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Alfalfa MsCBL4 enhances calcium metabolism but not sodium transport in transgenic tobacco under salt and saline–alkali stress

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MsCBL4 expression in tobacco enhanced its salt and saline–alkali stress tolerance by regulating calcium accumulation in roots, indicating the important role of calcium metabolism in plant saline–alkali stress tolerance

Abstract

The calcineurin B–like (CBL) family of proteins play important roles in plant abiotic stress tolerance and signal transduction. CBL4 is known to participate in the Salt Overly Sensitive pathway; however, little is currently known regarding the mechanisms underlying the response of CBL4 to saline–alkali stress. In this study, we cloned and characterized the alfalfa MsCBL4 gene. We found that MsCBL4 showed the highest expression in root tissues and was induced by salt and saline–alkali stress, with the latter causing higher induction. Overexpression of MsCBL4 in tobacco enhanced salt and saline–alkali stress tolerance and reduced the Na+/K+ ratio in roots of transgenic lines. Salt (30 and 300 mM NaCl) and saline–alkali (30 mM NaHCO3) stress assays performed for MsCBL4 transgenic tobacco lines revealed a substantial influx of sodium ions in roots under saline–alkali stress and indicated that the expression of MsCBL4 had little influence on sodium ion content reduction. In contrast, in roots subjected to saline–alkali stress, calcium accumulation occurred and was significantly enhanced by the overexpression of MsCBL4. Physiological and biochemical analyses indicated that MsCBL4 plays an important role in saline–alkali stress tolerance via its influence on the regulation of calcium transport and accumulation. These results provide novel insights into the saline–alkali stress tolerance mechanisms of plants.

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The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  • An YM, Song LL, Liu YR, Guo CH et al (2016) De novo transcriptional analysis of alfalfa in response to saline-alkaline stress. Front Plant Sci 7:931

    PubMed  PubMed Central  Google Scholar 

  • Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44(1):276–287

    CAS  PubMed  Google Scholar 

  • Bergmeyer HU, Bergmeyer J, Grassl M (1974) Methods of enzymatic analysis. Vol. 2, Samples, reagents, assessment of results[M]. Verlag Chemie

  • Bertorello AM, Zhu JK (2009) SIK1/SOS2 networks: decoding sodium signals via calcium-responsive protein kinase pathways. Pfluegers Arch 458(3):613–619

    CAS  Google Scholar 

  • Bickerton P, Pittman J (2015) Role of Cation/Proton Exchangers in Abiotic Stress Signaling and Stress Tolerance in Plants. Elucidation of Abiotic Stress Signaling in Plants: 95–177

  • Caland LB, Silveira ELC, Tubino M (2011) Determination of sodium, potassium, calcium and magnesium cations in biodiesel by ion chromatography. Anal Chim Acta 718:116–120

    Google Scholar 

  • Chen K, Kurgan L, Rahbari M (2007) Prediction of protein crystallization using collocation of amino acid pairs. Biochem Biophys Res Commun 335:764–769

    Google Scholar 

  • Chen L, Ren J, Shi H, Peng J et al (2015) TdCBL6, a calcineurin B-like gene from wild emmer wheat (Triticum dicoccoides), is involved in response to salt and low-K+ stresses. Mol Breed 35(1):35–50

    Google Scholar 

  • Dong LH, Qian W, Manik S, Liu HB et al (2015) Nicotiana sylvestris calcineurin B-like protein NsylCBL10 enhances salt tolerance in transgenic Arabidopsis. Plant Cell Rep 34:2053–2063

    CAS  PubMed  Google Scholar 

  • El-Gebali S, Mistry J, Bateman A et al (2019) The Pfam protein families database in 2019: Finn. Nucleic Acids Res 47:D427–D432

    CAS  PubMed  Google Scholar 

  • Gong B, Wen D, Vandenlangenberg K, Wang XF et al (2013) Comparative effects of NaCl and NaHCO3 stress on photosynthetic parameters, nutrient metabolism, and the antioxidant system in tomato leaves. Sci Hortic 157:1–12

