Accepted Manuscript Title: Comprehensive structural, interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice Author: Poonam Kanwar Sibaji K. Sanyal Indu Tokas Akhilesh K. Yadav Amita Pandey Sanjay Kapoor Girdhar K. Pandey PII: DOI: Reference:

S0143-4160(14)00092-X http://dx.doi.org/doi:10.1016/j.ceca.2014.05.003 YCECA 1567

To appear in:

Cell Calcium

Received date: Revised date: Accepted date:

9-12-2013 13-5-2014 27-5-2014

Please cite this article as: P. Kanwar, Comprehensive structural, interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice, Cell Calcium (2014), http://dx.doi.org/10.1016/j.ceca.2014.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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*Graphical Abstract

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*Highlights (for review)

Comprehensive structural, interaction and expression analysis of CBL and CIPK complement during abiotic stresses and

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development in rice

Research Highlight Genome-wide analysis

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Calcium signaling

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CBL-CIPK networking in rice

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Comprehensive structural, interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice Poonam Kanwar, Sibaji K. Sanyal, Indu Tokas, Akhilesh K. Yadav, Amita Pandey, Sanjay

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Kapoor, Girdhar K. Pandey*

Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez

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Road, Dhaula Kuan, New Delhi-110021, India

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*Author for correspondence: Tel. +91 11-24116615, Fax. +91 11-24115270

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Email: [email protected]

Author’s Email:

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Poonam Kanwar: [email protected], Sibaji K. Sanyal: [email protected], Indu Tokas: [email protected], Akhilesh K. Yadav: [email protected],

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Amita Pandey: [email protected], Sanjay Kapoor: [email protected],

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Abstract Calcium ion is involved in diverse physiological and developmental pathways. One of the important roles of calcium is a signaling messenger, which regulates signal transduction in plants. CBL (calcineurin B-like protein) is one of the calcium sensors that specifically interact with a family of serine-threonine protein kinases designated as CBL-interacting

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protein kinases (CIPKs). The coordination of these two gene families defines complexity of the signaling networks in several stimulus-response-coupling during various environmental

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stresses. In Arabidopsis, both of these gene families have been extensively studied. To understand in-depth mechanistic interplay of CBL-CIPK mediated signaling pathways,

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expression analysis of entire set of CBL and CIPK genes in rice genome under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages) were done using microarray expression data. Interestingly,

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expression analysis showed that rice CBLs and CIPKs are not only involved in the abiotic stress but their significant role is also speculated in the developmental processes.

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Chromosomal localization of rice CBL and CIPK genes reveals that only OsCBL7 and OsCBL8 shows tandem duplication among CBLs whereas CIPKs were evolve by many

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tandem as well as segmental duplications. Duplicated OsCIPK genes showed variable expression pattern indicating the role of gene duplication in the extension and functional diversification of CIPK gene family in rice. Arabidopsis SOS3/CBL4 related genes in rice

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(OsCBL4, OsCBL5, OsCBL7 and OsCBL8) were employed for interaction studies with rice and Arabidopsis CIPKs. OsCBLs and OsCIPKs are not only found structurally similar but likely to be functionally equivalent to Arabidopsis CBLs and CIPKs genes since SOS3/CBL4 related OsCBLs interact with more or less similarly to rice and Arabidopsis CIPKs and

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exhibited an interaction pattern comparable with Arabidopsis SOS3/CBL4. Key words: abiotic stress; reproductive development; expression; interaction; signaling

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1. Introduction Gene networking plays a major role in regulating the diverse biological responses in the cell. In context to this, CBL and CIPK are two gene families, which work together in pair in signaling networks [1-6]. During adverse environmental and developmental condition, plants respond by initiating a series of signaling processes. Elevation of cytosolic calcium

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concentration is a primary event in this response. Specificity of the signal itself depends on the calcium signature and/or on the various calcium binding proteins [7-13]. Calcineurin B-

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like (CBL) protein, a unique and novel group of calcium sensors present in plants believed to perceive fluctuation in cellular calcium levels [14]. CBLs are known to be providing the

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specificity of signal by associating and regulating specific member of serine-threonine protein kinase family, CBL-interacting protein kinases (CIPKs) [10, 15-17]. CIPKs generate specificity and overlap in signaling response by interacting with diverse array of proteins in

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the cell. Functional analysis of several CBL and CIPK family members in Arabidopsis have shown that various signaling processes depends on the structure, expression, localization and

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interaction between CBL and CIPK proteins [3, 4, 10, 18-23]. Previously, role of CBL and CIPK members have been implicated in abiotic stress and nutrient signaling but recently,

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CBL and CIPK genes have also been projected to be involved in developmental processes, ROS signaling and biotic stress [6, 24-28]. The CBL-CIPK signaling pathway is also evident in rice and others agronomically important crops. Rice genome also comprised of a large

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complement of CBL and CIPK genes, 10 CBLs and 30 CIPKs [13], with some of the CIPKs recently identified [29, 30]. Like Arabidopsis, a few of the rice CBL and CIPK members have also been implicated in abiotic stress signaling [29, 31-33]. However, knowledge pertaining to most of the CBL and CIPK genes is still not well worked out in agronomically important

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cereal crops.

Similarity in the sequence and structure may result in functional similarity of the CBLs and CIPKs within intra and inter-species. Like in Arabidopsis, role of CBL1 and CBL9 in ABA and osmotic stress signaling [12, 18, 20]; CBL2, CBL3 in ion homeostasis in vacuole [34]; CIPK3, CIPK15, and CIPK26 in ABA signaling [4, 19, 35, 36]; CIPK9, CIPK23 in lowpotassium signaling [3, 21-23] are found to be apparently similar. Not only this, interaction of CBL9 with CIPK3, and CBL1 with CIPK15 also hints towards interlinking of structure, function and interaction in CBL and CIPK networking [4, 35].

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Moreover, CBLs and CIPKs from Arabidopsis, pea and rice revealed a high degree of structural and functional conservation [17, 37]. Arabidopsis AtAKT1 (Arabidopsis

K+

transporter 1) counterpart in Vitis venifera VvK1.1 can be stimulated by co-expression of AtCIPK23-AtCBL1 in Xenopus oocytes [38]. Not only this, VvCIPK4-VvCBL1 and VvCIPK3-VvCBL2 pairs can stimulate another potassium channel VvK1.2 [39]. The wellstudied CBL-CIPK signaling pathway in Arabidopsis is SOS (salt overly sensitive) pathway.

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SOS3 (CBL4)-SOS2 (CIPK24) interactions recruit SOS2 to the plasma-membrane leading to activation of SOS1 (Na+/H+-antiporter), thereby maintaining ion homeostasis during salt

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stress [40-45]. Functional relatedness of this pathway has also been discovered in various other species [46-51]. In poplar, putative SOS genes i.e. PtSOS1, PtSOS2, and PtSOS3 were

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reported to be working in SOS pathway [48]. Similarly, MdSOS2 and MdSOS3, in case of Malus Domestica [49]. SlSOS3, SlSOS2 and SlSOS1 related to salt stress in tomatoes [45,

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47, 50]. Not only this, NAF motif which mediates CBL-CIPK physical interactions share extreme conservation among all identified CIPKs in green algae and early diverging land plants further support that homolog of CBLs and CIPKs are functionally linked [51]. These

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findings suggest that CBL-CIPK network is not only conserved among different plant species but CBL and CIPK can complement the signaling pathway in other species.

