value ≥ 50%, all the different
calcium transport element groups were divided into
different sub-clades, and members from both rice and
Arabidopsis were aligned, suggesting a high degree of
evolutionary relatedness and their evolution through a
common path and ancestor (Fig. 2). However, in the
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
case of GLRs, group IV was specifically composed of
rice genes, excluding Arabidopsis GLRs. Furthermore,
phylogenetic analysis also helped in classifying these
elements into different functional classes, e.g. in the
case of calcium exchangers, two major clades could be
easily distinguished into calcium/proton exchangers
and cation/calcium exchangers. Similarly, calcium
ATPases can be demarcated into P-IIA type/ER type
ATPases (ECAs) and P-IIB type/auto-inhibited calcium ATPases (ACAs).
Chromosomal localization and gene duplication
Chromosomal localization revealed that all the genes
were variously distributed on all the 12 chromosomes.
A maximum of 17 genes were found on chromosome
6, whereas only one gene was observed on chromosome 8. Amongst the calcium exchanger genes a maxi897
Calcium transport elements in rice
A. Singh et al.
Calcium exchangers
91
79
100
100
100
99
100
100
99
100
94
99
100
100
51
100
99
80
96
82
100
100
100
89
100
100
100
68
99
III
100
100
90
II
87
100
53
82
100
100
100
100
AtACA7
AtACA2
OsACA3
AtACA1
I
OsACA4
OsACA10
AtACA4
AtACA11
OsACA1
II
OsACA6
OsACA8
100
OsACA9
AtACA13
III
AtACA11
OsACA7
AtACA9
OsACA2
IV
OsACA5
AtACA10
AtACA8
OsECA2
AtECA3/AtACA6
AtECA2/AtACA5
OsECA1
AtECA4
100
AtECA1/AtACA3
100
63
84
100
100
100
99
100
100
64
100
44
92
38
46
100
99
100
98
100
87
100
100
95
89
86
100 85
100
85
100
97
100
41
73
85
100
AtGLR2.3
AtGLR2.2
AtGLR2.4
AtGLR2.1
AtGLR2.7
AtGLR2.9
AtGLR2.8
AtGLR2.6
AtGLR2.5
OsGLR 2.2
OsGLR 2.3
OsGLR 2.1
OsGLR 2.4
OsGLR 1.2
OsGLR 1.3
OsGLR 1.1
AtGLR1.1
AtGLR1.4
AtGLR1.2
AtGLR1.3
OsGLR 4.7
OsGLR 4.2
OsGLR 4.3
100
OsGLR 4.5
OsGLR 4.8
OsGLR 4.9
OsGLR 4.6
OsGLR 3.3
AtGLR5
AtGLR3.3
AtGLR3.6
AtGLR3.1
AtGLR3.2
OsGLR 3.1
OsGLR3.2
OsGLR 3.4
OsGLR 3.8
OsGLR3.6
OsGLR 3.5
OsGLR 3.7
AtGLR3.6
AtGLR3.5
74
93
100
OsANN5
OsANN1
AtANN8
AtANN3
OsANN4
OsANN6
OsANN3
AtANN4
94
89
99
98
100
AtANN5
OsANN7
100
100
I
II
III
OsANN9
OsANN10
OsANN8
0.1
Glutamate receptor homologs
100
AtANN1
OsANN2
100
97
0.05
0.1
AtANN6
AtANN7
AtANN2
97
100
93
ECA
100
100
IB
100
88
99
Cation/Calcium exchanger
100
IA
Annexins
ACA
100
Calcium ATPases
OsCAX1
OsCAX3
AtCAX1
AtCAX3
AtCAX4
OsCAX4
OsCAX2
OsCAX5
OsCAX6
AtCAX2
AtCAX6
AtCAX5
AtEFCAX
OsEFCAX1
OsEFCAX2
OsNCKX1
OsMHX1
OsMHX2
AtMHX1
OsCAX7
OsCAX8
OsCCX2
OsCCX4
OsCCX3
OsCAX2
AT5G17850
AtCAX7
OsCCX1
AtCCX4
AtCCX3
Calcium/Proton exchanger
100
Cyclic nucleotide gated channels
AtCNGC11
AtCNGC12
AtCNGC3
AtCNGC10
AtCNGC13
AtCNGC1
OsONGC2
OsONGC12
OsONGC6
OsONGC17
OsONGC8
100
95
99
II
100
100
75
98
100
100
100
100
69
I
100
99
100
100
100
IV
50
99
95
100
61
52
71
100
100
100
III
0.05
100
100
100
100
100
AtCNGC7
AtCNGC8
AtCNGC5
AtCNGC6
AtCNGC9
OsONGC3
AtCNGC15
AtCNGC18
OsCNGC13
OsONGC16
OsONGC15
AtCNGC14
AtCNGC17
OsONGC5
OsONGC10
OsONGC4
OsONGC11
AtCNGC19
AtCNGC20
OsONGC7
AtCNGC2
AtCNGC4
OsONGC1
OsONGC9
I
II
III
IV A
IV B
0.05
Fig. 2. Phylogenetic relationship of rice and Arabidopsis Ca2+ transporter gene families. The un-rooted neighbor-joining phylogenetic tree was
constructed from the protein sequences of each Ca2+ transporter family in rice and Arabidopsis. Multiple sequence alignment was performed
with CLUSTALX 2.0.8 and the trees were generated using MEGA5. Clustering of rice and Arabidopsis calcium transporters was done on the basis
of significant bootstrap value (> 50%). The scale bar indicates 0.05 and 0.1 amino acid substitutions per site for different families.
mum number of genes were present on chromosomes 2
and 3, while a minimum were on chromosomes 10 and
12. The maximum Ca2+-ATPase genes were located
898
on chromosome 3. Chromosomes 6, 7, 8 and 9 did not
have any member from these two gene families. The
sole member of the TPC family was located on
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
Calcium transport elements in rice
chromosome 1. Annexin family genes were observed
mainly on chromosome 9 and absent on chromosomes
3, 4, 10, 11 and 12. The maximum members from the
CNGC and GLR gene families were located on chromosome 6 (Fig. S1). Cautious analysis for the chromosomal duplication of genes revealed seven gene pairs
of the calcium transport elements located on duplicated segments of chromosomes, including three pairs
from CNGC, two pairs from annexins and one pair
each from the EFCAX and ACA groups (Table 2).
Meeting the criteria of separation by less than five
intervening genes, six groups of genes exhibited tandem duplication. Out of the six groups, two groups
had two genes each duplicated; the remaining groups
had more than two genes duplicated, thus forming
clusters on the chromosomes. Interestingly, members
from the GLR and CNGC families formed clusters
only. All the tandemly duplicated genes were present
on chromosomes 5, 6 and 9, with chromosome 9 bearing the maximum duplicated genes.
Expression profile of calcium transport elements
in rice under abiotic stress
The expression profile of a gene under a particular
condition provides a clue for its further functional
characterization. Keeping this fact in mind, we generated global expression profiles of the rice calcium
transport elements under three abiotic stress conditions
(salt, cold and drought) together with 7-day-old
untreated seedlings as control (Fig. 3). With strictly
defined parameters of a fold change ≥ 2 and a P
Table 2. Duplicated calcium transport element genes in rice genome.