    CAS  Google Scholar 

  • Guo R, Yang ZZ, Li F, Yan CR, Zhong XL, Zhao L et al (2015) Comparative metabolic responses and adaptive strategies of wheat (Triticum aestivum) to salt and alkali stress. BMC Plant Biol 15:170

    PubMed  PubMed Central  Google Scholar 

  • Hadi MR, Karimi N (2011) The role of calcium in plant salt tolerance. J Plant Nutr 35:2037–2054

    Google Scholar 

  • Hadi MR, Khiyam-Nekoie KR, Yavari P et al (2008) Accumulation and role of ions (Ca2+, Mg2+, SO42-) on salt tolerance in Triticum turgidum L. J Biol Sci 8(1):143–148

    CAS  Google Scholar 

  • Held K, Pascaud F, Kudla J et al (2011) Calcium-dependent modulation and plasma membrane targeting of the AKT2 potassium channel by the CBL4/CIPK6 calcium sensor/protein kinase complex. Cell Res. https://doi.org/10.1038/cr.2011.50

    Article  PubMed  PubMed Central  Google Scholar 

  • Hobbs SL, Kpodar P, DeLong CM (1990) The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol Biol 15:851–864

    CAS  PubMed  Google Scholar 

  • Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604–611

    CAS  Google Scholar 

  • Hu LX, Zhang PP, Jiang Y, Fu JM (2015) Metabolomic analysis revealed differential adaptation to salinity and alkalinity stress in kentucky bluegrass (Poa pratensis). Plant Mol Biol Rep 33:56–68

    CAS  Google Scholar 

  • Kang HK, Nam KH (2016) Reverse function of ROS-induced CBL 10 during salt and drought stressresponses. Plant Sci 243:49–55

    CAS  PubMed  Google Scholar 

  • Kolukisaoglu U, Weinl S, Blazevic D, Batistic O, Kudla J (2004) Calcium sensors and their interacting protein kinases: genomics of the arabidopsis and rice CBL-CIPK signaling networks. Plant Physiol 134:43–58

    CAS  PubMed  PubMed Central  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948

    CAS  PubMed  Google Scholar 

  • Li RL, Shi FC, Fukuda KJ, Yang YL (2011) Effects of salt and alkali stresses on germination, growth, photosynthesis and ion accumulation in alfalfa (Medicago sativa L.). Soil Sci Plant Nutr 56(5):725–733

    Google Scholar 

  • Liu F, Pang SJ (2010) Stress tolerance and antioxidant enzymatic activities in the metabolisms of the reactive oxygen species in two intertidal red algae Grateloupia turuturu and Palmaria palmata. J Exp Mar Biol Ecol 382:82–87

    CAS  Google Scholar 

  • Liu JY, Niu YF, Zhang JJ, Huang X et al (2018) Ca2+ channels and Ca2+ signals involved in abiotic stress responses in plant cells: recent advances. Plant Cell Tissue Organ Cult 132:413–424

    CAS  Google Scholar 

  • Liu LL, Ren HM, Chen LQ, Wang Y, Wu WH (2013) A protein kinase, calcineurin B-like protein-interacting protein kinase 9, interacts with calcium sensor calcineurin B-like protein3 and regulates potassium homeostasis under low-potassium stress in arabidopsis. Plant Physiol 161(1):266–277

    CAS  PubMed  Google Scholar 

  • Ma DM, Xu WD, Li HW, Xu X et al (2014) Co-expression of the Arabidopsis SOS genes enhances salt tolerance in transgenic tall fescue (Festuca arundinacea Schreb.). Protoplasma 251:219–231

    CAS  PubMed  Google Scholar 

  • Mahajan S, Pandey G, Tuteja N (2008) Calcium- and salt-stress signaling in plants: Shedding light on SOS pathway. Arch Biochem Biophys 471(2):146–158