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Considering these facts, we have examined mechanism of rice CBL and CIPK genes networking by comprehending information store in genomic organization, protein structure, expression profiling. We have also performed comprehensive interaction analysis via yeast

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two-hybrid for four orthologs of SOS3/AtCBL4 in rice (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) with CIPKs of rice and Arabidopsis.

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2. Results and discussion

2.1 Chromosomal localization and gene duplication A total of ten CBL genes were identified in Oryza sativa [13]. Chromosomal localization revealed that all rice CBL genes are distributed on six chromosomes (Chromosome 1, 2, 3, 5, 10, and 12) (Fig. 1). A maximum of three CBL genes were located on the largest rice chromosome (chromosome 1). On chromosome 2, one pair of a tandemly duplicated pair (OsCBL7:OsCBL8) was found, which suggest that duplication might have played much less role in the evolution of rice CBL genes (Fig. 1). None of the segmental duplications were found in the rice CBL gene family as compared to the Arabidopsis that has two segmental

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duplicated CBL pairs [13]. OsCBL3 exhibits maximum number of ESTs among all OsCBLs (Table 1A). Splice variants were found only in OsCBL6 and OsCBL9. Maximum numbers of rice CBL orthologs were found for Arabidopsis CBL4/SOS3 i.e OsCBL4, OsCBL5, OsCBL7 and OsCBL8. CIPK genes were found to be variably distributed among all on the rice chromosomes, except

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chromosomes 4. Maximum numbers of OsCIPK genes and segmental duplications were found on chromosomes 1 and 5 (Fig. 1). In contrast to CBLs, a large extent of gene duplication in CIPKs might be responsible for evolution of this family (Fig. 1). In our

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analysis, we found four tandem duplications and eleven segmental duplications (Fig. 1, Table

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2). Out of that, three tandem duplications (OsCIPK5:OsCIPK13, OsCIPK12:OsCIPK30 and OsCIPK2:OsCIPK29) and segmental duplication (OsCIPK14:OsCIPK15) were earlier reported by Kolukisaoglu et al [13] . OsCIPK10 exhibits 244 ESTs, maximum among all

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OsCIPKs (Table 1B). OsCIPK1, OsCIPK3, OsCIPK8, OsCIPK9, OsCIPK10, OsCIPK11, OsCIPK14, OsCIPK23, OsCIPK26, OsCIPK31, OsCIPK32 and OsCIPK33 have splice

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variants as mentioned in the Table 1B. OsCIPK31, which have maximum of eight splice variants among all these OsCIPKs. Functional impact of these splice variants is still not clear

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and requires additional molecular characterization.

2.2. Structural analysis of rice CBLs and CIPKs proteins

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CBLs possess four EF-hands and newly identified PFPF motif, which is required for phosphorylation of CBLs by CIPKs [52]. Among all OsCBLs, OsCBL6, OsCBL9 and OsCBL10 were found to be with extended N terminal region (Fig. 2A). Distance between the EF-hands was found to be same in all OsCBLs. In addition, five OsCBLs (OsCBL1,

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OsCBL4, OsCBL5, OsCBL7 and OsCBL8) contain a conserved N-myristoylation motif. MEME tool [53] was used to compare conserve motifs in the all CBLs in Arabidopsis and rice protein sequences. All the motifs found were highly conserved in both rice and Arabidopsis. Similar to Arabidopsis CBLs, OsCBLs also possess conserved serine residue in the PFPF motif, which is phosphorylated by CIPKs [52]. Ser residue is conserved in the PFPF motif for all ten OsCBLs proteins (Fig. 2B). Whereas, Arabidopsis CBL6 do not have this conserved serine residue in PFPF motif and CBL5 contains threonine as putative site of phosphorylation in the PFPF motif [52] CIPK proteins contain conserved N-terminal catalytic kinase and C-terminal regulatory domain [15, 54]. Like typical of protein kinases, the N-terminal catalytic domain has ATP 5

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binding site and activation loop (Takahashi 2011). The C-terminal regulatory domain contains FISL or NAF motifs that mediates interaction with CBLs [1, 54-56] and other motif called as PPI motif, mediates interaction with type 2C protein phosphates [57]. All of the OsCIPKs contained catalytic kinase domain and NAF domain. Surprisingly, OsCIPK4 does not possess NAF as well as PPI motifs. OsCIPK28 has the longest C-terminal regulatory domain whereas OsCIPK21 and OsCIPK28 have the longest N-terminal region among all the

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OsCIPKs. OsCIPK27 is the smallest CIPK because of shorter C-terminal regulatory region and the important PPI domain responsible for interaction with protein phosphatases is absent

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that they are highly conserved across these two species (Fig. 3B).

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in this protein (Fig. 3A). Comparison of these motifs between Arabidopsis and rice showed

2.3 Rice CBLs-CIPKs show distinct differential expression during abiotic stress and

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development

Preliminary reports on some of the CBL and CIPK genes in rice showed the differential expression in various abiotic stresses [31, 58, 59]. These previous reports enticed us to

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generate a complete genome wide expression profile of CBL-CIPKs under different abiotic stresses (salt, drought and cold). Genome-wide expression profile of rice CBLs and CIPKs

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was generated using Affymetrix rice genome arrays data for 7 d old rice seedlings treated with three abiotic stress conditions (salt, cold and drought). The corresponding probe sets were available for all the OsCBLs and OsCIPKs on the microarray gene chip (Table S1).

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However, the probe set for two pairs of OsCIPKs {(OsCIPK14 and OsCIPK15) &(OsCIPK31 and OsCIPK33)} turn out to be same. Genes, either up- or down- regulated with an expression fold change of ≥ 2 w.r.t control were considered to be differentially regulated.

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In comparison to control (untreated 7 d old seedlings), a total of four CBL members out of ten expressed differentially (either up- or down-regulated) under three abiotic stress conditions with several fold changes in expression (Fig. 4A, 5A, Table S1). OsCBL3, OsCBL10 were commonly up-regulated whereas OsCBL8 was down-regulated under both salt and drought stress conditions while OsCBL4 was exclusively down-regulated under cold stress condition (Fig. 5A). In OsCIPK gene family, most of the genes were differentially regulated by abiotic stresses (Fig. 4B, 5B, Table S1). Out of 33 genes, none of the gene is commonly expressed differentially under all the abiotic stresses tested. Most of the genes, which were exclusively differentially expressed in the drought and cold stresses, were downregulated, except OsCIPK9 in the drought and OsCIPK26 in the cold stress showing

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upregulation. OsCIPK32 and OsCIPK33 were up-regulated exclusively in salt stress whereas OsCIPK6, OsCIPK16, OsCIPK17, OsCIPK19, OsCIPK25, and OsCIPK29 are commonly up-regulated in the salt and drought stresses. OsCIPK21 was up-regulated in salt stress conditions, and down-regulated under cold stress. OsCIPK4 was found commonly downregulated in both drought and cold stresses (Fig. 4B, 5B, Table S1). Upon relaxing our analysis stringency to 1.2 and 1.5 fold, many of the OsCBL and OsCIPK genes were found to

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be differentially regulated by abiotic stresses (Table S2).