Segmental duplication
RGAP locus ID
Gene
LOC_Os01g11414
LOC_Os01g57370
LOC_Os02g15580
LOC_Os02g51750
LOC_Os02g53340
LOC_Os08g32970
LOC_Os11g04460
OsEFCAX1
OsCNGC1
OsCNGC2
OsANN2
OsCNGC4
OsANN7
OsACA8
Chromosome
1
1
2
2
2
8
11
RGAP locus ID
Duplicated partner
LOC_Os02g14980
LOC_Os05g42250
LOC_Os06g33570
LOC_Os06g11800
LOC_Os06g10580
LOC_Os09g23160
LOC_Os12g04220
OsEFCAX2
OsCNGC9
OsCNGC12
OsANN5
OsCNGC11
OsANN9
OsACA9
Chromosome
2
5
6
6
6
9
12
Tandem duplication
RGAP Locus ID
Gene
Duplication group
Chromosome
LOC_Os05g31750
LOC_Os05g31760
OsANN3
OsANN4
1
5
5
LOC_Os06g08880
LOC_Os06g08890
LOC_Os06g08900
LOC_Os06g08910
LOC_Os06g08930
OsGLR4.1
OsGLR4.2
OsGLR4.3
OsGLR4.4
OsGLR4.5
2
6
6
6
6
6
LOC_Os06g09090
LOC_Os06g09120
LOC_Os06g09130
OsGLR4.7
OsGLR4.8
OsGLR4.9
3
6
6
6
LOC_Os06g33570
LOC_Os06g33600
LOC_Os06g33610
OsCNGC12
OsCNGC13
OsCNGC14
4
6
6
6
LOC_Os09g25960
LOC_Os09g25980
LOC_Os09g25990
LOC_Os09g26000
OsGLR2.1
OsGLR2.2
OsGLR2.3
OsGLR2.4
5
9
9
9
9
LOC_Os09g26144
LOC_Os09g26160
OsGLR1.3
OsGLR1.1
6
9
9
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
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Calcium transport elements in rice
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A. Singh et al.
L R SL P1 P2 P3 P4 P5 P6 S1 S2 S3 S4 S5 C D S
B
L R SL P1 P2 P3 P4 P5 P6 S1 S2 S3 S4 S5 C D S
OsCNGC5
OsCAX6
OsCNGC17
OsCCX1
OsCNGC11
OsCCX2
OsCNGC8
OsEFCAX1
OsCNGC2
OsCAX1
OsCNGC4
OsCAX5
OsCNGC7
OsMHX1
OsEFCAX2
OsCNGC12
OsCAX8
OsCNGC10
OsCAX3
OsCNGC15
OsCAX7
OsCNGC13
OsCCX3
OsCNGC6
OsCAX2
OsCNGC16
OsCNGC1
OsCAX4
2.16
6.6
11.05
OsCNGC3
OsCNGC9
2.18
C
L R SL P1 P2 P3 P4 P5 P6 S1 S2 S3 S4 S5 C D S
6.28
10.39
D
OsGLR3.2
L R SL P1 P2 P3 P4 P5 P6 S1 S2 S3 S4 S5 C D S
OsGLR4.7
OsANN7
OsGLR3.1
OsGLR4.9
OsANN8
OsGLR3.6
OsANN1
OsGLR3.5
OsANN6
OsGLR3.7
OsANN10
OsGLR1.1
OsANN2
OsGLR4.1
OsANN9
OsGLR3.3
OsANN5
OsGLR3.8
OsANN3
OsGLR4.3
OsANN4
OsGLR2.1
OsGLR2.4
2.28
6.49
10.7
OsGLR2.2
OsGLR4.2
OsGLR3.4
F
OsGLR1.3
OsGLR1.2
OsGLR4.8
L R SL P1 P2 P3 P4 P5 P6 S1 S2 S3 S4 S5 C D S
Os ECA1
Os ACA4
2.28
7.6
12.92
Os ACA6
Os ACA3
Os ACA1
E
Os ACA5
Os ACA10
L R SL P1 P2 P3 P4 P5 P6 S1 S2 S3 S4 S5
C D S
OsTPC1
Os ACA8
Os ACA7
3.37
6.67
9.97
2.61
7.11
11.62
Fig. 3. Microarray expression profile of rice calcium transporter gene families. (A)–(F) The separate heat maps for the expression profiles of
all the different Ca2+ transporter gene families. Development includes three vegetative stages (L, mature leaf; R, root; SL, 7-day-old
seedling) and 11 reproductive stages, comprising six panicle developmental stages [P1 (0–3 cm), P2 (3–5 cm), P3 (5–10 cm), P4 (10–
15 cm), P5 (15–22 cm) and P6 (22–30 cm)] and five stages of seed development [S1 (0–2 DAP), S2 (3–4 DAP), S3 (4–10 DAP), S4 (11–20
DAP) and S5 (21–29 DAP)]. The abiotic stress treatments are denoted by C, cold; D, drought; S, salt; and SL, 7-day-old untreated seedling
as control. The color scale at the bottom of each heat map is given in log2 intensity value.
value < 0.05 in treated samples with respect to
untreated controls, 19 rice genes exhibited significant
differential regulation with eight of them being overall
upregulated and 11 being downregulated. This set of
differentially regulated genes included members from
almost all the groups (except a single TPC member).
Of four differentially expressed calcium exchanger
genes, two were upregulated (OsCCX2 and OsCAX8)
while two were downregulated (OsCAX1 and OsMHX2). Four calcium ATPases were differentially
900
expressed (OsACA3, OsACA5, OsACA7 and OsECA1)
and all were upregulated. Similarly, three OsCNGCs,
seven OsGLRs and a single annexin (OsANN2) gene
showed differential regulation of expression with members exhibiting upregulation and downregulation in
varying numbers (Table S1). The sole member of the
TPC family did not show change in expression under
any of the abiotic stress conditions. Expression profiling for selected candidates by qPCR showed that
most of the calcium transport element genes exhibited
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
Calcium transport elements in rice
Microarray
(r = 0.734)
OsCAX1
1200
(r = 0.828)
OsCAX8
45
OsECA1
qPCR
(r = 0.726)
100
40
1000
80
35
30
800
60
25
600
20
400
40
15
10
200
20
5
0
R e la tiv e tr a n s c r ip t a b u n d a n c e
0
0
Control
Salt
Cold
(r = 0.768)
OsACA3
60
Drought
Control
Salt
Cold
OsACA5
4000
Drought
(r = 0.927)
20
10
400
1500
300
1000
200
500
100
0
0
Control
Salt
Cold
OsGLR1.1
Drought
Control
(r = 0.803)
Salt
Cold
Drought
(r = 0.840)
OsANN2
12000
100
(r = 0.822)
500
2000
0
OsACA7
900
Drought
600
2500
30
Cold
700
3000
40
Salt
800
3500
50
Control
Control
Salt
Cold
OsCNGC7
700
Drought
(r = 0.837)
600
10000
80
500
8000
400
60
6000
40
300
4000
200
20
2000
100
0
0
Control
Salt
Cold
Drought
0
Control
Salt
Cold
Drought
Control
Salt
Cold
Drought
Fig. 4. Validation of the microarray expression profile for rice Ca2+ transporters by qPCR under abiotic stresses. Three and two biological
replicates were used to generate microarray and qPCR expression profiles, respectively. Standard error bars are shown for the data from
both the techniques. The normalized expression values are plotted on the y-axis while the x-axis represents different experimental
conditions. Dark and light grey columns depict the expression values from microarray and qPCR, respectively. The Pearson correlation
coefficient r for all the genes indicates the statistical significance of the data.
similar expression patterns as observed by microarray
analysis with significantly strong correlation (Fig. 4).