    CAS  PubMed  Google Scholar 

  • Meneguzzo S, Navari-Izzo F, Izzo R (2000) NaCI effects on water relations and accumulation of mineral nutrients in shoots, roots and cell sap of wheat seedlings. J Plant Physiol 156:711–716

    CAS  Google Scholar 

  • Midhat U, Ting MKY, Teresinski HJ, Snedden WA (2018) The calmodulin-like protein, CML39, is involved in regulating seed development, germination, and fruit development in Arabidopsis. Plant Mol Biol https://doi.org/10.1007/s11103-018-0703-3

  • Mouttet P, Escobar-Gutiérrez A, Esquibet M et al (2014) Banning of methyl bromide for seed treatment: could Ditylenchus dipsaci again become a major threat to alfalfa production in Europe. Pest Manag Sci 70(7):1017–1022

    CAS  PubMed  Google Scholar 

  • Mukherjee SP, Choudhuri MA (1983) Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Plant Physiol 58:166–170

    CAS  Google Scholar 

  • Oosten M, Sharkhuu A, Batelli G, Bressan R, Maggio A (2013) The Arabidopsis thaliana mutant air1 implicates SOS3 in the regulation of anthocyanins under salt stress. Plant Mol Biol 83:405–415

    PubMed  Google Scholar 

  • Pandey GK, Kanwar P, Luan S et al (2015) Calcineurin B-Like protein-interacting protein kinase CIPK21 regulates osmotic and salt stress responses in arabidopsis. Plant Physiol 169:780–792

    CAS  PubMed  PubMed Central  Google Scholar 

  • Passonneau JV, Lowry OH (1993) Enzymatic analysis: a practical guide. Springer Science & Business Media, Berlin

    Google Scholar 

  • Peng YL, Gao ZW, Gao Y, Wang DL et al (2008) Eco-physiological characteristics of alfalfa seedlings in response to various mixed salt-alkaline stresses. J Integr Plant Biol 50(1):29–39

    CAS  PubMed  Google Scholar 

  • Rodríguez-Rosales MP, Galvez FJ, Venema K et al (2009) Plant NHX cation/proton antiporters. Plant Signal Behav 4(4):265–276

    PubMed  PubMed Central  Google Scholar 

  • Sanchez-Barrena MJ, Martinez-Ripoll M, Zhu JK, 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

    CAS  PubMed  Google Scholar 

  • Sathee L, Sairam RK, Chinnusamy V, Jha SK (2015) Differential transcript abundance of salt overly sensitive (SOS) pathway genes is a determinant of salinity stress tolerance of wheat. Acta Physiologiae Plantarum. https://doi.org/10.1007/s11738-015-1910-z

    Article  Google Scholar 

  • Shi HZ, Kin YS, Guo Y, Stevenson B, Zhu JK (2003) The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion. Plant Cell 15:19–32

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shi HZ, Xiong LM, Stevenson B, Lu T, Zhu JK (2002) The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for Vitamin B6 in plant salt tolerance. Plant Cell 14:575–588

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shi J, Fu XZ, Peng T, Liu JH et al (2009) Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response. Tree Physiol 30:914–922

    Google Scholar 

  • Shukla PS, Agarwal PK, Jha B (2012) Improved salinity tolerance of arachis hypogaea (L.) by the interaction of halotolerant plant-growth-promoting rhizobacteria. J Plant Growth Regul 31:195–206

    CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    CAS  PubMed  Google Scholar 

  • Viladevall L, Serrano R, Ruiz A, Domenech G, Arino J et al (2004) Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae. J Biol Chem 279(42):43614–43624

    CAS  PubMed  Google Scholar 

  • Wang H, Liang Y, Zhang B, Zheng W, Xing LJ, Li MC (2011) Alkaline stress triggers an immediate calcium fuctuation in Candida albicans mediated by Rim101p andCrz1p transcription factors. Yeast Res. https://doi.org/10.1111/j.1567-1364.2011.00730.x

