Cloning of VaCBL01 Gene from Vitis amurensis and Analysis of Its Interaction with VaCIPKs Proteins
SHEN Wei1,2, YAO Wen-Kong1, ZHENG Qiao-Ling1,2, ZHANG Ning-Bo1,2, XU Wei-Rong1,2,3*
1 College of Agronomy, Ningxia University, Yinchuan 750021, China; 2 Engineering Research Center of Grape and Wine, Ministry of Education, Ningxia University / Ningxia Institute of Grape &Wine / Ningxia Engineering and Technology Research Center of Grape and Wine, Yinchuan 750021, China; 3 Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia, Yinchuan 750021, China
Abstract：Calcineurin B-like proteins (CBLs) represent a family of newly emerging plant-specific Ca2+ sensors, which could selectively interplay with their respective kinase effectors (CBL-interacting protein kinases, CIPKs), thus playing critical roles in response to signal transduction of various abiotic stresses. To identify the functional characteristics of the CBLs gene involved in stress response in Vitis plants, the cold-tolerant species of Vitis amurensis 'Zuoshan-1' was used as the material to isolate one calcineurin B protein family member VaCBL01 by homologous cloning. Analysis of its sequence and structure indicated that the ORF of VaCBL01 gene (GenBank No. MH921999) was 642 bp, encoding 213 amino acids, with four EF-conserved domains, and a myristoylation site at the N-terminus. Chromosome location prediction indicated that VaCBL01 could be mapped to locus of 5 571 989~5 581 623 in chromosome 2. Sequence alignment revealed that VaCBL01 shared high sequence homology with Vitis vinifera VvCBL01 (100%), followed by tomato (Solanum lycopersicum) SlCBL01 (95.3%), Arabidopsis AtCBL01 (95.3%) and PtCBL01 (94.8%). Phylogenetic analysis of CBL01 gene families across a variety of species suggested VaCBL01 protein had the closest evolutionary relationship with proteins clustered in GroupⅡ, including V. vinifera, tomato, Arabidopsis and Populus trichocarpa. Furthermore, the expression levels of VaCBL01 genes either in different tissue and organs or in response to abscisic acid (ABA) and cold stress were examined by qRT-PCR. Expression of VaCBL01 was found to be ubiquitous in all tissues in V. amurensis, but its expression was relatively high in tendril. VaCBL01 gene was found to respond to ABA and cold stress, suggesting that the grapevine CBL-CIPK network may be a point of convergence for several different signaling pathways. Also, a comparison of interaction patterns of VaCBL01 and 19 VaCIPKs was performed using yeast two-hybrid system. The results showed that none of the 19 VaCIPKs had autotoxicity and self-activation in yeast cells. VaCBL01 exhibited a significant interaction only with a subset of 12 CIPKs (VaCIPK01, 02, 04, 06, 09, 10, 11, 12, 13,16, 17 and 18). For 5 additional CIPKs (VaCIPK03, 07, 15, 19 and 20), a weak tendency of complex formation was observed, whereas 2 CIPKs (VaCIPK08 and 14) did not show any affinity toward AtCBL01. The results would provide an important foundation for further functional dissection of the CBL-CIPK signaling network in grapevine.
[1] 贺普超, 王跃进, 王国英, 等. 1991. 中国葡萄属野生种抗病性的研究[J]. 中国农业科学, 24(3): 50-56. (He P C, Wang Y J, Wang G Y, et al.1991. Studies on disease resistance of wild grape species in China[J]. Scientia Agricultura Sinica, 24(3): 50-56.) [2] 沈金秋, 郑仲仲, 潘伟槐, 等. 2014. 植物CBL-CIPK信号系统的功能及其作用机理[J]. 植物生理学报, 50(4): 641-650. (Shen J Q, Zheng Z Z, Pan W H, et al.2014. Functions and action mechanisms of CBL-CIPK signaling system in plants[J]. Plant Physiology Journal, 50(4):641-650.) [3] 闫朝辉, 李桂荣, 扈岩松, 等. 2017. 欧洲葡萄中CIPK基因的克隆及表达分析[J]. 园艺学报, 44(8): 1463-1476. (Yan C H, Li G R, Hu Y S, et al.2017. Cloning and expression analysis of CIPK genes in grapevine[J]. Acta Horticulturae Sinica, 44(8): 1463-1476) [4] 余义和, 李秀珍, 郭大龙, 等. 2016. 葡萄类钙调磷酸酶B亚基互作蛋白激酶VvCIPK10的特性与表达[J]. 