However, the magnitude of expression was variable in
some samples and genes, which can be attributed to
differences in the sensitivity to detect the transcripts by
these two techniques. This kind of variation in transcript levels has been observed previously in various
studies [39–42]. These findings suggest a possible role
of various calcium transport elements in signaling triggered by abiotic stress conditions in crop plant rice.
Expression profile of rice calcium transport
elements during development
Comparison with three vegetative developmental stages
revealed that a total of 41 calcium transport element
genes expressed differentially during various stages of
reproductive development including members from all
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
the different groups. A subset of 24 genes was found
to be upregulated and composed of six CNGCs, four
GLRs, four annexins, five exchangers (two CAX, two
EFCAX and one CCX) and five ACA members, while
17 members including four CNGCs, four GLRs, two
annexins, three exchangers (two CAX and one MHX),
three ACAs and a single TPC member exhibited
downregulation (Fig. 2, Table S1). Among the differentially expressed genes, 29 members were found to be
commonly expressed during both the phases of reproductive development, i.e. panicle and seed. Observation
for specific and unique expression revealed that a
total of eight genes are specifically expressed during
panicle development stages with seven members exhibiting upregulation while a single exchanger member
OsEFCAX2 was downregulated. Similarly, nine calcium transport element genes expressed specifically
during seed development where seven and two
901
Calcium transport elements in rice
members exhibited downregulation and upregulation,
respectively.
Overlapping expression pattern in abiotic
stresses and development
We keep in mind that abiotic stresses and reproductive
development are an interconnected phenomenon in the
plant life cycle as is evident from previous work [41,43–
45]. We investigated a similar connection involving calcium transport elements. In-depth analysis revealed
that a significant proportion of 14 genes exhibited pronounced differential expression under abiotic stresses
and stages of panicle and seed development where nine
and five calcium transport element encoding genes were
upregulated and downregulated, respectively. Here, the
genes which were commonly upregulated included
OsCCX2, OsCAX8, OsACA3, OsACA5, OsACA7, OsECA1, OsANN2, OsCNGC7 and OsGLR1.1. Downregulated genes included OsCAX1, OsMHX2, OsGLR3.5,
OsGLR3.6 and OsCNGC11. None of the genes was
exclusively upregulated under stress conditions but two
genes, OsCNGC3 and OsGLR4.7, were specifically
downregulated. Also, none of the genes showed overlapping upregulation during seed development and
stresses but three members of GLRs – OsGLR3.1,
OsGLR3.2 and OsGLR4.9 exhibited overlapping downregulation under these stages/conditions. It is noteworthy that none of the genes showed expression during
panicle development and abiotic stresses. Strikingly,
there were genes which exhibited upregulation during
panicle development but downregulation under abiotic
stresses and vice versa. Genes with this type of unique
expression pattern included OsCNGC3, OsGLR3.1, OsGLR3.2 and OsGLR4.7.
Expression profile of duplicated genes
To analyze the expression behavior of duplicated pairs
of genes, expression profiles were generated for segmental and tandemly duplicated pairs/clusters under
abiotic stresses and during all stages of development
(vegetative and reproductive). The average signal value
from a microarray for all the samples is presented as
an area diagram (Fig. 5). Among the seven segmentally duplicated pairs, an expression profile could be
generated for six pairs because a common probe set
represented OsACA8 and OsACA9. Among the segmentally duplicated genes three pairs, OsCNGC2:
OsCNGC12, OsCNGC4:OsCNGC11, OsANN2:OsANN5, showed retention of expression as both the
paired partners had a similar expression pattern in
most of the conditions and stages; however, the magni902
A. Singh et al.
tude of the expression varied. One of the paired partners from OsCNGC1:OsCNGC9 and OsANN7:
OsANN9 seems to have lost its expression during most
of the conditions and hence the pairs exhibited
pseudo-functionalization. Another pair OsEFCAX1:
OsEFCAX2 exhibited neo-functionalization as both
the partners had a diverse expression pattern. Similarly,
among the tandemly duplicated gene pairs/groups,
three groups showed retention of expression while the
other three exhibited pseudo-functionalization.
Subcellular localization of calcium transport
element proteins
A total of six members, namely OsANN2, OsACA7,
OsECA2, OsTPC1, OsGLR1.1 and OsCNGC7, were
analyzed for their subcellular location together with
the empty green fluorescent protein (GFP) vector as
control. It was observed that all the proteins have a
distinct cellular localization pattern and reside at
diverse subcellular compartments. OsANN2 was
detected throughout the cytoplasm. GFP fluorescence
of OsTPC1 was observed as globular structures on the
periphery of the cell (Fig. 6), which are clearly visible
in the magnified view (Fig. S2). The fluorescent signal
approaches the nucleus and chloroplast only on the
side facing the interior of the cell and completely
merged with the tonoplast marker vac-rk (CD3-975,
ABRC) (Fig. 7), which confirmed its tonoplast localization. OsACA7 appears as small, nearly round, spots
in the cell. These spots completely merged with the
Golgi marker G-rk (CD3-967, ABRC) as observed in
the overlay image. OsECA2 was distributed at the
periphery of the cell and finally confirmed to be membrane localized by its complete co-localization with the
plasma membrane marker (CBL1n-OFP). OsGLR1.1
was observed as a thread-like network and was concluded to be ER localized by co-localization with the
ER marker ER-rk (CD3-959, ABRC), which could be
clearly seen also in the magnified view (Fig. S3).
Expression of the OsCNGC7 protein was detected as a
network-like structure in the whole cell and preferentially localized around the nucleus (Fig. 6), as clearly
seen in the magnified view (Fig. S4). The differential
localization of calcium transport elements suggests
their possible site of action at respective subcellular
locations to maintain calcium homeostasis.