    Article  Google Scholar 

  • White PJ, Broadley MR (2003) Calcium in plants. Ann Bot 92:487–511

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wu WH, Wang Y, Lee SC, Lan WZ, Luan S (2010) Regulation of ion channels by the calcium signaling network in plant cells. Signal Commun Plants. https://doi.org/10.1007/978-3-642-10494-7_6

    Article  Google Scholar 

  • Xiong LM, Schumaker K, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 14:165–183

    Google Scholar 

  • Yadav NS, Shukla PS, Jha A, Agarval PK, Jha B (2012) The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na+ loading in xylem and confers salt tolerance in transgenic tobacco. BMC Plant Biol 12:188

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yang CW, Guo WQ, Shi DC (2010) Physiological roles of organic acids in alkali-tolerance of the alkali-tolerant halophyte Chloris virgata. Agron J 102(4):1081–1089

    CAS  Google Scholar 

  • Yang Q, Chen ZZ, Zhou XF, Gong ZZ et al (2009) Overexpression of SOS (salt overly sensitive) genes increases salt tolerance in transgenic arabidopsis. Molecular Plant 2(1):22–31

    CAS  PubMed  Google Scholar 

  • Yang YQ, Wu YJ, Ma L et al (2019) The Ca2+ sensor SCaBP3/CBL7 modulates plasma membrane H+-ATPase activity and promotes alkali tolerance in arabidopsis. Plant Cell 31(6):1367–1384

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang CX, Bian MD, Yu H, Liu Q, Yang ZM (2011) Identification of alkaline stress-responsive genes of CBL family in sweet sorghum (Sorghum bicolor L.). Plant Physiol Biochem 49:1306–1312

    CAS  PubMed  Google Scholar 

  • Zhang XG, Huang B, Hu WY et al (2013) Study on salinization characteristics of surface soil in western songnen plain. Soil 45(2):332–338

    CAS  Google Scholar 

  • Zhang YM, Linghu JJ, Wang D, Zhao TY et al (2017) Foxtail millet CBL4 (SiCBL4) interacts with SiCIPK24, modulates plant salt stress tolerance. Plant Mol Biol Rep 35:634–646

    Google Scholar 

  • Zhao YK, Wang T, Zhang WS, Li X (2011) SOS3 mediates lateral root development under low salt stress through regulation of auxin redistribution and maxima in Arabidopsis. New Phytol 189:1122–1134. https://doi.org/10.1111/j.1469-8137.2010.03545.x

    Article  CAS  PubMed  Google Scholar 

  • Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6(2):66–71

    CAS  PubMed  Google Scholar 

  • Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu S, Zhou XP, Wu XM, Jiang ZH (2013) Structure and function of the CBL–CIPK Ca2+-decoding system in plant calcium signaling. Plant Mol Biol Rep 31:1193–1202

    CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National and Science Foundation of China (grant numbers 31470571, 31972507); the National Major Project for Cultivation of Transgenic Crops (grant number 2016ZX08004-002-003); the National Key Research and Development Program of China (grant number 2017YFD0101303). Science and Technology Major Project of Heilongjiang Province (GA18B104); Science and Technology Achievement Cultivation Project of Heilongjiang Education Department (TSTAU-R2018008); Graduate Innovation Fund of Harbin Normal University (HSDSSCX2019-23); Research Fund of Beijing Advanced Innovation Center for Food Nutrition and Human Health (20182018).

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Correspondence to Changhong Guo.

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An, Y., Yang, XX., Zhang, L. et al. Alfalfa MsCBL4 enhances calcium metabolism but not sodium transport in transgenic tobacco under salt and saline–alkali stress. Plant Cell Rep 39, 997–1011 (2020). https://doi.org/10.1007/s00299-020-02543-x

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