The functional role of CBL and CIPKs in development still required to be investigated.

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Recently, a few reports presented functional clues about specific CBL and CIPKs, which

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were involved in the developmental processes [24, 26]. The holistic expression profile of OsCBLs-OsCIPKs during different vegetative and reproductive developmental stages will provide more insight into their critical roles in plant growth and development. From the

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expression profile of eleven reproductive developmental stages of rice including, six panicle stages (P1-P6) and five seed developmental stages (S1-S5) along with three vegetative stages

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namely; mature leaf, root and seedling, a total of 6 OsCBL genes expressed differentially during reproductive developmental stages when compared to three vegetative developmental stages (Fig. S1). Almost 22 CIPKs out of 33 were showing differential regulation during

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these two developmental processes (Fig. S2) conveying the elaborate role of CBL and CIPK in development, which is still unexplored. Since most of the OsCBL and OsCIPK transcripts

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were over or under expressed by both abiotic stresses and/or during developmental stages, differential overalapping expression pattern OsCBLs and OsCIPKs were studied. In case of OsCBLs, OsCBL1 and OsCBL5 were found to be up-regulated in seed stage whereas in panicle OsCBL6 was up-regulated and OsCBL5 downregulated (Fig. 4C, 5C, Table S2, S3).

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OsCBL4 was found to be down-regulated whereas OsCBL10 was up-regulated commonly under abiotic stresses and panicle development. OsCBL8 was commonly down-regulated in both reproductive development (panicle and seed stages) and stress condition (Fig. 4C, 5C, Table S2, S3). All the OsCIPK genes (OsCIPK11, OsCIPK14, OsCIPK18, OsCIPK20 and OsCIPK24), which were differentially expressed in the panicle stages were found to be upregulated. Whereas, OsCIPK2, OsCIPK25 and OsCIPK26 differentially expressing exclusively in the seed stages were downregulated (Fig. 4D, 5D, Table S3, S4). OsCIPK12 was commonly upregulated and OsCIPK3, OsCIPK17 and OsCIPK22 were commonly downregulated between panicle and seed developmental stages. OsCIPK6 and OsCIPK21 were found to be commonly up-regulated whereas OsCIPK4, OsCIPK5, OsCIPK7 and

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OsCIPK23 were commonly down-regulated in all the reproductive developmental stages and abiotic stresses. OsCIPK9, OsCIPK25 and OsCIPK32 were up-regulated during panicle development and abiotic stresses whereas; OsCIPK1 and OsCIPK10 were down-regulated under these conditions. Only OsCIPK18 was down-regulated during seed development and abiotic stresses (Fig. 4D, 5D, Table S3, S4). The commonly affected OsCBLs and OsCIPKs in abiotic stresses and development stages hint toward their possible connections in stress and

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development together in rice.

Such overlapping expression pattern was also reported by Tripathi et al., [24] where they

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showed involvement of CIPK6 in root development, as well as in the salt-stress response.

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Beside this an important link between development and abiotic stresses was unearthed by the discovery of involvement of SOS2/CIPK24 and GIGANTEA (GI) protein, which is an important component of a circadian clock-controlled signaling pathways regulating flower

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development [28]. Under normal condition (when plants are not challenged by stress), SOS2/CIPK24 is complexed with GI protein and is not available for activating SOS1. But

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during the salt stress, SOS2/CIPK24 is released from the SOS2/GI complex because GI protein is degraded and hence activate the SOS1 [28]. Not only lateral root development in sos3-1 mutant is highly affected particularly under low salt condition [60], it has been

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suggested that SOS3 gene modulates lateral root developmental plasticity and adaptation in response to low salt stress [60]. OsSOS3 (OsCBL4) and OsSOS2 (OsCIPK) exhibited a

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rhythmic and diurnal expression pattern suggesting the SOS pathway might be influenced by diurnal rhythm [61]. These important findings suggest the role of CBLs and CIPKs in bringing coordination between the abiotic stresses and the developmental conditions like flowering and root development.

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2.4 Functional divergence between duplicated OsCBLs and OsCIPKs Because some of the rice CBLs and CIPKs were found to be present as tandem and segmentally duplicated genes on chromosome, we have also generated the expression profile for these duplicated OsCBL and OsCIPK genes using microarray data for the three abiotic stresses and entire spectrum of developmental stages. The expression pattern of OsCBLs and OsCIPKs present in tandem (Fig. 6) and segmentally duplicated regions (Fig. 7) was analysed as an area-diagram. In the case of OsCBLs, only one tandemly duplicated pair (OsCBL7: OsCBL8) having 90.1% homology showed retention of function except for up- regulation of OsCBL8 under cold stress. Out of 5 tandemly duplicated pairs of OsCIPKs, two pairs (OsCIPK16:OsCIPK27 and OsCIPK2:OsCIPK29) showed retention of function as reflected 8

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by the expression pattern. However, the amplitude of expression varied in paired partners (Fig. 6). In another pairs OsCIPK5:OsCIPK13, one of the genes had almost negligible expression

exhibiting

pseudo-functionalization.

The

other

pair

of

genes

(OsCIPK30:OsCIPK12) depicting neo-functionalization because expression pattern was very divergent, exactly opposite for most of the tissue and condition tested (Fig. 6). In the case of segmentally duplicated OsCIPK genes, the expression pattern is variable. The duplicated

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genes group, (OsCIPK2:OsCIPK14:OsCIPK15) (OsCIPK32:OsCIPK33), showed similar expression pattern depicted that these duplicated genes may exhibit retention of functions Rest

of the

segmentally duplicated

(OsCIPK1:OsCIPK17),

(OsCIPK11:OsCIPK28), and

(OsCIPK4:OsCIPK7),

(OsCIPK6:OsCIPK27),

(OsCIPK5:OsCIPK12:OsCIPK20)

followed

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(OsCIPK3:OsCIPK31),

gene pairs

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(Fig. 7).

pseudo-

functionalization (Fig. 7). All the different functional attributes such as neofunctionalization,

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subfuctionalization as well as pseudofunctionalization of duplicated OsCBLs and OsCIPKs pairs hints toward their functional redundancy, relatedness and variability in their respective

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gene families.

To understand the relationship between evolution and functional divergence of duplicated OsCBLs and OsCIPKs pair, synonymous substitutions per synonymous site (Ks) was

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estimated to determine the age of comparable duplicate gene pairs (Table 3). Among CBLs, only single tandemly duplicated gene pair (OsCBL7: OsCBL8) was duplicated approximately MYA.

