Identification and comprehensive analyses of the CBL gene families in sweet orange (Citrus sinensisL.)

Document Type : Original research paper


1 Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University, PO Box 578, Sari

2 Crop and Horticultural Science Research Department, Mazandaran Agricultural and Natural Resources Research and Education Center, AREEO, Sari, Iran;

3 Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran, P. O. Box 578

4 3) National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran


Calcineurin B-like (CBL) proteins, as calcium sensors, serve roles in plant responses to varied abiotic stressors and in growth and development through interaction with CBL-interacting protein kinases (CIPKs). However, information on the roles and development of CBLs in sweet orange plants is limited. We surveyed the whole Citrus sinensis genome and found eight CBL genes. Domains features, position and distribution, and conserved motif revealed that the EF-hands domain was conserved across the eight CsCBLs. CsCBL proteins are classed as acidic CBL, and five myristoylation sites and six palmitoylation sites were predicted. Eight CsCBLs were distributed across chromosomes Chr01, Chr02, Chr04, and Chr05 and contig chrNW-006257104.1. In chromosome 05, tandem duplications likely gave rise to two CsCBL4 and CsCBL5 genes. The phylogeny tree of 37 CBL proteins from different plant species including Arabidopsis thaliana, Oryza sativa, Sesamum indicum, and C. sinensis showed that these CBLs are closely related. A meta-analysis of the CsCBL gene family's expression in different tissues/stresses revealed that CsCBL genes expressed differently in tissues, which could be evidence for CsCBL tissue/stress-specific expression. The results of this study highlight the functional properties of the CsCBL gene family and provide crucial data for future research on their functional activities.