Yeast Ca2+ transport activity complementation
To investigate and verify the functional behavior of
the identified calcium transport elements, one of the
candidates from the Ca2+-ATPases, namely OsACA7,
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
Calcium transport elements in rice
Segmental duplication
1500
1000
2000
OsERFCAX2
OsERFCAX1
1000
500
0
0
800
L R S P1
P2 P3 P4
P5 P6 S1 S2
S3 S4 S5 SS
CS DS
OsCNGC11
OsCNGC4
600
P5 P6 S1 S2
S3 S4 S5 SS
CS DS
150
OsCNGC1
OsCNGC9
100
400
50
200
0
0
L R S P1
P2 P3 P4
L R S P1
P2 P3 P4 P5
P5 P6 S1 S2
S3 S4 S5 SS
CS DS
OsANN5
OsANN2
8000
P6 S1 S2 S3
S4 S5 SS CS
DS
OsANN7
OsANN9
6000
6000
4000
4000
2000
Pseudofunctionalization
Retention of expression
L R S P1
P2 P3 P4
Neofunctionalization
OsCNGC2
OsCNGC12
2000
0
0
L R S P1
P2 P3 P4
L R S P1
P2 P3 P4
P5 P6 S1 S2
S3 S4 S5 SS
CS DS
P5 P6 S1 S2
S3 S4 S5 SS
CS DS
Tandem duplication
OsANN4
OsANN3
2000
1500
300
1000
200
500
100
0
L R S P1
P2 P3 P4
0
L R S P1
P2 P3 P4 P5
P5 P6 S1 S2
S3 S4 S5 SS
CS DS
OsGLR 4.1
OsGLR 4.2
OsGLR 4.5
6
P6 S1 S2 S3
S4 S5 SS
CS DS
OsGLR 1.3
OsGLR 1.1
200
4
100
2
0
0
L R S P1
P2 P3 P4 P5
OsGLR 2.2
OsGLR 2.3
OsGLR 2.4
5.1
5
L R S P1
P2 P3 P4
P6 S1 S2 S3
S4 S5 SS CS
DS
1500
Pseudofunctionalization
Retention of expression
OsGLR 4.6
OsGLR 4.7
OsGLR 4.5
400
P5 P6 S1 S2
S3 S4 S5
SS CS DS
OsCNGC13
OsCNGC12
1000
4.9
500
4.8
0
4.7
L R S P1
P2 P3 P4
P5 P6 S1 S2
S3 S4 S5 SS
CS DS
L R S P1
P2 P3 P4
P5 P6 S1 S2
S3 S4 S5 SS
CS DS
Fig. 5. Expression profiles of duplicated Ca2+ transporters. The expression pattern of duplicated gene pairs/clusters (segmental and tandem)
was analyzed during a spectrum of developmental stages and abiotic stresses. Due to variable expression pattern duplicated gene pairs/
clusters showed retention of expression, pseudo-functionalization and neo-functionalization. Each graph depicts mean normalized microarray
signal intensity value on the y-axis and different developmental stages and stress conditions on the x-axis.
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
903
Calcium transport elements in rice
Bright field
Merge
35S:GFP
GFP-OsCNGC7
GFP-OsGLR1.1
OsECA2-GFP
GFP-OsTPC1
OsACA7-GFP
GFP-OsANN2
GFP
A. Singh et al.
904
Fig. 6. Subcellular localization of Ca2+
transporter proteins in
Nicotiana benthamiana epidermal cells.
GFP-OsANN2 fusion protein is distributed
throughout the cytoplasm (first row)
whereas OsACA7-GFP appears as small,
nearly round, spots (second row) in the
cell. GFP-OsTPC1 appears as circular
vesicles inside the lumen of the cell (third
row). OsECA2-GFP fusion protein shows
preferential accumulation in the cell
periphery (fourth row) and GFP-OsGLR1.1
fusion protein shows network-like
structures (fifth row). Expressed GFPOsCNGC7 fusion protein in Nicotiana
epidermal cells expresses preferentially
around the nucleus (sixth row). Cells
transformed with CaMV35S-GFP were
used as a control. Fluorescence was
detected under a confocal laser-scanning
microscope (wavelength 488 nm). All the
images were taken in five different
sections in the z direction and merged
together. Scale bar 40 lm.
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
Fig. 7. Co-localization of Ca2+ transporter
proteins with organelle markers. GFP
signal merges completely with Golgi
marker (g-rk) for OsACA7-GFP in the
overlay (first row). In OsECA2-GFP green
GFP signal merges completely with
plasma membrane marker CBL1n-OFP
(second row). GFP-OsGLR1.1 was present
in a large bright spot that co-localizes with
endoplasmic reticulum marker (ER-rk)
(third row). The GFP-OsTPC1 localized
completely with globular vesicles as
shown by tonoplast markers (vac-rk)
(fourth row). GFP fusions to the calcium
transporter proteins are shown in green,
mCherry/OFP organelle markers are
shown in red and overlay of the two
mentioned proteins in dark field view. All
the images were taken in five different
sections in the z direction and merged
together. Scale bar 20 lm.
Calcium transport elements in rice
OsACA7-GFP
G-rk
Overlay
OsECA2-GFP
CBL1n-OFP
Overlay
GFP-OsGLR1.1
ER -rk
Overlay
GFP-OsTPC1
vac-rk
Overlay
was selected for a yeast complementation assay
because the yeast complementation system for this
group of transporters is well established and readily
available. To test its Ca2+-ATPase activity, yeast
mutant strain K616 [46] was selected. This mutant
lacks two endogenous Ca2+-ATPases (PMC1, PMR1)
and a Ca2+-dependent phosphatase CNB1, which are
involved in Ca2+ homeostasis [46,47]. The calcium
homeostasis of the K616 mutant strain is entirely
dependent on the H+/Ca2+ exchanger VCX1 activity.
The K616 mutant grows like wild-type at physiological
Ca2+ concentrations (≥ 1 mM Ca2+). In depleted or
below suboptimal Ca2+ concentrations, low affinity
H+/Ca2+ exchanger VCX1 is inactivated and hence
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
the K616 strain fails to grow and this forms the basis
for complementation assays of Ca2+-ATPases in the
K616 yeast mutant [35,46,48–52]. For complementation analysis, full-length OsACA7 (OsACA7/pYES2)
and N-terminal truncated protein of OsACA7 (ΔOsACA7/pYES2) were cloned into galactose inducible
pYES2 vector and expressed along with vector control
in K616 strain. Wild-type strain K601 transformed
with vector alone grows in all Ca2+ sufficient (10 mM
CaCl2) or Ca2+ depleted medium (10 mM EGTA, pH
5.5) while the K616 strain having only pYES2 vector
or full-length OsACA7 was able to grow on Ca2+ sufficient medium but unable to grow on Ca2+ depleted