Among

duplicated

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22.58

(OsCIPK32:OsCIPK33)

pair

was

OsCIPKs recently

gene

pairs,

duplicated

it (2.5

was

found

MYA)

that while

(OsCIPK3:OsCIPK31) pair shows oldest duplication event (65.8 MYA) (Table 3). By comparing the expression of gene pair (OsCIPK32:OsCIPK33) and (OsCIPK14:OsCIPK15)

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retention of functions was observed for both the gene pairs, which were found to be recently duplicated. Whereas, other duplicated OsCIPKs gene pairs, (OsCIPK4:OsCIPK7) (OsCIPK1:OsCIPK17), (OsCIPK6:OsCIPK27), (OsCIPK3:OsCIPK31), were estimated to have evolved, very early and because of this might have followed pseudofunctionalization (Table 3). Varying expression patterns of segmentally and tandemly duplicated CIPK genes are suggesting a role of chromosome gene duplication in the expansion and evolution of this gene family in rice. The Ka/Ks ratio (synonymous substitutions to non-synonymous substitutions) were found to be less than 1 for the duplicated gene pair indicating a purifying selection pressure on both OsCBLs and OsCIPKs duplicated genes pairs (Table 3). 2.5 Functional resemblances of CBLs and CIPKs between rice and Arabidopsis 9

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Plasma membrane-targeting calcium sensor, SOS3/CBL4; SOS3/CBL4 -interacting kinase, SOS2/CIPK24; and plasma membrane localized Na+/H+ exchanger SOS1 are three major components of SOS pathway in Arabidopsis [1, 44]. Ortholog comparison facilitates the finding of potential targets and functional resemblances by detecting genomic correlations among different organisms. To determine evolutionary relatedness, multiple sequence alignment of amino acid sequences of the Arabiodpsis SOS3/AtCBL4 and OsCBLs was

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performed using ClustalX2 and an un-rooted tree was generated using the neighbor-joining algorithm of MEGA5 (Fig. S3A). SOS3 associated closely with a group of four rice CBLs

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(OsCBL4, OsCBL5, OsCBL7 and OsCBL8) [13]. Structurally in terms of number and position of motif and protein length, all four genes were found to be similar (Fig. 2). Like

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SOS3, all four rice CBLs (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) were predicted to possess N-myristoylated site in their proteins sequence, a post-translational modification that

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is essential for SOS3/AtCBL4 function [41]. Evolutionary relationship of SOS3/AtCBL4 with rice CBLs led to identify homology of 66.2%, 60.6%, 67.1%, and 66.7% with OsCBL4, OsCBL5, OsCBL7, and OsCBL8 respectively. Among them OsCBL7 and OsCBL8 (90.1%);

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and OsCBL4 and OsCBL5 (69.5%) were more similar to each other and present closer also in phylogenetic clade (Fig. S3A).

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Rice also possesses the similar SOS pathway. The rice SOS pathway components; OsSOS3/OsCBL4, OsSOS2/OsCIPK24 and OsSOS1, were able to complements yeast salt sensitive mutants AXT3K and hence provide salt tolerance to the yeast when all the three

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component were co-transformed [46]. The AtSOS2/CIPK24 was also able to phosphorylate OsSOS1 in an in vitro phosphorylation assay. Beside this, Martı´nez-Atienza et al. [46] speculated OsCBL7 and OsCBL8 with high degree of sequence similarity might be functionally redundant genes. However upon overexpression of OsCBL8 in rice, the

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phenomenon of salt tolerance was observed in the transgenic plants [59]. OsCBL8 was also reported to localize to the plasma membrane like OsCBL4 and AtSOS3 [59]. Previous results have shown both SOS1 and SOS2/CIPK24 expressed in both roots and shoots, while SOS3/CBL4 is mainly expressed in root tissues and all of them induced by salt stress in roots [42, 62, 63]. We have analysed the stress, developmental and tissue specific expression pattern expression pattern of all rice orthologs of Arabidopsis SOS3/CBL4. Expression analysis has shown both striking similarities and differences. Expression of OsCBL4 is more in leaf and shoot, less in roots which is totally opposite to Arabidopsis SOS3/CBL4 and other rice OsCBLs (OsCBL5, OsCBL7 and OsCBL8), which showed higher

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expression in the roots (Fig. S3B, S4). Beside this OsCBL7 doesn’t exhibit any differential regulation in any developmental stages and abiotic stresses. In salt stress, OsCBL4 is upregulated and OsCBL8 is down-regulated (Fig. S3B). OsCBL5 was found to be down regulated in all five stages of panicle and first three stages of seed development, but upregulated in last two stages of seed development. Upon comparing the tissue specific expression of OsCIPK24 with OsCBL4, OsCBL5, OsCBL7 and OsCBL8, it was found that

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the expression pattern of OsCIPK24 was shared by all four OsCBLs. Higher expression was seen in the stigma for OsCIPK24, OsCBL7 and OsCBL8 and in the stamen in case of

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OsCIPK24 and OsCBL4 (Fig. S4). All of these aforementioned points hint toward the possibility that there might be alternative calcium sensors beside SOS3/CBL4 regulating salt

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stress pathways in rice, in addition to the canonical SOS pathway. Therefore, other members of CBLs and CIPKs could possibly be explored in rice for a detail understanding of salt stress

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signaling by different behaviour of orthologs of SOS pathway.

2.6 Interaction analysis of rice SOS3 related genes with CIPKs of rice and Arabidopsis

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Previous investigations have established that the specific CBL-CIPK complex formation contributes to generating specificity in the CBL-CIPK signaling network [3, 4, 16, 23, 54].

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To get more insight about interaction strength, and interaction specificity, we employed yeast two-hybrid matrix method, which involved a subset of 6 OsCBL and 5 OsCIPK proteins to uncover selective and distinctive interactions between the rice CBL and CIPK

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proteins. Among the six CBLs, four of the SOS3/AtCBL4 orthologs i.e. OsCBL4, OsCBL5, OsCBL7 and OsCBL8 were used and rest two OsCBL1, which has N-myristoylated site and OsCBL2, which do not possess this site were included in the yeast two-hybrid based protein interaction analysis of OsCBLs from different clades as control (Fig. 8, S5). Five OsCIPKs;

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OsCIPK1, OsCIPK12, OsCIPK17, OsCIPK26 and OsCIPK30 were used to determine their interaction with rice CBLs in this study. Of all the six OsCBL proteins examined, OsCBL7 and OsCBL8 are the closest members, tandemly duplicated pair and closest ortholog of SOS3/AtCBL4. Both, OsCBL7 and OsCBL8 interact strongly with OsCIPK1 and weakly with OsCIPK17 and OsCIPK26 (Fig. 8). Similar kind of interaction was seen in the case of OsCBL5 but there was no interaction with OsCIPK17 (Fig. 8). Meanwhile, OsCBL4 did not show strong interaction with any of the OsCIPKs tested in this study. Nonetheless, OsCBL1 interacted strongly with OsCIPK1 and OsCBL2 interacted with both OsCIPK1 and OsCIPK26 (Fig. S5).

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Previously in various studies cross species interaction were performed between CBL and CIPK to understand the functional relationship among species [24, 49]. It is already known that rice CBL can interact with the Arabidopsis CIPKs [64, 65]. Interactions of Arabidopsis SOS3/CBL4 and its all four rice orthologs (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) were checked with all the 26 Arabidopsis CIPKs by yeast two-hybrid assay to investigate if the ortholog interactions match the pattern of interaction of Arabidopsis SOS3/CBL4 (Fig. 9). It

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was found that SOS3/CBL4 and three rice orthologs OsCBL5, OsCBL7 and OsCBL8 interacted with the same Arabidopsis CIPKs (CIPK1, CIPK2, CIPK5, CIPK6, CIPK8,

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CIPK12, CIPK16, CIPK17, CIPK18, CIPK24 and CIPK25) except CIPK6 and CIPK25, which was specific to CBL4/SOS3. On contrary, CIPK5, CIPK9, CIPK15, CIPK16, CIPK24

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and CIPK26 interacted only with OsCBL4. Based on the interaction analysis, it has been observed that most of the rice SOS3/CBL4 orthologues, interacted with the different CIPKs

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in a similar way as Arabidopsis SOS3/CBL4 with Arabidopsis CIPKs.