Arab, M., Najafi Zarrini, H., Nematzadeh, G., Heidari, P., Hashemipetroudi, S.H., and Kuhlmann, M. (2023). analysis of calcium sensor families, CBL and CIPK, in Aeluropus littoralis and their expression profile in response to salinity. Genes 14(3): 753.
Blaskovic, S., Blanc, M., and van der Goot, F.G. (2013). What does palmitoylation do to membrane proteins? FEBS J 280(12): 2766-2774.
Cannon, S.B., Mitra, A., Baumgarten, A., Young, N.D., and May, G. (2004). The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol 4(1): 10. doi: 10.1186/1471-2229-4-10.
Cheong, Y.H., Pandey, G.K., Grant, J.J., Batistic, O., Li, L., Kim, B.G., Lee, S.C., Kudla, J., and Luan, S. (2007). Two calcineurin B-like calcium sensors, interacting with protein kinase CIPK23, regulate leaf transpiration and root potassium uptake in Arabidopsis. Plant J 52(2): 223-239. doi: 10.1111/j.1365-313X.2007.03236.x.
Chow, C.N., Zheng, H.Q., Wu, N.Y., Chien, C.H., Huang, H.D., Lee, T.Y., Chiang-Hsieh, Y.F., Hou, P.F., Yang, T.Y., and Chang, W.C. (2016). PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants. Nucleic Acids Res 44(D1): D1154-1160. doi: 10.1093/nar/gkv1035.
Gao, P., Zhao, P.-M., Wang, J., Wang, H.-Y., Du, X.-M., Wang, G.-L., and Xia, G.-X. (2008). Co-expression and preferential interaction between two calcineurin B-like proteins and a CBL-interacting protein kinase from cotton. Plant Physiol Biochem 46(10): 935-940.
Gu, Z., Ma, B., Jiang, Y., Chen, Z., Su, X., and Zhang, H. (2008). Expression analysis of the calcineurin B-like gene family in rice (Oryza sativa L.) under environmental stresses. Gene 415(1-2): 1-12. doi: 10.1016/j.gene.2008.02.011.
Guo, Y., Halfter, U., Ishitani, M., and Zhu, J.-K. (2001). Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance. Plant Cell 13(6): 1383-1400.
Hashemipetroudi, S., and Bakhshandeh, E. (2020). Expression analysis of SiSOD gene family during Sesamum indicum L. seed germination under various abiotic stresses. J Plant Mol Breed 8(2): 50-60.
Hashemipetroudi, S.H., Arab, M., Heidari, P., and Kuhlmann, M. (2023). Genome-wide analysis of the laccase (LAC) gene family in Aeluropus littoralis: A focus on identification, evolution and expression patterns in response to abiotic stresses and ABA treatment. Front Plant Sci 14: 1112354. doi: 10.3389/fpls.2023.1112354.
Ikai, A. (1980). Thermostability and aliphatic index of globular proteins. J Biochem 88(6): 1895-1898.
Ishitani, M., Liu, J., Halfter, U., Kim, C.S., Shi, W., and Zhu, J.K. (2000). SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding. Plant Cell 12(9): 1667-1678. doi: 10.1105/tpc.12.9.1667.
Kanchiswamy, C.N., Mohanta, T.K., Capuzzo, A., Occhipinti, A., Verrillo, F., Maffei, M.E., and Malnoy, M. (2013). Differential expression of CPKs and cytosolic Ca 2+ variation in resistant and susceptible apple cultivars (Malus x domestica) in response to the pathogen Erwinia amylovora and mechanical wounding. BMC Genom 14(1): 1-14.
Kim, B.G., Waadt, R., Cheong, Y.H., Pandey, G.K., Dominguez-Solis, J.R., Schultke, S., Lee, S.C., Kudla, J., and Luan, S. (2007). The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis. Plant J 52(3): 473-484. doi: 10.1111/j.1365-313X.2007.03249.x.
Kleist, T.J., Spencley, A.L., and Luan, S. (2014). Comparative phylogenomics of the CBL-CIPK calcium-decoding network in the moss Physcomitrella, Arabidopsis, and other green lineages. Front Plant Sci 5: 187. doi: 10.3389/fpls.2014.00187.
Kolukisaoglu, Ü., Weinl, S., Blazevic, D., Batistic, O., and Kudla, J. (2004). Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks. Plant Physiol 134(1): 43-58.
Kudla, J., Becker, D., Grill, E., Hedrich, R., Hippler, M., Kummer, U., Parniske, M., Romeis, T., and Schumacher, K. (2018). Advances and current challenges in calcium signaling. New Phytol 218(2): 414-431. doi: 10.1111/nph.14966.
