MdbHLH106-like transcription factor enhances apple salt tolerance by upregulating MdNHX1 expression

Abstract

Soil salinization is one of the most important abiotic stresses adversely affecting plant growth and yield. In this study, we cloned and characterized a putative salt tolerance gene encoding a bHLH (basic helix-loop-helix) transcription factor, MdbHLH106L (MdbHLH106-like), in the genome of apple (Malus domestica Borkh.). The expression level and promoter activity of MdbHLH106L are increased under salt stress. Overexpressing MdbHLH106L in ‘Orin’ calli can promote the expression level of MdNHX1 (Na+/H+ exchanger 1). Further analysis showed that MdbHLH106L is able to bind the MdNHX1 promoter. Moreover, we also confirmed the interaction between MdbHLH106L and MdZAT10. In conclusion, MdbHLH106L participates in the salt-stress signaling pathway and may mediate salt tolerance by activating the MdNHX1 promoter and interacting with MdZAT10. Our findings may serve as a theoretical basis for further investigating molecular mechanisms of stress responses and for enriching genetic resources of stress-tolerant apple cultivars.

Key Message

  1. 1.

    MdbHLH106-like transcription factor enhances apple salt tolerance

  2. 2.

    MdbHLH106L binds to the MdNHX1 promoter

  3. 3.

    MdbHLH106L interacts with MdZAT10 in vitro and in vivo

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63–78

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Ahmad A, Niwa Y, Goto S, Ogawa T, Shimizu M, Suzuki A, Kobayashi K, Kobayashi H (2015) bHLH106 Integrates functions of multiple genes through their G-Box to confer salt tolerance on arabidopsis. PLoS One 10:e0126872

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  3. Alizadeh M, Singh SK, Patel VB, Bhattacharya RC, Yadav BP (2010) In vitro responses of grape rootstocks to NaCl. Biol Plant 54:381–385

    Article  Google Scholar 

  4. Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285:1256–1258

    CAS  PubMed  Article  Google Scholar 

  5. Babitha KC, Ramu SV, Pruthvi V, Mahesh P, Nataraja KN, Udayakumar M (2013) Co-expression of AtbHLH17 and AtWRKY28 confers resistance to abiotic stress in Arabidopsis. Transgenic Res 22:327–341

    CAS  PubMed  Article  Google Scholar 

  6. Bassil E, Blumwald E (2014) The ins and outs of intracellular ion homeostasis: NHX-type cation/H(+) transporters. Curr Opin Plant Biol 22:1–6

    CAS  PubMed  Article  Google Scholar 

  7. Blumwald E, Poole RJ (1985) Na/H antiport in isolated tonoplast vesicles from storage tissue of beta vulgaris. Plant Physiol 78:163–167

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Britto DT, Kronzucker HJ (2006) Futile cycling at the plasma membrane: a hallmark of low-affinity nutrient transport. Trends Plant Sci 11:529–534

    CAS  PubMed  Article  Google Scholar 

  9. Carretero-Paulet L, Galstyan A, Roig-Villanova I, Martínez-García JF, Bilbao-Castro JR, Robertson DL (2010) Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in arabidopsis, poplar, rice, moss, and algae. Plant Physiol 153:1398–1412

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Chen XS, Han MY, Su GL, Liu FZ, Guo GN, Jiang YM, Mao ZQ, Peng FT, Shu HR (2010) Discussion on today’s world apple industry trends and the suggestions on sustainable and efficient development of apple industry in China. J Fruit Sci 27(4):598–604

    Google Scholar 

  11. Ciftci-Yilmaz S, Morsy MR, Song L, Coutu A, Krizek BA, Lewis MW, Warren D, Cushman J, Connolly EL, Mittler R (2007) The EAR-motif of the Cys2/His2-type zinc finger protein Zat7 plays a key role in the defense response of Arabidopsis to salinity stress. J Biol Chem 282:9260–9268

    CAS  PubMed  Article  Google Scholar 

  12. Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ et al (2005) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17:268–281

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Ding Z, Li S, An X, Liu X, Qin H, Wang D (2009) Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana. J Genet Genom 36:17–29

    CAS  Article  Google Scholar 

  14. Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55:307–319

    CAS  PubMed  Article  Google Scholar 

  15. Furrer EM, Ronchetti MF, Verrey F, Pos KM (2007) Functional characterization of a NapA Na+/H+ antiporter from Thermus thermophilus. FEBS Lett 581:572–578

    CAS  PubMed  Article  Google Scholar 

  16. Golldack D, Li C, Mohan H, Probst N (2014) Tolerance to drought and salt stress in plants: unraveling the signaling networks. Front Plant Sci 5:151

    PubMed  PubMed Central  Article  Google Scholar 

  17. Guo Y, Halfter U, Ishitani M, Zhu JK (2001) Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance. Plant Cell 13:1383–1399

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463–499

    CAS  PubMed  Article  Google Scholar 

  19. Hichri I, Heppel SC, Pillet J, Léon C, Czemmel S, Delrot S, Lauvergeat V, Bogs J (2010) The basic Helix-Loop-Helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine. Mol Plant 3:509–523