OsCBL4, OsCBL5, OsCBL7 and OsCBL8 were able to interact with AtSOS2/CIPK24, thus

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indicating a conservation of SOS pathway even in a cross species interaction analysis. In Arabidopsis beside CBL4, CBL10 also interact with CIPK24/SOS2 leading to a novel Ca2+signaling pathway for salt tolerance in Arabiodpsis [63, 66]. Unlike, CBL4–CIPK24/SOS2

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complex, which take place and function at plasma membrane, CBL10–CIPK24/SOS2 complex is associated with the vacuolar compartments and speculated to be responsible for

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Na+ ion sequestration and detoxification in the plant cell [66]. This fact suggests the intricate and complex nature of salt stress signaling pathways regulated by multiple CBL-CIPK in plants. Since salt tolerance is a complex trait in plant and we truly believe that multiple signaling pathways might be operating to regulate this complex trait. To further explore

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possibility of an alternate pathway regulating this salt stress signaling process in rice and to determine which rice SOS3/AtCBL4 ortholog-OsCIPK24 complex functionally work in the spatial and temporal manner require detail functional analysis. Similar to Arabidopsis, the existence of multiple members of CBL and CIPK gene family in rice adds to the complexity of CBL-CIPK function in plants. All four rice orthologs (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) of Arabidopsis CBL4/SOS3 have more or less similar CIPKs as interacting partner (both in rice and Arabidopsis). The interaction analysis also brought forward some very specific rice and Arabidopsis CIPK interactors. When we generated the phylogenetic tree with NAF domain of the all Arabidopsis CIPKs, it was found that those CIPKs, which were interacting in the yeast two-hybrid analysis, fall in the specific 12

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clades that possibly suggest functional relatedness (Fig. S6). This kind of study will help in understanding the specificity of CBL-CIPK complex formation, though in planta, the specificity of the interaction might also be guided by temporal and spatial expression pattern, and subcellular localization of the interacting OsCBL and OsCIPK. Moreover, the change in CBLs subcellular locations upon the perception of extra-cellular signals might serve as an additional control in regulating the interaction of CBLs and CIPKs. Cross-spices interaction

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analysis of CBLs and CIPKs shows that interacting CIPK partners of SOS3/CBL4 are shared by its orthologs in rice also. This analysis possibly conveys that rice CBLs and CIPKs are not

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only structurally similar but also hints toward functional conservation based on their

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interaction analysis.

2.7 OsCBL7 and OsCBL8 might not be functionally redundant

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OsCBL7 and OsCBL8, a tandemly duplicated gene pair has high degree of sequence similarity among OsCBLs. Interestingly, most of the CIPK interacting partners of OsCBL7 and OsCBL8 were found similar but some unique CIPK interactors were also found, which

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implies that they might not be functionally redundant. For example, OsCIPK26 showed strong interaction with OsCBL7 while with OsCBL8 the interaction is weak. When the

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interaction analysis was extrapolated to Arabidopsis CIPKs, some specific CIPKs were identified, which interacted specifically with either of them. OsCBL7 specifically interacted with Arabidopsis CIPK14 whereas OsCBL8 interacted specifically with CIPK10, CIPK11,

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CIPK17 and CIPK18 (Fig. 9). In this regard, the case of Arabidopsis CBL1 and CBL9 present a good example. CBL1 and CBL9, which are segmentally duplicated pair with 89% similarity, but are not considered to be redundant proteins [13]. In-planta characterization of CBL1 and CBL9, implicated them to be regulating salt/osmotic and ABA signaling,

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respectively [2, 20]. Both CBL1 and CBL9 interact and target CIPK23 to plasma membrane in regulating both K+ tolerance and the stomatal closure [3]. Again both CBL1 and CBL9 were shown to interact with CIPK1 in BiFC analysis and regulating the salt and osmotic stress signaling disparately [67]. Recently, CBL1 and CBL9 were also shown to interacted with CIPK26 and regulating the reactive oxygen species (ROS) pathway [6]. They may have functional role in different signaling pathways and choose different interacting partners to propagate the signals downstream. At the same time, they might also be bringing signals from different pathways and channelling them into a single pathway by using the same interactor. In order to understand the slight differences in interacting partners of OsCBL7 and OsCBL8, we closely examine the amino acid sequences of these two proteins and found that in EF13

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hand 1, 3 and 4 there is variation of a single amino acid, whereas EF2 is almost similar in amino acid sequence (Fig. S7). Single amino acid substitution at a very important Ca2+ coordinating position on EF-hand 3 may indicate a differential Ca2+ sensing ability between the homologs [13]. From previous studies on investigation of interacting domain on CBLs responsible for interaction with CIPKs, it has been postulated that the CBLs provide a hydrophobic crevice, which is the site that binds the NAF domain of CIPKs [68]. Detailed

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structural analysis of free AtCBL2 and AtCBL2-AtCIPK14 complex suggested a conformational change in N-lobe (formed by EF-1 and EF-2) and C-lobe (formed by EF-3

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and EF-4) [69]. This was specifically observed in the region containing alpha helices C, D, G and H. SOS2-SOS3 complex also showed that the helices connecting the EF-hands in SOS3

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undergo position change in order to accommodate the NAF of SOS2 [70]. Again, when the amino acid sequences of OsCBL7 and OsCBL8 were compared, a single amino acid

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substitution was seen in this region (Fig. S7). Also the helices E and F connecting the EFhand 2 and 3 also showed some variation in the sequence. Based on this information, a mutagenesis study of these target amino acids can be done in future to dissect the motif or

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domain containing the specific amino acids responsible for determining the specificity

3. Material and Methods

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between very close homologs in selecting interacting CIPK partners.

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3.1 Identification and nomenclature of CBLs and CIPKs in rice genome Kolukisaoglu et al. [13] evoked the presence of 10 OsCBL and 30 OsCIPKs genes in the rice genome. We followed the same nomenclature during our study, although we added three more OsCIPK genes (OsCIPK3, OsCIPK32 and OsCIPK33) and replaced OsCIPK3 with OsCIPK31 (Table 1B) [71]. Genome Annotation Project [RICECHIP.ORG: Support for

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Annotation & Functional Analysis of the Oryza sativa (Rice) Genome (http:// www.ricechip.org /)] was used to identify the number of Expressed Sequence Tags (EST). Various gene attributes such as locus ID, protein (aa) size, introns, were extracted from RGAP ver. 6.1 and knowledge based oryza molecular biological encyclopedia (KOME) (http://cdna01.dna.affrc.go.jp/cDNA). 3.2 Multiple Sequence Alignment, phylogenetic analysis, generation and identification of motifs To mark the conserved domains and motifs, the amino acid sequences of all the rice CBLs were aligned using ClustalW method in MegAlign software of DNASTAR. For phylogenetic