Li, J., Jiang, M.M., Ren, L., Liu, Y., and Chen, H.Y. (2016). Identification and characterization of CBL and CIPK gene families in eggplant (Solanum melongena L.). Mol Genet Genomics 291(4): 1769-1781. doi: 10.1007/s00438-016-1218-8.
Li, L., Kim, B.G., Cheong, Y.H., Pandey, G.K., and Luan, S. (2006). A Ca(2)+ signaling pathway regulates a K(+) channel for low K response in Arabidopsis. Proc Natl Acad Sci USA 103(33): 12625-12630. doi: 10.1073/pnas.0605129103.
Linder, M.E., and Deschenes, R.J. (2007). Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol 8(1): 74-84. doi: 10.1038/nrm2084.
Liu, J., Ishitani, M., Halfter, U., Kim, C.-S., and Zhu, J.-K. (2000). The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proc Natl Acad Sci 97(7): 3730-3734.
Liu, L.L., Ren, H.M., Chen, L.Q., Wang, Y., and Wu, W.H. (2013). A protein kinase, calcineurin B-like protein-interacting protein Kinase9, interacts with calcium sensor calcineurin B-like Protein3 and regulates potassium homeostasis under low-potassium stress in Arabidopsis. Plant Physiol 161(1): 266-277. doi: 10.1104/pp.112.206896.
Lu, T., Zhang, G., Sun, L., Wang, J., and Hao, F. (2017). Genome-wide identification of CBL family and expression analysis of CBLs in response to potassium deficiency in cotton. PeerJ 5: e3653. doi: 10.7717/peerj.3653.
Luan, S. (2009). The CBL-CIPK network in plant calcium signaling. Trends Plant Sci 14(1): 37-42. doi: 10.1016/j.tplants.2008.10.005.
Ma, X., Li, Q.H., Yu, Y.N., Qiao, Y.M., Haq, S.U., and Gong, Z.H. (2020). The CBL-CIPK Pathway in Plant Response to Stress Signals. Int J Mol Sci 21(16): 5668. doi: 10.3390/ijms21165668.
Mao, J., Manik, S.M., Shi, S., Chao, J., Jin, Y., Wang, Q., and Liu, H. (2016). Mechanisms and Physiological Roles of the CBL-CIPK Networking System in Arabidopsis thaliana. Genes 7(9): 62. doi: 10.3390/genes7090062.
Martı́n, M.L., and Busconi, L. (2001). A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature. Plant Physiol 125(3): 1442-1449.
Mohanta, T.K., Mohanta, N., Mohanta, Y.K., Parida, P., and Bae, H. (2015). Genome-wide identification of Calcineurin B-Like (CBL) gene family of plants reveals novel conserved motifs and evolutionary aspects in calcium signaling events. BMC Plant Biol 15(1): 189. doi: 10.1186/s12870-015-0543-0.
Mohanta, T.K., and Sinha, A.K. (2016). "Role of calcium-dependent protein kinases during abiotic stress tolerance," in Abiotic stress response plants, eds. N. Tuteja & S.S. Gill.  (USA: Wiley), pp. 185-206.
Mount, S.M. (1982). A catalogue of splice junction sequences. Nucleic Acids Res 10(2): 459-472. doi: 10.1093/nar/10.2.459.
Nagae, M., Nozawa, A., Koizumi, N., Sano, H., Hashimoto, H., Sato, M., and Shimizu, T. (2003). The crystal structure of the novel calcium-binding protein AtCBL2 from Arabidopsis thaliana. J Biol Chem 278(43): 42240-42246. doi: 10.1074/jbc.M303630200.
Sanchez-Barrena, M.J., Martinez-Ripoll, M., Zhu, J.K., and Albert, A. (2005). The structure of the Arabidopsis thaliana SOS3: molecular mechanism of sensing calcium for salt stress response. J Mol Biol 345(5): 1253-1264. doi: 10.1016/j.jmb.2004.11.025.
Sanyal, S.K., Pandey, A., and Pandey, G.K. (2015). The CBL-CIPK signaling module in plants: a mechanistic perspective. Physiol Plant 155(2): 89-108. doi: 10.1111/ppl.12344.
Sarwat, M., Ahmad, P., Nabi, G., and Hu, X. (2013). Ca(2+) signals: the versatile decoders of environmental cues. Crit Rev Biotechnol 33(1): 97-109. doi: 10.3109/07388551.2012.672398.
Sathyanarayanan, P.V., and Poovaiah, B.W. (2004). Decoding Ca2+ signals in plants. Crit Rev Plant Sci 23(1): 1-11. doi: 10.1080/07352680490273310.
Sharafi, E., Dehestani, A., and Farmani, J. (2017). Bioinformatics evaluation of plant chlorophyllase, the key enzyme in chlorophyll degradation. Appl Food Biotech 4(4): 167-178.
Smotrys, J.E., and Linder, M.E. (2004). Palmitoylation of intracellular signaling proteins: regulation and function. Annu Rev Biochem 73(1): 559-587. doi: 10.1146/annurev.biochem.73.011303.073954.