    CAS  PubMed  Article  Google Scholar 

  20. Hu DG, Sun CH, Zhang QY, An JP, You CX, Hao YJ (2016) Glucose sensor MdHXK1 phosphorylates and stabilizes MdbHLH3 to promote anthocyanin biosynthesis in apple. PLoS Genet 12:e1006273

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  21. Husaini AM, Abdin MZ (2008) Development of transgenic strawberry (Fragaria × ananassa Duch.) plants tolerant to salt stress. Plant Sci 174:446–455

    CAS  Article  Google Scholar 

  22. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions, betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Ji XH, Wang YT, Zhang R, Wu SJ, An MM, Li M, Wang CZ, Chen XL, Zhang YM, Chen XS (2015) Effect of auxin, cytokinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell Tissue Organ Cult 120:325–337

    CAS  Article  Google Scholar 

  24. Jiang Y, Yang B, Deyholos MK (2009) Functional characterization of the Arabidopsis bHLH92 transcription factor in abiotic stress. Mol Gent Genom 282:503–516

    CAS  Article  Google Scholar 

  25. Kazan K (2006) Negative regulation of defence and stress genes by EAR-motif-conta9268ining repressors. Trends Plant Sci 11:109–112

    CAS  PubMed  Article  Google Scholar 

  26. Kim J, Kim HY (2006) Molecular characterization of a bHLH transcription factor involved in Arabidopsis abscisic acid-mediated response. Biochim Biophys Acta 1759:191–194

    CAS  PubMed  Article  Google Scholar 

  27. Li H, Sun J, Xu Y, Jiang H, Wu X, Li C (2007) The bHLH-type transcription factor AtAIB positively regulates ABA response in Arabidopsis. Plant Mol Biol 65:655–665

    CAS  PubMed  Article  Google Scholar 

  28. Liu XM, Nguyen XC, Kim KE, Han HJ, Yoo J, Lee K, Kim MC, Yun DJ, Chung WS (2013) Phosphorylation of the zinc finger transcriptional regulator ZAT6 by MPK6 regulates Arabidopsis seed germination under salt and osmotic stress. Biochem Biophys Res Commun 430:1054–1059

    CAS  PubMed  Article  Google Scholar 

  29. Liu X, Liu X, An X, Han P, You C, Hao Y (2017) An apple protein kinase MdSnRK1.1 interacts with MdCAIP1 to regulate ABA sensitivity. Plant Cell Physiol 58:1631–1641

    CAS  PubMed  Article  Google Scholar 

  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    CAS  Article  Google Scholar 

  31. Meng CM, Zhang TZ, Guo WZ (2009) Molecular cloning and characterization of a Novel Gossypium hirsutum L. bHLH Gene in response to ABA and drought stresses. Plant Mol Biol Rep 27:381–387

    CAS  Article  Google Scholar 

  32. Miller G, Shulaev V, Mittler R (2008) Reactive oxygen signaling and abiotic stress. Physiol Plant 133:481–489

    CAS  PubMed  Article  Google Scholar 

  33. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410

    CAS  PubMed  Article  Google Scholar 

  34. Mittler R, Kim YS, Song L, Coutu J, Coutu A, Ciftci-Yilmaz S, Lee H, Stevenson B, Zhu JK (2006) Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress. FEBS Lett 580:6537–6542

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Nguyen XC, Kim SH, Lee K, Kim KE, Liu XM, Han HJ, Hoang MHT, Lee SW, Hong JC, Moon YH, Chungal WS (2012) Identification of a C2H2-type zinc finger transcription factor (ZAT10) from Arabidopsis as a substrate of MAP kinase. Plant Cell Rep 31:737–745

    CAS  PubMed  Article  Google Scholar 

  36. Ohta M, Matsui K, Hiratsu K, Shinshi H, Ohme-Takagi M (2001) Repression domains of class II ERF transcriptional repressors share an essential motif for active repression. Plant Cell 13:1959–1968

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Quintero FJ, Martinez-Atienza J, Caros I, Jiang XY, Kim Y, Ali Z, Fujii H, Mendoza I, Yun DJ, Zhu JK, Pardo JM (2011) Activation of the plasma membrane Na+/H+ antiporter SOS1 by phosphorylation of an auto-inhibitory C-terminal domain. Proc Natl Acad Sci U S A 108:2611–2616

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Ramsay NA, Glover BJ (2005) MYB–bHLH–WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci 10(2):63–70

    CAS  PubMed  Article  Google Scholar 

  39. Rana M (2010) Genes and salt tolerance: bringing them together. New Phytol 167:645–663

    Google Scholar 

  40. Sahi C, Singh A, Blumwald E, Grover A (2006) Beyond osmolytes and transporters: novel plant salt-stress tolerance-related genes from transcriptional profiling data. Physiol Plant 127:1–9