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analysis, OsCBL and OsCIPK protein sequences were used for multiple sequence alignment employing clustalX (version 2.0). From this, an unrooted tree was generated using the neighbor-joining algorithm of MEGA5. A bootstrap statistical analysis was performed with 1000 replicates to test the phylogeny. To identify conserved motifs within position and composition of conserved motifs in Arabidopsis and rice CBLs and CIPKs, MEME Suite-

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GLAM2 version 4.8.0 [53] was employed (http://meme.nbcr.net/meme/intro.html). 3.3 Chromosomal localization and gene duplication

The location of rice CBL and CIPK genes was determined using chromosomal map tool as

Oryzabase-Integrated

Science

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available

Database

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(http://viewer.shigen.info/oryzavw/maptool/MapTool.do) (Fig. 1). The RGAP (rice genome annotation project) version 6.1 (http://rice.plantbiology.msu.edu/) segmental duplication database was explored to find out the segmentally duplicated genes at a maximum length

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distance permitted between collinear gene pairs of 100 kb (Table 2). Genes separated by five or fewer genes on a chromosome were considered to be tandemly duplicated. and amino acid

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sequence homology of these gene products was computed using MegAlign software 5.07©. 3.4 Plant material, growth conditions and abiotic stress treatment

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The tissue samples were collected from field grown rice plants (Oryza sativa ssp. indica var IR64), at different panicle and seed development stages. To avoid wounding, collected panicles were instantly frozen in liquid nitrogen. Stress treatment was given to IR64 rice

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seeds, which were first sterilized with 0.1% HgCl2 and grown in culture room conditions at 28 ± 1°C with a daily photoperiodic cycle of 14 h light and 10 h dark. After seven days of growth, seedlings were subjected to different stress treatments [72].

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3.5 Microarray expression analysis

To perform the microarray based expression analysis, the total RNA was isolated from the three replicates of rice tissues representing different stages of plant development, which included three vegetative stages (mature leaf, 7 d old seedling and their roots), 11 reproductive stages (P1–P6 and S1–S5; representing panicle and seed developmental stages, respectively) and three abiotic stress conditions, i.e., cold, salt, and drought. Microarray experiments were then performed using 51 Affymetrix GeneChip Rice Genome Arrays (Gene Expression Omnibus, GEO, platform accession number GPL2025). The raw data (*.cel) files generated from all the chips were imported to Array Assist 5.0 software (Stratagene, USA) for detailed analysis [73]. The microarray expression data have been deposited in the gene

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expression omnibus (GEO) database at NCBI under the series accession numbers GSE6893 and GSE6901. 3.6 Estimating the age of duplicated gene pairs To calculate the age of segmentally duplicated CIPK pair, the pairwise alignment of gene pairs was performed using Clustal X 2.0.12. The duplication age was estimated by number of

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synonymous substitution per synonymous site (Ks). The Ks values of the duplicate gene pairs were estimated by the program K-Estimator 6.1 [74]. Based on the synonymous substitutions per year (λ) of 6.5×10−9 for rice [75] and by substituting the calculated Ks values, the

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approximate age of duplicated events of the duplicate CBL and CIPK gene pairs was

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estimated (T = Ks/2λ). The selection pressure for these duplicate gene pairs was calculated as Ka/Ks ratio.

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3.7 Cloning of OsCBLs and OsCIPKs

Coding region of respective OsCBL and OsCIPK were amplified from stress treated cDNA

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of rice (IR64) or KOME clone with gene specific primer using Phusion polymerase (Finnzyme, Finnland). The amplified respective CDS were cloned into the linearized blunt end CloneJET1.2 vector (Fermentas, Germany). OsCBL1, OsCBL2, OsCBL4, OsCBL5,

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OsCBL7 and OsCBL8 were cloned into pGAD.GH vector. OsCIPK1, OsCIPK12, OsCIPK17, OsCIPK26 and OsCIPK30 were cloned into pGBT9.BS. Arabidopsis

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SOS3/CBL4 was cloned in pGBT9.BS and all the Arabidopsis CIPKs in both pGAD.GH and pGBT9.BS. All the constructs were confirmed by sequencing. The primer sequences are provided in the Table S5.

3.8 Yeast two-hybrid assays

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A GAL4-based yeast two-hybrid system was used in this study. The lithium acetate (LiAc) method was used to transform yeast cells [76]. Each combination of OsCBLs with OsCIPKs plasmids were co-transformed into yeast strain AH109 and cotransformants were selected on synthetic complete medium lacking leucine and tryptophan (SC–LW). Potential interactors were identified by examining the expression of His3 reporter genes under the control of GAL4 transcriptional factor. Spot dilution assays were performed by inoculating transformed yeast cells in a medium lacking leucine and tryptophan (SC–LW) and allowed to grow at 28°C (at 200 rpm overnight). These cultures were diluted to obtain an A600 of 0.5 OD. Series of dilutions from 10−1-10−4 were performed for every culture. 5µl of each dilution were

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Page 18 of 42

spotted on synthetic complete medium lacking leucine, tryptophan and histidine, and supplemented with 1 and 3 mM 3-amino-1, 2, 4-triazole (Sigma, Saint Louis) to score growth as an indicator of the protein-protein interaction. Plates were incubated at 28◦C for 6 days followed by documentation of the results.

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Acknowledgements This work is partially supported by grants from University of Delhi, Department of

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Biotechnology (DBT), Department of Science and Technology (DST), and Council of Scientific and Industrial Research (CSIR), India to GKP. PK, IT, AKY acknowledges CSIR

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for their research fellowship.

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including cold, light, cytokinins, sugars and salts, Plant Mol Biol. 52 (2003) 1191-1202. [59] Z. Gu, B. Ma, Y. Jiang, Z. Chen, X. Su, H. Zhang, Expression analysis of the calcineurin B-like gene family in rice (Oryza sativa L.) under environmental stresses, Gene 415 (2008) 1-

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[63] R. Quan, H. Lin, I. Mendoza, Y. Zhang, W. Cao, Y. Yang, M. Shang, S. Chen, J.M. Pardo, Y. Guo, SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress, Plant Cell 19 (2007) 1415-1431. [64] Y.S. Hwang, P.C. Bethke, Y.H. Cheong, H.S. Chang, T. Zhu, R.L. Jones, A gibberellinregulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function, Plant Physiol. 138 (2005) 1347-1358. [65] S. Yoon, J. Park, M. Ryu, I.S. Yoon, K.N. Kim, Calcineurin B-like proteins in rice can bind with calcium ion and associate with the Arabidopsis CIPK family members, Plant Science 177 (2009) 577-583.

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[66] B.G. Kim, R. Waadt, Y.H. Cheong, G.K. Pandey, J.R. Dominguez-Solis, S. Schultke, S.C. Lee, J. Kudla, S. Luan, The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis, Plant J. 52 (2007) 473-484. [67] R. Waadt, L.K. Schmidt, M. Lohse, K. Hashimoto, R. Bock, J. Kudla, Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative

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conditions in rice (Oryza sativa L. ssp. indica), Mol Genet Genomics. 278 (2007) 493-505. [73] R. Arora, P. Agarwal, S. Ray, A.K. Singh, V.P. Singh, A.K. Tyagi, S. Kapoor, MADSbox gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress, BMC Genomics 8 (2007) 242. [74] J.M. Comeron, K-Estimator: calculation of the number of nucleotide substitutions per site and the confidence intervals, Bioinformatics 15 (1999) 763-764. [75] J. Yu, J. Wang, W. Lin, S. Li, H. Li, J. Zhou, P. Ni, W. Dong, S. Hu, C. Zeng, J. Zhang, Y. Zhang, R. Li, Z. Xu, X. Li, H. Zheng, L. Cong, L. Lin, J. Yin, J. Geng, G. Li, J. Shi, J.