中国农业科学, 49(19): 3798-3806. (Yu Y H, Li X Z, Guo D L, et al.2016. Characteristics and expression of calmodulin like B Subunit interaction protein VvCIPK10 in grapevine[J]. Scientia Agricultura Sinica, 49(19):3798-3806.) [5] Albrecht V, Ritz O, Linder S, et al.2001. The NAF domain defines a novel protein-protein interaction module conserved in Ca2+-regulated kinases[J]. Embo Journal, 20(5): 1051-1063. [6] Batistic O, Rehers M, Akerman A, et al.2012. Sacylation-dependent association of the calcium sensor CBL2 with the vacuolar membrane is essential for proper abscisic acid responses[J]. Cell Research, 22(7): 1155-1168. [7] Batistic O, Sorek N, Schültke S, et al.2008. Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca2+ signaling complexes in Arabidopsis[J]. The Plant Cell, 20(5): 1346-1362. [8] Chen X, Zhimin G U, Liu F, et al.2010. Molecular analysis of rice involved in biotic and abiotic stress responses[J]. Rice Science, 18(1): 1-9. [9] Cheong Y H, Pandey G K, Grant J J, et al.2007. Two calcineurin B-like calcium sensors, interacting with protein kinase CIPK23, regulate leaf transpiration and root potassium uptake in Arabidopsis[J]. Plant Journal, 52(2): 223-239. [10] D'Angelo C, Weinl S, Batistic O, et al.2010. Alternative complex formation of the Ca-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis[J]. Plant Journal, 48(6): 857-872. [11] Eckert C, Offenborn J N, Heinz T, et al.2014. The vacuolar calcium sensors CBL2 and CBL3 affect seed size and embryonic development in Arabidopsis thaliana[J]. Plant J, 78(1): 146-156. [12] Egea I, Pineda B, Ortíz-Atienza A, et al.2017. The SlCBL10 calcineurin B-like protein ensures plant growth under salt stress by regulating Na+ and Ca2+ homeostasis[J]. Plant Physiology, 176 (2): pp.01605.02017. [13] Fuglsang A, Guo Y, Cuin T, et al.2007. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein[J]. The Plant Cell, 19(5): 1617-1634. [14] Held K, Pascaud F, Eckert C, et al.2011. Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex[J]. Cell Research, 21(7): 1116. [15] Huang C, Ding S, Zhang H, et al.2011. CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana[J]. Plant Science, 1811): 57-64. [16] Jiang X, Hao D L, Li Q C, et al.2006. A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis[J]. Cell, 125(7): 1347-1360. [17] Kanwar P, Sanyal S K, Tokas I, et al.2014. Comprehensive structural, interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice[J]. Cell Calcium, 56(2): 81-95. [18] Kim B G, Waadt R, Cheong Y H, et al.2007. The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis[J]. The Plant Journal, 52(3): 473-484. [19] Kolukisaoglu U, Weinl S, Blazevic D, et al.2004. Calcium sensors and their interacting protein kinases: Genomics of the Arabidopsis and rice CBL-CIPK signaling networks[J]. Plant Physiology, 134(1): 43-58. [20] Kudla J, Xu Q, Harter K, et al.1999. Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals[J]. Proceedings of the National Academy of Sciences of the USA, 96(8): 4718-4723. [21] Lan W Z, Lee S C, Che Y F, et al.2011. Mechanistic analysis of AKT1 regulation by the CBL-CIPK-PP2CA interactions[J]. Molecular plant, 4(3): 527-536. [22] Lewitbentley A, Rety S.2000. EF-hand calcium-binding proteins[J]. Current Opinion in Structural Biology, 10(6): 637-643. [23] Li J, Wang Y.2014. The Os-AKT1 channel is critical for K+ uptake in rice roots and is modulated by the rice CBL1-CIPK23 complex[J]. Plant Cell, 26(8): 3387. [24] Li Z Y, Xu Z S, Chen Y, et al.2013. A novel role for Arabidopsis CBL1 in affecting plant responses to glucose and gibberellin during germination and seedling development[J]. PLoS ONE, 8(2): e56412. [25] Li Z Y, Xu Z S, He G Y, et al.2012. Overexpression of soybean GmCBL1 enhances abiotic stress tolerance and promotes hypocotyl elongation in Arabidopsis[J]. Biochemical Biophysical Research Communications, 427(4): 731-736. [26] Ligaba-Osena A, Fei Z, Liu J, et al.2017.Loss-of-function mutation of the calcium sensor CBL1 increases aluminum sensitivity in Arabidopsis[J]. New Phytologist, 214(2): 830-841. [27] Liu J, Zhu J.1997. An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt?tolerance[J]. Proceedings of the National Academy of Sciences of the USA, 94(26): 14960-14964. [28] Liu J, Zhu J.1998. A calcium sensor homolog required for plant salt tolerance[J]. Science, 280(5371): 1943-1945. [29] Liu L L, Ren H M, Chen L Q, et al.