medium. The K616 strain expressing N-terminal
905
Calcium transport elements in rice
A. Singh et al.
A
B
Fig. 8. Functional activity of rice Ca2+-ATPase OsACA7 in yeast. (A) Complementation of yeast mutant K616 Ca2+ transport activity. The
wild-type K601strain was transformed with pYES2 (pYES2-K601). The yeast mutant K616 (Δpmr1Δpmc1Δcnb1) was transformed with
empty pYES2 vector as vector control (pYES2-K616), full-length OsACA7 (OsACA7-pYES2-K616) or N-terminal modified OsACA7 (ΔOsACA7pYES2-K616). Dotting assay was done by taking a single colony on SC-Ura/Glu and SC-Ura/Gal plates containing either 10 mM CaCl2 or
10 mM EGTA (pH 5.5) and incubated for 4 day at 30 °C. Expression of the OsACA7 gene was regulated by the galactose-inducible GAL1
promoter. An N-terminal truncated protein of OsACA7 was able to complement yeast mutant K616 in the absence of calcium (+10 mM
EGTA) whereas full-length OsACA7 could not complement the Ca2+ transport activity. (B) Salinity stress tolerance analysis of K616 yeast
mutant, complemented with OsACA7. Growth of yeast mutant transformed with different constructs of OsACA7 and vector pYES2 on SC
-Uracil with galactose having different concentrations of Ca2+, i.e. 20 lM, 100 lM, 680 lM and 10 mM with (lower panel) and without (upper
panel) 400 mM NaCl. The gradually decreasing bars below the panels show serial dilution of 10 1, 10 2 and 10 3 fold of cells adjusted to an
A600 of 1.0. The N-terminal truncated ΔOsACA7/pYES2-K616 transformants show better growth at lower concentrations of calcium in the
absence of NaCl (upper panel) while in the presence of NaCl full-length OsACA7/pYES2-K616 grows better.
truncated protein (ΔOsACA7/pYES2) was able to
restore the growth on Ca2+ depleted medium in the
presence of galactose (Fig. 8A). Therefore it was concluded that full-length OsACA7 could not restore
growth of K616 on Ca2+ depleted medium while
N-terminal truncated ΔOsACA7 supported the growth
of the mutant strain in the same conditions. Growth
restoration of K616 during the complementation assay
was achieved only in the presence of galactose while
growth was absent in the presence of glucose that
represses the GAL1 promoter, which rules out vectorencoded complementation.
OsACA7 provides salt tolerance to K616 yeast
mutant
To assess the salt tolerance behavior of OsACA7, a
stress assay was performed in yeast at 400 mM NaCl
concentration [52]. The K616 yeast mutant was transformed with vector pYES2-K616 and full-length
OsACA7 and showed poor growth in 20 lM free Ca2+
906
while N-terminal truncated OsACA7 (ΔOsACA7/
pYES2-K616) was able to grow and complement the
K616 mutant at the very low calcium concentration.
As the concentration of calcium was gradually
increased (from 20 lM to 10 mM), both K616 mutant
and full-length OsACA7 showed prominent growth. In
the medium supplemented with 400 mM NaCl, better
growth was observed for full-length OsACA7 with
increasing concentration of calcium whereas vector
control K616 and N-terminal truncated OsACA7
showed comparatively lesser growth (Fig. 8B). This
observation suggests that OsACA7 confers salinity
stress tolerance to K616 yeast mutant and might perform a similar function in plants.
Discussion
Exhaustive exploration of various available databases
and a homology search with different approaches and
tools have resulted in the identification of 81 calcium
transport elements in the rice genome. This set of
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
calcium transporters is composed of Ca2+-ATPases,
Ca2+ exchangers, channels, annexins, CNGCs and
GLRs (Table 1). The presence of a variety of Ca2+
transport elements, with different modes of action,
indicates the existence of a diverse and complex calcium transport system in plants. Mapping all the genes
on the 12 chromosomes of rice revealed a variable distribution and localization on the chromosomes. This
widespread chromosomal distribution of genes can be
attributed to a high degree of segmental and tandem
duplication amongst the calcium transport element
genes. Seven pairs of genes belonging to different
groups were located on a duplicated segment of the
chromosomes. A large set of genes exhibited tandem
duplication and organized themselves in pairs or
groups of genes forming clusters on the chromosomes.
Among the duplicated genes most members belong to
GLRs (14 members) and CNGC groups (nine members). These findings suggest that chromosomal duplication has been the potent driving force for the
evolution and expansion of GLRs, CNGCs and other
Ca2+ transport element groups in the rice genome.
Phylogenetic analysis of different calcium transport
elements from rice and Arabidopsis revealed that most
members of the different groups from these two species form common sub-clades with high bootstrap values (Fig. 2). This phylogenetic trend has suggested a
high degree of sequence similarity among the members
of the respective Ca2+ transporter groups and indicates the conserved evolution of these groups of genes
in monocots and eudicots through a common origin
and ancestors. However, rice group IV GLRs with
nine members forms a unique clade, excluding any
Arabidopsis GLR member. Here, it can be speculated
that the pioneer members of this group initially
diverged from their common origin and later in time
they might have expanded into a large group through
chromosomal duplication, as eight of the nine members of this group are in tandem duplication (Table 2).
To get a clue about the functional role of the rice
calcium transport elements, detailed genome-wide
expression profiling was done using gene chip microarray data. In this analysis, a set of Ca2+ transport
genes was significantly and differentially regulated
under three abiotic stresses (salt, cold and drought).
This set of differentially expressed genes included
members from all groups of Ca2+ transport elements
and most affected genes belong to three groups: ATPases, exchangers and GLRs. Furthermore, qPCR
analysis validated the microarray expression pattern
for a few interesting candidates. This expression pattern information emphasizes the involvement of various Ca2+ transport elements in abiotic stress triggered
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
Calcium transport elements in rice
signaling. This is quite coherent also as fluctuations in
the cytosolic calcium levels are one of the primary
responses to various stimuli and Ca2+ transport elements actively participate in maintaining this flux and
homeostasis. Previously, it was reported that NaCl
treatment leads to upregulation of the Ca2+-ATPase
transcript level in different plant species such as
tomato, tobacco, soybean and Arabidopsis [50,53–56]
and it was speculated that increased capacity of Ca2+ATPase may help in lowering the cytosolic calcium
level, which was elevated due to NaCl stress and might
be involved in maintenance of Ca2+ homeostasis.