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Liu, H. Lv, J. Li, Y. Deng, L. Ran, X. Shi, X. Wang, Q. Wu, C. Li, X. Ren, D. Li, D. Liu, X. Zhang, Z. Ji, W. Zhao, Y. Sun, Z. Zhang, J. Bao, Y. Han, L. Dong, J. Ji, P. Chen, S. Wu, Y. Xiao, D. Bu, J. Tan, L. Yang, C. Ye, J. Xu, Y. Zhou, Y. Yu, B. Zhang, S. Zhuang, H. Wei, B. Liu, M. Lei, H. Yu, Y. Li, H. Xu, S. Wei, X. He, L. Fang, X. Huang, Z. Su, W. Tong, Z. Tong, J. Ye, L. Wang, T. Lei, C. Chen, H. Chen, H. Huang, F. Zhang, N. Li, C. Zhao, Y. Huang, L. Li, Y. Xi, Q. Qi, W. Li, W. Hu, X. Tian, Y. Jiao, X. Liang, J. Jin, L. Gao, W.

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carrier DNA/polyethylene glycol method. Methods Enzymol. 350 (2002) 87-96.

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Table 1A. Features of CBLs in the rice genome. Total ESTs

Protein length

Intron

spliced variant

LOC_Os10g41510

AK243065

19

214

7

1

Arabidopsis homolog TAIR Locus Gene Percentage Name homology At5g47100.1 CBL9 0.79

OsCBL2

LOC_Os12g40510

AK111887

35

226

7

1

At4g26570.1

CBL3

0.92

OsCBL3

LOC_Os03g42840

AK112092

57

226

7

1

At4g26570.1

CBL3

0.94

OsCBL4

LOC_Os05g45810

AK101368

12

211

7

1

At1g64480.1

CBL4

0.64

OsCBL5

LOC_Os01g41510

AK111570

3

219

7

1

At5g24270.2

CBL4

0.61

OsCBL6

LOC_Os12g06510

AK111505

34

295

7

3

At4g26570.1

CBL3

0.81

OsCBL7

LOC_Os02g18880

n/a

1

214

7

1

At5g24270.2

CBL4

0.66

OsCBL8

LOC_Os02g18930

n/a

1

214

7

1

At5g24270.2

CBL4

0.66

OsCBL9

LOC_Os01g39770

AK111936

34

291

8

3

At4g33000.2

CBL10

0.78

OsCBL10

LOC_Os01g51420

n/a

4

267

8

1

At4g33000.1

CBL10

0.75

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FL cDNA

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Rice genes TIGR Locus

Gene Name OsCBL1

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Table 1B. Features of CIPKs in the rice genome. Protein length

Intron

spliced variant

OsCIPK1

LOC_Os01g18800

AK061640

100

462

12

5

Arabidopsis homolog TAIR Locus Gene Percentage Name homology At3g17510.1 CIPK1 0.66

OsCIPK2

LOC_Os07g48100

AK072868

174

444

2

1

At5g58380.1

CIPK10

0.7

OsCIPK3

LOC_Os07g48760

n/a

3

446

14

1

At2g26980.4

CIPK3

0.65

OsCIPK4

LOC_Os12g41090

n/a

5

440

0

1

At4g14580.1

CIPK4

0.64

OsCIPK5

LOC_Os01g10890

AK069681

67

462

0

1

At5g58380.1

CIPK10

0.71

OsCIPK6

LOC_Os08g34240

AK108225

13

452

0

1

At4g30960.1

CIPK6

0.73

OsCIPK7

LOC_Os03g43440

AK111510

44

448

0

1

At4g14580.1

CIPK4

0.55

OsCIPK8

LOC_Os01g35184

AK120431

20

447

13

2

At4g24400.1

CIPK8

0.79

OsCIPK9

LOC_Os03g03510

n/a

49

457

13

2

At1g01140.1

CIPK9

0.78

OsCIPK10

LOC_Os03g22050

AK062464

244

452

0

4

OsCIPK11

LOC_Os01g60910

AK103032

8

503

1

2

OsCIPK12

LOC_Os01g55450

AK101442

17

541

1

1

OsCIPK13

LOC_Os01g10870

n/a

0

512

OsCIPK14

LOC_Os12g02200

AK243050

76

440

OsCIPK15

LOC_Os11g02240

AB264037

100

435

OsCIPK16

LOC_Os09g25090

AK061493

89

457

OsCIPK17

LOC_Os05g04550

AK059282

27

OsCIPK18

LOC_Os05g26820

AK101355

25

OsCIPK19

LOC_Os05g43840

AK066778

OsCIPK20

LOC_Os05g11790

AK107068

OsCIPK21

LOC_Os07g44290

AK107137

OsCIPK22

LOC_Os05g26940

AK107763

OsCIPK23

LOC_Os07g05620

OsCIPK24

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Total ESTs

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0

1

At4g18700.1

CIPK12

0.7

0

2

At5g01810.2

CIPK15

0.71

1

1

At5g01810.2

CIPK15

0.71

0

1

At5g10930.1

CIPK5

0.66

455

11

1

At3g17510.1

CIPK1

0.64

458

1

1

At5g58380.1

CIPK10

0.71

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0.68

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FL cDNA

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Rice genes Gene Name TIGR Locus

At5g07070.1

CIPK2

At5g07070.1

CIPK2

0.7

At4g18700.1

CIPK12

0.72

509

0

1

At4g18700.1

CIPK12

0.74

11

467

0

1

At5g45820.1

CIPK20

0.65

9

526

12

1

At3g17510.1

CIPK1

0.64

0

452

0

1

At3g23000.1

CIPK7

0.48

AK069726

105

451

13

2

At1g30270.1

CIPK23

0.85

LOC_Os06g40370

AK102270

44

454

13

1

At5g35410.1

CIPK24

0.69

OsCIPK25

LOC_Os06g35160

AK065374

40

515

0

1

At4g18700.1

CIPK12

0.48

OsCIPK26

LOC_Os02g06570

AK111660

13

494

1

2

At5g07070.1

CIPK2

0.69

OsCIPK27

LOC_Os09g25100

n/a

2

405

0

1

At4g30960.1

CIPK6

0.73

OsCIPK28

LOC_Os05g39870

n/a

0

648

6

1

At5g07070.1

CIPK2

0.64

OsCIPK29

LOC_Os07g48090

AK111746

14

444

0

1

At2g30360.1

CIPK11

0.68

OsCIPK30

LOC_Os01g55440

AK069231

59

477

0

1

At5g45820.1

CIPK20

0.68

OsCIPK31

LOC_Os03g20380

AK111752

82

450

14

8

At2g26980.4

CIPK3

0.71

OsCIPK32

LOC_Os12g03810

AK111953

43

439

14

3

At2g26980.4

CIPK3

0.79

OsCIPK33

LOC_Os11g03970

AK112043

29

455

14

2

At2g26980.4

CIPK3

0.8

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Table 2. Segmental and tandem duplicated genes in rice CBL and CIPK gene families.

