Genome-wide expressional and functional analysis of calcium transport elements during abiotic stress and development in rice.

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.

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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


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 896

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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

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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


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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

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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


899


A

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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


6.28

10.39

D

OsGLR3.2


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


F


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

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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


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

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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

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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.


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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.


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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.


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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


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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

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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


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


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 908

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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

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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-

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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


Vacuolar lumen

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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].

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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/

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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)


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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


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



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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