Also, overexpression of N-terminal modified ACA4 in
Arabidopsis seedlings resulted in increased salt tolerance in comparison with wild-type plants [18]. This
gene was also reported to enhance the salt tolerance in
yeast, shown by the K616 mutant complementation
[50]. In addition, transcript level was also escalated for
Physcomitrella patens P-IIB Ca2+-ATPase PCA1 in
response to dehydration, salt and abscisic acid, and
PCA1 knockout mutant displayed enhanced sensitivity
to high salinity [35]. Arabidopsis Ca2+ exchanger
CAX3 expression was found to be strongly induced
under salt stress [56]. Genetic analysis revealed that
cax3 mutant and cax1/cax3 double mutants showed
strong sensitivity on 50 mM and 100 mM NaCl media
[57]. Surprisingly, cax1 knockout mutant showed
increased tolerance to freezing stress [58]. qPCR based
expression analysis showed that multiple members of
the Arabidopsis annexin family exhibit differential
expression under abiotic stresses such as salt and
drought [59]. In a study in Arabidopsis, annexin 1 (AnnAt1) was highly induced by abiotic stresses such as
salt and drought and subsequent phenotypic analysis
clearly revealed that annexin 1 overexpressing plants
showed enhanced tolerance to drought stress while the
knockout mutant was highly sensitive [60]. Apart from
abiotic stresses, it is important to understand the regulation of various genes during plant development,
especially reproductive stages such as panicle and seed
development which ultimately determine crop productivity. In our study, several Ca2+ transport element
genes were found to be expressed significantly and differentially during various stages of panicle and seed
development (Fig. 3, Table S1). These are very critical
developmental stages in rice and include floral organ
development (P1), meiosis (P2–P3), young microspore
(P4) to mature pollen (P6), while seed development
stages represent early globular embryo (S1), middle
and late globular embryo (S2), embryo morphogenesis
(S3), embryo maturation (S4) to dormancy and desiccation (S5). Ca2+ level fluctuations are also critical
events in many developmental processes and regulate
907
Calcium transport elements in rice
the developmental physiology of the plant [1,61]. Since
these changes in calcium level and overall calcium
homeostasis are regulated by various Ca2+ transport
elements, spatio-temporal changes in the expression of
these genes might affect specific developmental stages
and in turn the overall yield and productivity of the
plant. Bock and co-workers in their expression analysis
for transporter genes at different stages of male gametophyte development in Arabidopsis showed that at
least four CNGCs (CNGC7, 8, 16 and 18) expressed
significantly and preferentially at different pollen
developmental stages, and knockout mutant of AtCNGC18 resulted in sterile male plants [29,62].
Recently, Goel et al. (2012) have proposed a possible
role of calcium transporters and exchangers during rice
seed development, and through in silico database and
MPSS library expression analysis they concluded that
several Ca2+ exchangers significantly expressed during
early seed developmental stages while Ca2+-ATPases
were highly expressed throughout the seed developmental stages [38]. Interestingly, a few rice Ca2+ transport element genes had overlapping expression under
abiotic stresses and during developmental stages. Such
genes might be involved at the conjuncture of abiotic
stress triggered and developmental signaling and result
in the ‘cross-talk’ of these signaling pathways. Previous
studies have suggested that some common components
such as Ca2+ and abscisic acid might connect two signaling cascades triggered by different stimuli [1,4,61].
Moreover, in the promoter, the presence of a cis-regulatory element such as ABRE, which regulates both
abiotic stress and development, can be attributed to
such overlapping expression [39,44]. Arabidopsis
CAX1, which had been previously implicated in abiotic
stress responses, was also reported to mediate plant
development. Knockout cax1 mutant plants exhibited
significant reduction in primary root length and lateral
roots. Transition from the vegetative to the flowering
phase was delayed by 5–7 days and the length of the
primary inflorescence was greatly reduced [56]. In
another study, Arabidopsis GLR1.1 was shown to regulate abscisic acid signaling and control drought stress
response by controlling stomatal movement and the
overall growth and development of plants [63]. Expression profiling for the duplicated Ca2+ transporter
genes in a spectrum of developmental stages and abiotic stresses showed that duplicated partners exhibited
variable expression patterns, and retention of expression, pseudo-functionalization and neo-functionalization were observed (Fig. 5). This variable expression
pattern for the duplicated partners might have been
the result of lack of intensive selection pressure and
might have been required for functional diversification
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A. Singh et al.
of the various Ca2+ transporters in rice. Moreover,
segmentally duplicated genes are also known to display
functional divergence quite often [64].
To understand the function of a gene at protein
level, it is important to know about its possible site of
residence in a cell. In particular, in plant cells calcium
is stored and released from different intracellular and
extracellular storage compartments such as the vacuole, ER, mitochondria, chloroplasts and cell wall
[12,65,66]. Therefore, understanding of the subcellular
localization of various types of Ca2+ transporters
becomes more relevant. Confocal microscopic analysis
revealed that one of the P-IIB Ca2+-ATPases (ACAs)
OsACA7 was localized in the Golgi bodies. Previous
studies have reported variable localization of these
types of ATPases to different organelles such as vacuole, ER and plasma membrane [18,49,67]. However, in
yeast PMR1, a P-type ATPase was localized to Golgi
bodies and was required for normal secretory processes [68]. Similar to localization of OsECA2 in the
plasma membrane in our study, one of the Lycopersicon ECAs was detected at the plasma membrane [69].
However, Arabidopsis ECA1 was ER localized [70]
suggesting a diverse localization pattern for the plant
ECAs. Localization of OsCNGC7 at the nuclear
periphery suggests its association with the nuclear
envelope, which remains connected with the endomembrane system. Arabidopsis CNGC10 was shown to be
localized in ER, Golgi and vesicles, which are trafficking intermediates in the secretory pathway of plasma
membrane proteins [71]. OsTPC1 was detected in
tonoplast and tonoplast vesicles, which is supported by
the prior study in Arabidopsis where AtTPC1 was
localized to tonoplast [72]. Kurusu and co-workers
recently expressed OsTPC1-GFP in tobacco BY-2 cells
and it was found to be localized to the vacuolar membrane [73]. However, Arabidopsis TPC was also
detected partly in the plasma membrane when it was
expressed in tobacco [74]. Also, OsTPC1 has been
found to be localized in the plasma membrane in prior
studies [75,76]. This variable localization pattern of
OsTPC1 might be due to different plant systems used
for the localization analysis. Plant annexins have been
found to localize primarily in the cytosol [77,78]. This
observation agreed with our study as OsANN2 was
detected in the cytosol. Rice GLR member OsGLR1.1
was localized in the ER in this study. Earlier dual
localization was observed for Arabidopsis AtGLR3.4
and it was detected in plastids and plasma membrane
[79]. Chloroplast localization was seen in the case of
spinach for iGLR3 [80]. These observations suggest
that GLR can be localized to plasma membrane and
organelle also. ER is one of the major Ca2+ reservoirs
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
Abiotic
stress
OsECA2
OsCNGC7
?
rice genome. A detailed and comprehensive expression
profiling indicated their possible involvement in abiotic
stress and development triggered signaling. A great
amount of chromosomal duplication was observed
amongst the various calcium transporter genes which
has supposedly played a significant role in the expansion of various Ca2+ transporter gene families in rice.
Successful localization in different cellular compartments provides a clue about their possible site of
action. Complementation and salt stress tolerance
behavior of K616 yeast mutant verify the functional
activity of one of the Ca2+-ATPases, OsACA7. We
are proposing a hypothetical model for the activity of
different calcium transport elements in rice (Fig. 9).
Abiotic stresses and developmental stimuli lead to
increase in [Ca2+]cyt, which in turn triggers various signaling cascades. Various calcium transporters/channels/exchangers that have been found to be localized
in different subcellular compartments become activated
and complex functional coordination of these transporters regulates Ca2+ homeostasis. This study pro-
Cytoplasm
ATP
ADP + Pi
Ca2+
OsANN2
OsCAX1a
H+
Ca2+
OsTPC1
ADP + Pi
Nucleus
OsACA7
Golgi
lumen
OsGLR1.1
CaM
ATP
in the animal cell and this channel might play a vital
role in calcium homeostasis during certain processes.