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[J]. Plant Physiology, 161(1): 266-277. [30] Lyzenga W J, Liu H, Schofield A, et al.2013. Arabidopsis CIPK26 interacts with KEG, components of the ABA signalling network and is degraded by the ubiquitin-proteasome system[J]. Journal of Experimental Botany, 64(10): 2779-2791. [31] Monihan S M, Magness C A, Yadegari R, et al.2016. Arabidopsis CALCINEURIN B-LIKE10 functions independently of the SOS pathway during reproductive development in saline conditions[J]. Plant Physiology, 171(1): 369. [32] Nagae M, Nozawa A, Koizumi N, et al.2003. The crystal structure of the novel calcium-binding protein AtCBL2 from Arabidopsis thaliana[J]. Journal of Biological Chemistry, 278(43): 42240-42246. [33] Nozawa A, Koizumi N, Sano H.2001. An Arabidopsis SNF1-related protein kinase, AtSR1, interacts with a calcium-binding protein, AtCBL2, of which transcripts respond to light[J]. Plant Cell Physiology, 42(9): 976. [34] Pandey G K.2008. Emergence of a novel calcium signaling pathway in plants: CBL-CIPK signaling network[J]. Physiology and Molecular Biology of Plants, 14(14): 51-68. [35] Pandey G K, Kanwar P, Singh A, et al.2015. CBL-interacting protein kinase, CIPK21, regulates osmotic and salt stress responses in Arabidopsis[J]. Plant Physiology, 169(1): 780-792. [36] Qiu Q S, Guo Y, Dietrich M A, et al.2002. Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3[J]. Proceedings of the National Academy of Sciences of the USA, 99(12): 8436-8441. [37] Ren X L, Qi G N, Feng H Q, et al.Calcineurin B-like protein CBL 10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis[J]. The Plant Journal, 2013, 74(2): 258-266. [38] Sagi M, Fluhr R.2006. Production of reactive oxygen species by plant NADPH oxidases[J]. Plant Physiology, 141(2): 336-340. [39] Steinhorst L, Mähs A, Ischebeck T, et al.2015. Vacuolar CBL-CIPK12 Ca2+-sensor-kinase complexes are required for polarized pollen tube growth[J]. Current Biology, 25(11): 1475-1482. [40] Tang R, Liu H, Yang Y, et al.2012. Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis[J]. Cell Research, 22(12): 1650-1665. [41] Tang R J, Yang Y, Yang L, et al.2014. Poplar calcineurin B-like proteins PtCBL10A and PtCBL10B regulate shoot salt tolerance through interaction with PtSOS2 in the vacuolar membrane[J]. Plant Cell & Environment, 37(3): 573-588. [42] Tang R J, Zhao F G, Garcia V J, et al.2015. Tonoplast CBL-CIPK calcium signaling network regulates magnesium homeostasis in Arabidopsis[J]. Proceedings of theNational Academy of Sciences, 112(10): 3134-3139. [43] Weinl S, Kudla J.2009. The CBL-CIPK Ca2+-decoding signaling network: Function and perspectives[J]. New Phytologist, 184(3): 517-528. [44] Xi Y, Liu J, Dong C, et al.2017. The CBL and CIPK gene family in grapevine (Vitis vinifera): Genome-wide analysis and expression profiles in response to various abiotic stresses[J]. Frontiers in Plant Science, 8: 978. [45] Ye N H, Wang F Z, Shi L, et al.2018. Natural variation in the promoter of rice calcineurin B-like protein10 (OsCBL10) affects flooding tolerance during seed germination among rice subspecies[J]. Plant Journal for Cell & Molecular Biology, 94(4): 612-625. [46] Yong H C, Sun J S, Kim B G, et al.2010. Constitutive overexpression of the calcium sensor CBL5 confers osmotic or drought stress tolerance in Arabidopsis[J]. Molecules, 29(2): 159-165. [47] Yu Q, An L, Li W.2013. The CBL-CIPK network mediates different signaling pathways in plants[J]. Plant Cell Reports, 33(2): 203-214. [48] Zhang H, Lv F, Han X, et al.2013. The calcium sensor PeCBL1, interacting with PeCIPK24/25 and PeCIPK26, regulates Na(+)/K(+) homeostasis in Populus euphratica[J]. Plant Cell Reports, 32(5): 611-621. [49] Zhang H, Yin W, Xia X.2008. Calcineurin B-Like family in Populus: Comparative genome analysis and expression pattern under cold, drought and salt stress treatment[J]. Plant Growth Regulation, 56(2): 129-140. [50] Zhao J, Sun Z, Zheng J, et al.2009. Cloning and characterization of a novel CBL-interacting protein kinase from maize[J]. Plant Molecular Biology, 69(6): 661-674.