Steinhorst, L., Mähs, A., Ischebeck, T., Zhang, C., Zhang, X., Arendt, S., Schültke, S., Heilmann, I., and Kudla, J. (2015). Vacuolar CBL-CIPK12 Ca2+ sensor kinase complexes are required for polarized pollen tube growth. Curr Biol 25(11): 1475-1482.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30(12): 2725-2729. doi: 10.1093/molbev/mst197.
Tang, R.J., Wang, C., Li, K., and Luan, S. (2020). The CBL-CIPK calcium signaling network: unified paradigm from 20 years of discoveries. Trends Plant Sci 25(6): 604-617. doi: 10.1016/j.tplants.2020.01.009.
Thoday-Kennedy, E.L., Jacobs, A.K., and Roy, S.J. (2015). The role of the CBL–CIPK calcium signalling network in regulating ion transport in response to abiotic stress. Plant Growth Regul 76(1): 3-12.
Tuteja, N., and Mahajan, S. (2007). Calcium signaling network in plants: an overview. Plant Signal Behav 2(2): 79-85. doi: 10.4161/psb.2.2.4176.
Wang, J.P., Xu, Y.P., Munyampundu, J.P., Liu, T.Y., and Cai, X.Z. (2016). Calcium-dependent protein kinase (CDPK) and CDPK-related kinase (CRK) gene families in tomato: genome-wide identification and functional analyses in disease resistance. Mol Genet Genomics 291(2): 661-676. doi: 10.1007/s00438-015-1137-0.
Wang, Y., Tang, H., Debarry, J.D., Tan, X., Li, J., Wang, X., Lee, T.H., Jin, H., Marler, B., Guo, H., Kissinger, J.C., and Paterson, A.H. (2012). MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res 40(7): e49. doi: 10.1093/nar/gkr1293.
Wu, G.A., Terol, J., Ibanez, V., Lopez-Garcia, A., Perez-Roman, E., Borreda, C., Domingo, C., Tadeo, F.R., Carbonell-Caballero, J., Alonso, R., Curk, F., Du, D., Ollitrault, P., Roose, M.L., Dopazo, J., Gmitter, F.G., Rokhsar, D.S., and Talon, M. (2018). Genomics of the origin and evolution of Citrus. Nature 554(7692): 311-316. doi: 10.1038/nature25447.
Xu, J., Li, H.D., Chen, L.Q., Wang, Y., Liu, L.L., He, L., and Wu, W.H. (2006). A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell 125(7): 1347-1360. doi: 10.1016/j.cell.2006.06.011.
Yaghobi, M., and Heidari, P. (2023). Genome-wide analysis of Aquaporin gene family in Triticum turgidum and its expression profile in response to salt stress. Genes 14(1): 202. doi: 10.3390/genes14010202.
Yin, X., Wang, Q., Chen, Q., Xiang, N., Yang, Y., and Yang, Y. (2017). Genome-wide identification and functional analysis of the Calcineurin B-like protein and Calcineurin B-like protein-Interacting Protein Kinase gene families in Turnip (Brassica rapa var. rapa). Front Plant Sci 8: 1191. doi: 10.3389/fpls.2017.01191.
Yu, Q., An, L., and Li, W. (2014). The CBL-CIPK network mediates different signaling pathways in plants. Plant Cell Rep 33(2): 203-214. doi: 10.1007/s00299-013-1507-1.
Zhang, C., Bian, M., Yu, H., Liu, Q., and Yang, Z. (2011). Identification of alkaline stress-responsive genes of CBL family in sweet sorghum (Sorghum bicolor L.). Plant Physiol Biochem 49(11): 1306-1312. doi: 10.1016/j.plaphy.2011.08.010.
Zhang, F., Li, L., Jiao, Z., Chen, Y., Liu, H., Chen, X., Fu, J., Wang, G., and Zheng, J. (2016). Characterization of the calcineurin B-Like (CBL) gene family in maize and functional analysis of ZmCBL9 under abscisic acid and abiotic stress treatments. Plant Sci 253: 118-129. doi: 10.1016/j.plantsci.2016.09.011.
Zhang, H., Yang, B., Liu, W.Z., Li, H., Wang, L., Wang, B., Deng, M., Liang, W., Deyholos, M.K., and Jiang, Y.Q. (2014). Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.). BMC Plant Biol 14(1): 8. doi: 10.1186/1471-2229-14-8.
Zhang, H., Yin, W., and Xia, X. (2008). Calcineurin B-Like family in Populus: comparative genome analysis and expression pattern under cold, drought and salt stress treatment. Plant Growth Regul 56(2): 129-140.
Volume 10, Issue 2
December 2022
Pages 76-91
  • Receive Date: 07 June 2022
  • Revise Date: 03 January 2024
  • Accept Date: 04 January 2024
  • First Publish Date: 04 January 2024