We have extended our analysis to the assessment of
functional activity by yeast complementation of the
K616 mutant (ΔPMC1 ΔPMR1 ΔCNB1) by one of the
Ca2+-ATPases because high affinity Ca2+-ATPases
and low affinity H+/Ca2+ exchanger are thought to
be the major players for regulating [Ca2+]cyt homeostasis in yeast. Complementation of K616 mutant with
OsACA7 showed that the rice P-IIB type ATPase
could restore calcium transport activity of this yeast
mutant even in Ca2+ depleted medium (Fig. 8A).
According to our anticipation, N-terminal modified
OsACA7 (ΔOsACA7) but not the full OsACA7 was
able to complement the Ca2+ transport activity
because P-IIB type Ca2+-ATPases contain a characteristic auto-inhibitory domain at the N-terminal and
removal of this domain activates this pump. Successful
complementation of K616 mutant provided strong
genetic evidence that OsACA7 is a functional Ca2+
pump. Various possible factors such as low Ca2+
availability, high expression level of plant pumps and
hence less availability of endogenous CaM in yeast,
and differences in the canonical CaM binding site of
ACA pumps might hamper the Ca2+ pump activity of
full-length OsACA7 [49,81]. The regulatory mechanism
of OsACA7 is apparently similar to Arabidopsis
ACA2, ACA4, ACA9 and Physcomitrella patens
Ca2+-ATPase PCA1 [18,34,49,51,81]. Interestingly, salt
stress analysis revealed that expression of the Ca2+ATPase OsACA7 confers salt tolerance to hypersensitive K616 mutant (Fig. 8B). Since transcript analysis
by microarray and qPCR showed no differential
expression of OsACA7 under salt stress, a post-translational regulation mechanism might be involved in
salinity tolerance. It can also be speculated that under
salt stress conditions the auto-inhibitory constraint of
full-length OsACA7 has been relieved, which contributes towards salt tolerance to yeast; however, this
assumption needs further experimental verification.
Based on the salt stress tolerance imparted by
OsACA7 in yeast complementation assays, we also
analyzed the functional activity of this transporter in
osmotic stress response mediated by sorbitol but could
not find any significant osmotic stress tolerance (Fig.
S5), which suggests the specificity of OsACA7 in
cation transport during abiotic stress conditions. Plant
Ca2+-ATPases have been previously implicated in the
fine tuning of [Ca2+]cyt and in the suppression of the
salt hypersensitive phenotype of yeast mutant K616
[50–52].
In conclusion, this study presents a genome-wide
survey of various Ca2+ transport element genes in the
Calcium transport elements in rice
Vacuolar
lumen
ER
lumen
Fig. 9. Proposed model for the activity of different calcium
transporters in rice. Abiotic stresses lead to increase in [Ca2+]cyt
which triggers a number of signaling cascades in plant cells.
Various calcium transporters/channels/exchangers get activated
upon perception of the stimulus and they function to maintain the
physiological [Ca2+]cyt by exporting Ca2+ outside the cell and/or
through sequestration to various cellular organelles. Rice OsACA7
resides at Golgi bodies, OsECA2 was localized at plasma
membrane and rice glutamate receptor, OsGLR1.1 was localized at
the endoplasmic reticulum. OsCNGC2 forms a network-like
structure around the nucleus and is suspected to be localized at
the endoplasmic reticulum. OsTPC1 was present at the vacuolar
membrane. Rice calcium exchanger OsCAX1a was detected at the
vacuolar membrane [87] while annexin OsANN2 is generally
expressed in the cytosol and translocates to various cell organelles
and membrane to perform diverse functions [88].
909
Calcium transport elements in rice
vides a critical platform for the detailed functional
characterization of potential candidate Ca2+ transporters to ascertain their physiological function. With
a detailed characterization of these calcium transport
elements in rice, a future goal of generating crops
which can grow and sustain a higher degree of abiotic
stresses with higher calcium levels as a better nutritional aspect in edible parts of the plants such as seeds
and fruits to meet with calcium deficiency, especially
in developing countries, can be achieved.
Materials and methods
Identification of calcium transport elements in
rice and Arabidopsis
To identify rice calcium transporting elements, the Rice
Genome Annotation Project – The Institute of Genomic
Research (RGAP-TIGR) version 6.1 was searched using
different keywords such as ‘calcium transporter’, ‘calcium
exchanger’, ‘calcium pump’, ‘calcium channel’, ‘cyclic nucleotide gated channel’, ‘glutamate receptor’ and ‘annexin’.
Hidden Markov model profiles were obtained for all the
groups of genes by seed alignment with default parameters
from the Pfam database and were then used as query to
search various protein databases such as RGAP-TIGR,
Superfamily and PlantsT. All the unique entries obtained
from the homology searches were scanned through domain
and motif analysis tools such as SMART, INTERPRO and
Pfam to confirm the characteristic domains and motifs.
Arabidopsis calcium transport elements were also searched
employing similar approaches mainly using the Arabidopsis
Information Resource (TAIR) database.
Phylogenetic analysis of calcium transport
elements
The non-redundant protein sequences of rice and
Arabidopsis calcium transport elements were used to generate multiple sequence alignments employing CLUSTALX
version 2.0.8. Phylogenetic trees were constructed by the
neighbor-joining algorithm with the p-distance method and
pairwise deletion of gaps, employing MEGA version 5 with
default parameters. Statistical analysis was performed by
bootstrapping of 1000 replicates to test the phylogeny.
Chromosomal localization and gene duplication
All the 81 members from different groups of rice genes
involved in calcium transport were mapped on the 12 rice
chromosomes as per the respective coordinates mentioned
in the RGAP database (http://rice.plantbiology.msu.edu/
pseudomolecules/info.shtml). The RGAP segmental duplication database (ftp://ftp.plantbiology.msu.edu/pub/data/
910
A. Singh et al.
Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_6.1/all.dir/) was searched to find the segmentally duplicated genes. Genes separated by five or fewer
genes on a chromosome were considered to be tandemly
duplicated.
Plant material, growth conditions and stress
treatment
Tissue at different developmental stages of panicle and seed
were harvested from field-grown Oryza sativa ssp. Indica
var. IR64 and immediately frozen in liquid nitrogen to
avoid wounding. For abiotic stress treatment, 7-day-old
rice seedlings were treated with cold, dehydration and salinity stresses for 3 h along with untreated control samples
according to Singh et al. [39]. Treated seedlings were frozen
in liquid nitrogen immediately.
Microarray experiment and expression profiling
Expression profiles were generated using microarray data
which were submitted to GEO NCBI under the series
accession GSE6893 and GSE6901. Raw expression data
files (.cel) for three vegetative stages (mature leaf, 7-day-old
seedlings and their roots), 11 reproductive stages (P1–P6
panicle stages and S1–S5 seed stages) and three abiotic
stress conditions, namely, cold, drought and salt stresses,
were downloaded and further analysis was carried out
according to Ray et al. [82].