    CAS  Article  Google Scholar 

  41. Sakamoto H, Maruyama K, Sakuma Y, Meshi T, Iwabuchi M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol 136:2734–2746

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Seo JS, Joo JS, Kim MJ, Kim YK, Nahm BH, Song SI, Cheong JJ, Lee JS, Kim JK, Choi YD (2011) OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice. Plant J 65:907–921

    CAS  PubMed  Article  Google Scholar 

  43. Shinozaki K (1999) Plant response to drought and salt stress: overview. Tanpakushitsu Kakusan Koso Protein Nucleic Acid Enzyme 44:2186–2187

    CAS  Google Scholar 

  44. Silva P, Facanha AR, Tavares RM, Gerós H (2010) Role of tonoplast proton pumps and Na+/H+ antiporter system in salt tolerance of Populus euphratica Oliv. Plant Growth Regul 29:23–34

    CAS  Article  Google Scholar 

  45. Toledo-Ortiz G, Huq E, Quail PH (2003) The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell 15:1749–1770

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40:428–438

    CAS  PubMed  Article  Google Scholar 

  47. Wang YJ, Zhang ZJ, He XJ, Zhou HL, Wen YX, Dai JX, Zhang SJ, Chen SY (2003) A rice transcription factor OsbHLH1 is involved in cold stress response. Theor Appl Genet 107:1402–1409

    CAS  PubMed  Article  Google Scholar 

  48. Wang N, Qu CZ, Wang YC, Xu HF, Jiang SH, Fang HC, Liu JX, Zhang ZY, Chen XS (2017) MdMYB4 enhances apple callus salt tolerance by increasing MdNHX1 expression levels. Plant Cell Tissue Organ Cult 131:283–293

    Article  CAS  Google Scholar 

  49. Wang N, Liu WJ, Yu L, Guo ZW, Chen ZJ, Jiang SH, Xu HF, Fang HC, Wang YC, Zhang ZY, Chen XS (2020) Heat shock factor A8a modulates flavonoid synthesis and drought tolerance. Plant Physiol 183:1273–1290

    Article  CAS  Google Scholar 

  50. Xu HF, Wang N, Liu JX, Qu CZ, Wang YC, Jiang SH, Lu NL, Wang DY, Zhang ZY, Chen XS (2017a) The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Mol Biol 94:149–165

    CAS  PubMed  Article  Google Scholar 

  51. Xu D, Jiang Y, Li J, Holm M, Deng XW (2017b) The B-box domain protein BBX21 promotes photomorphogenesis. Plant Physiol 176:2365–2375

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. Xu HF, Wang N, Wang YC, Jiang SH, Fang HC, Zhang J, Su MY, Zuo WF, Xu L, Zhang ZY, Chen XS (2018) Overexpression of the transcription factor MdbHLH33 increases cold tolerance of transgenic apple callus. Plant Cell Tissue Organ Cult 134:131–140

    CAS  Article  Google Scholar 

  53. Yadav S, Irfan M, Ahmad A, Hayat S (2011) Causes of salinity and plant manifestations to salt stress: a review. J Environ Biol 32:667

    PubMed  Google Scholar 

  54. Ye CY, Yang X, Xia X, Yin W (2013) Comparative analysis of cation/proton antiporter superfamily in plants. Gene 521:245–251

    CAS  PubMed  Article  Google Scholar 

  55. Zhang HX, Blumwald E (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol 19:765

    CAS  PubMed  Article  Google Scholar 

  56. Zhang X-H, Rao X-L, Shi H-T, Li R-J, Lu Y-T (2011) Overexpression of a cytosolic glyceraldehyde-3-phosphate dehydrogenase gene OsGAPC3 confers salt tolerance in rice. Plant Cell Tissue Organ Cult (PCTOC) 107(1):1–11

    CAS  Article  Google Scholar 

Download references

Acknowledgments

We thank Yujin Hao Laboratories for providing the vectors, and Shujing Wu for comments on the manuscript.

Funding

This study was supported by the Agricultural Variety Improvement Project of Shandong Province (2019LZGC007) and the National Natural Science Foundation of China (31730080).

Author information

Affiliations

Authors

Contributions

QZ and XC devised and supervised the project. GY, and QZ performed the experiments. HX, LY, HJ, ZM and JH contributed to the preparation of all figures and tables. ZZ and NW analyzed the data. QZ and GY wrote the manuscript. All authors reviewed, revised, and approved the manuscript.

Corresponding author

Correspondence to Xuesen Chen.

Ethics declarations

Conflict of interest

All authors have no competing financial interests to declare.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by Ming-Tsair Chan.

Supplementary Information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zou, Q., Xu, H., Yang, G. et al. MdbHLH106-like transcription factor enhances apple salt tolerance by upregulating MdNHX1 expression. Plant Cell Tiss Organ Cult (2021). https://doi.org/10.1007/s11240-021-02011-8

Download citation

Keywords

  • Apple
  • Salt tolerance
  • Callus
  • bHLH106L
  • NHX1