Pair No.

Amino acid % homology

Gene name

Duplicated gene name

Duplication

OsCBL7

OsCBL8

Tandom

1

OsCIPK14

OsCIPK02

Segmental

60.30%

2

OsCIPK15

OsCIPK14

Segmental

98.40%

3

OsCIPK15

OsCIPK02

Segmental

59.60%

4

OsCIPK04

OsCIPK07

Segmental

39%

5

OsCIPK05

OsCIPK20

Segmental

64.20%

6

OsCIPK05

OsCIPK12

Segmental

55.30%

7

OsCIPK06

OsCIPK27

Segmental

65.60%

8

OsCIPK01

OsCIPK17

Segmental

77%

9

OsCIPK031

OsCIPK03

Segmental

79.60%

10

OsCIPK11

OsCIPK28

Segmental

64.50%

11

OsCIPK33

OsCIPK32

Segmental

99.30%

12

OsCIPK13

OsCIPK5

Tandem

51.20%

13

OsCIPK30

OsCIPK12

14

OsCIPK16

OsCIPK27

15

OsCIPK29

OsCIPK2

OsCBLs 1

90.10%

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42%

Tandem

45%

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Tandem

38.60%

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Tandem

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OsCIPKs

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Gene name

Duplicated gene name

Ka

Ks

Ka/Ks

Age (MYA)

1

OsCBL7

OsCBL8

0.0518

0.2935

0.176491

22.58

2

OsCIPK15

OsCIPK14

0.0033

0.0403

0.081886

3.10

3

OsCIPK04

OsCIPK07

0.1477

0.2682

0.550708

20.63

4

OsCIPK06

OsCIPK27

0.1811

0.436

0.415367

33.54

5

OsCIPK01

OsCIPK17

0.1468

0.6162

0.238234

47.40

6

OsCIPK031

OsCIPK03

0.1156

0.8561

0.135031

65.85

7

OsCIPK33

OsCIPK32

0.0034

0.033

0.10303

2.54

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Pair No.

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Table 3. Estimation of the age of duplicated OsCBLs and OsCIPKs

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Figure Legends Fig. 1. Chromosomal localization of rice CBL and CIPK genes. OsCBLs and OsCIPKs have been mapped by their position on 12 chromosomes. Segmental duplications have been marked by green lines while the tandemly duplicated genes are joined with vertical red lines. Chromosomes are grouped randomly to show the duplication with clarity. Respective

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chromosome numbers are mentioned at the top. Fig. 2. Schematic representations of the domain and motif organization of OsCBLs. (A) Five

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OsCBLs contain a conserved N-myristoylation motif shown by grey round box. Green round boxes represent four EF-hand motifs and red round box represents PFPF motif. Scale bar

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represents 0.05 amino acid substitutions per site. (B) Detailed comparison of Nmyristoylation motif sequences, EF-hand and PFPF motifs of CBLs. Upper panel represent

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rice sequences while the lower panel is from Arabidopsis .

Fig. 3: Schematics of the domain and motif organization of OsCIPKs. (A) CIPK proteins contain a conserved N-terminal catalytic kinase domain (light purple rectangular box), which

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have ATP binding site (red round box) and activation loop (yellow round box). The Cterminal regulatory domain contains FISL or NAF motifs (blue round box) and PPI motif

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(grey round box). Scale bar represents 0.1 amino acid substitutions per site. (B) Detailed comparison of ATP binding site, activation loop, NAF motifs and PPI motif of CIPKs

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between rice and Arabidopsis.

Fig. 4. Expression profile of rice CBLs and CIPKs under abiotic stress and reproductive development. Microarray expression profile of rice CBLs (A, C) and CIPKs (B, D) in three abiotic stress conditions (CS: cold stress, DS: drought stress, and SS: salt stress) and in three

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vegetative stages (leaf, root, and seedling), 11 reproductive development stages (P1–P6 panicle development stages, and S1–S5 seed development stages). Colour bar represents log2 expression values, green represents low level of expression, black shows medium expression, and red shows high expression. A gene is considered differentially regulated if it is at least two-fold up- or down-regulated w.r.t. control. In case of abiotic stress, untreated 7-day-old seedling was used as control, whereas all the three vegetative stages were considered as control for the different reproductive stages. Clustering of the expression profile was done with log transformed average values taking mature leaf as base line.

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Fig. 5. Venn diagram for differentially expressed OsCBLs and OsCIPKs. OsCBL (A) and OsCIPK (B) genes up- and down-regulated under different abiotic stress conditions. OsCBL (C) and OsCIPK (D) genes up- and down-regulated under abiotic stress conditions and development stages. Different compartments showing genes specific to either a particular stress/developmental stage or common to more than one stress and/or developmental stage.

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Genes with red colour are up-regulated whereas green are down-regulated. Fig. 6. Expression profiles of tandemly duplicated OsCBLs and OsCIPKs. Expression profiles of tandemly duplicated gene pairs from microarray data were compared in various

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developmental stages including leaf (L), root (R) and seven day old seedling (S) tissue, in

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various stages of panicle development (P1-P6), seed development (S1-S5) and under cold stress (CS), drought stress (DS) and salt stress (SS). Each area graph represents compilation of the mean normalized signal intensity values. The duplicated genes have been grouped into

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retention of function, pseudo-functionalization and neo- functionalization.

Fig. 7. Expression profile of segmentally duplicated OsCIPKs. Expression profiles of

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segmentally duplicated gene pairs/clusters from microarray data were compared in various developmental stages including leaf (L), root (R) and seven day old seedling (S) tissue, in

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various stages of panicle development (P1-P6), seed development (S1-S5) and under cold stress (CS), drought stress (DS) and salt stress (SS). Each area graph represents compilation of the mean normalized signal intensity values. The duplicated genes have been grouped into

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retention of function and pseudo-functionalization. Fig. 8. Yeast two-hybrid interaction between OsCBL4, OsCBL5, OsCBL7 and OsCBL8 with OsCIPKs. The OsCBLs and OsCIPKs cDNAs were cloned into the pGAD.GH prey vector

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and pGBT9 bait vector, respectively. Each plasmid combination was introduced into AH109 yeast cells and their growth was monitored on SC-Leu-Trp-His medium containing 1mM and 3mM 3-AT.

Fig. 9. Yeast two-hybrid interactions between Arabidopsis SOS3/CBL4, OsCBL4, OsCBL5, OsCBL7 and OsCBL8 with all Arabidopsis CIPKs. Interaction was examined for OsCBLs in the pGAD.GH prey vector and Arabidopsis CIPKs in pGBT9 bait vector. Arabidopsis SOS3/CBL4 was cloned in pGBT9 bait vector and its interaction was analysed with Arabidopsis CIPKs in the pGAD.GH prey vector. Each plasmid combination was introduced into AH109 yeast cells and their growth was monitored on SC-Leu-Trp-His medium containing 1mM 3-AT. 31

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