Expression analysis by qPCR
Microarray expression data under abiotic stress conditions
for a few selected genes, which showed significant differential regulation, were validated by qPCR using two biological replicates according to Singh et al. [39]. Primers used
for qPCR analysis are listed in Table S2.
Statistical analysis
All the expression data are presented as mean SD. A twotailed Student’s t test was performed to determine the statistical significance among the samples. A P value of < 0.05 was
considered statistically significant and differentially expressed
genes were selected on this criterion along with a fold change
value ≥ 2. Statistical correlation between the expression
patterns from two methods (microarray and qPCR) was calculated with the Pearson correlation coefficient r.
Preparation of constructs for subcellular
localization and yeast complementation
The ORF of calcium transporters lacking a stop codon
were amplified from stress treated cDNA of rice (IR64)
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
A. Singh et al.
with gene specific primers using iProof high-fidelity DNA
polymerase (Bio-Rad, Hercules, CA, USA). The constructs
for the translation fusion of GFP-OsCNGC7, GFP-OsANN2, GFP-OsTPC1 and GFP-OsGLR1.1 were prepared
by cloning the respective ORF in Gateway entry vector
pENTR-D/TOPO (Invitrogen) and mobilized to pSITE2CA, a gateway cloning vector [83]. To prepare OsACA7-GFP and OsECA2-GFP constructs, the respective
coding sequences were cloned in the binary vector
pGPTVII.GFP.Kan [84]. Expression of the cloned genes in
both the vectors was regulated by CaMV 35S promoter.
OsACA7 complete coding sequence and deletion fragment
(ΔOsACA7) were cloned in pYES2 vector for the yeast
complementation experiment. All the constructs were verified by sequencing. A list of primers used in these experiments is given in Table S3.
Agrobacterium infiltration of
Nicotiana benthamiana and confocal microscopy
Agrobacterium tumefaciens (GV3101: pMP90) was transformed with the plasmids of GFP constructs of calcium
transport element genes according to Singh et al. [85]. GFP
was detected by excitation at 488 nm and scanning at 500–
535 nm; mCherry was excited at 543 nm and scanned at
600–630 nm; OFP was excited at 543 nm and scanned at
565–595 nm. Auto-fluorescence of plastids was detected at
650–720 nm. For co-localization experiments sequential
scanning was done for both the channels and the data were
then merged together to show overlapping signals. All the
images were further processed using LEICA LAS AF LITE software.
Yeast transformation, complementation and
growth
Yeast complementation assay was performed in Saccharomyces cerevisiae wild-type strains K601/W303-1A (MATa,
leu2, his3, ade2, trp1 and ura3) and triple mutant K616
(MATa pmr1::HIS3 pmc1::TRP1cnb1::LEU2, ura3). Yeast
strains K601 and K616 were transformed with empty vector pYES2 as control, OsACA7/pYES2 and ΔOsACA7/
pYES2 (Δ2-68 OsACA7) by the LiAc/ss carrier DNA/PEG
method [86]. Transformants were selected for uracil prototrophy by plating on synthetic medium minus uracil (SCUracil; 6.7 gL 1 yeast nitrogen base without ammonium
sulfate, without amino acids, 5 gL 1 ammonium sulfate,
1.92 gL 1 of dropout mix without uracil, 100 mgL 1 adenine, 10 mM CaCl2, 2% glucose and 2% agar). For complementation studies, a single colony of each transformant
was grown in SC-Uracil with 10 mM CaCl2 to mid log
phase; cultures were pelleted and washed thrice with 10 mM
EGTA, pH 5.5; A600 of 0.5 in water was adjusted and a
dot assay was performed on SC-Uracil-glucose/galactose
with either 10 mM CaCl2 or 10 mM EGTA pH 5.5. To
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
Calcium transport elements in rice
observe the growth, plates were incubated at 30 °C for
4 days.
Salt stress assay in yeast
To perform salt tolerance assay for OsACA7, the free
Ca2+ concentration was maintained by Ca2+ EGTA buffer
in the uracil dropout SC medium (buffered with 5 mM
MES, pH 6.0) containing 2% galactose. Different yeast cultures grown to mid log phase were pelleted and washed
with 10 mM EGTA (pH 5.5) for dot assay. Yeast cells were
serially diluted with MilliQ water to obtain 10 1, 10 2 and
10 3 fold dilutions; 5 lL of each serial dilution was dotted
on the media plates having different concentrations of free
Ca2+, i.e. 20 lM, 100 lM, 680 lM and 10 mM, in the
absence or presence of 400 mM NaCl. Plates were incubated at 30 °C and growth was recorded after 4 days of
incubation.
Acknowledgements
We are grateful to Professor J€
org Kudla (Universit€
at
M€
unster, Germany) for providing the plasmid CBL1nOFP and pGPTVII.GFP.Kan vector; Dr Michael
Goodin (University of Kentucky, USA) for the pSITE
2CA vectors; and Professor Kyle W. Cunningham
(Johns Hopkins University, Baltimore, MD, USA) for
yeast wild-type strains K601/W303-1A and triple
mutant K616. Arabidopsis Biological Resource Center,
Ohio, is acknowledged for providing the organelle
tracker plasmids. This work was partially supported
by grants from the University of Delhi, Department of
Biotechnology, Department of Science and Technology, and the Council of Scientific and Industrial
Research (CSIR), India, to GKP. AS, PK, AKY and
VB acknowledge CSIR for their research fellowship.
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Supporting information
Additional supporting information may be found in
the online version of this article at the publisher’s web
site:
Fig. S1. Chromosomal localization of calcium transport elements on 12 rice chromosomes.
Fig. S2. GFP-OsTPC1 expressing only on one side of
the chloroplast (arrow, first row) and nucleus (arrow,
second row) and not on the side facing the plasma
membrane. The magnified view of epidermal cells
FEBS Journal 281 (2014) 894–915 ª 2013 FEBS
Calcium transport elements in rice
expressing GFP-OsTPC1 depicts tonoplast circular
extensions into the lumen of the vacuole (third row).
The arrow denotes the cell-wall space between two
adjacent cells.
Fig. S3. Expressed GFP-OsGLR1.1 in epidermal cells
does not co-localize with the plasma membrane marker protein CBL1n-OFP (first row). A magnified view
of epidermal cells expressing GFP-OsGLR1.1 clearly
shows a network-sheet-like structure (second row).
Fig. S4. Magnified view of the nucleus of Nicotiana
epidermal cells expressing GFP-OsCNGC7 fusion protein, showing preferential localization around the
nucleus (scale bar 10 lm). (a)–(f) Different sections in
the z direction. The arrow represents the position of
the nucleus.
Fig. S5. Osmotic stress tolerance analysis of K616
yeast mutant, complemented with OsACA7. Growth
of yeast mutant transformed with different constructs
of OsACA7 and vector pYES2 on SC-Uracil with
galactose is shown.
Table S1. Microarray expression data for rice calcium
transport elements during development and abiotic
stresses.
Table S2. Primers used for qPCR expression analysis
of rice calcium transport elements.
Table S3. Primers used in subcellular localization and
yeast complementation of rice calcium transport elements.
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