Heterologous overexpression of the Arabidopsis SnRK2.8 gene enhances drought and salt tolerance in Populus × euramericana cv ‘Nanlin895’

  • Hui Wei
  • Ali Movahedi
  • Chen Xu
  • Pu Wang
  • Weibo Sun
  • Tongming Yin
  • Qiang ZhugeEmail author
Original Article


The SnRK (sucrose non-fermenting-1 related protein kinase) families respond to salt tolerance, nutritional stress, disease stress, and carbon metabolic regulation. In this study, the SnRK2.8 gene was cloned from Arabidopsis thaliana and nominated AtSnRK2.8. Bioinformatics characterization of AtSnRK2.8 showed that it contained an ATP binding site and a serine/threonine protein kinase binding site at the N-terminal catalytic domain. In addition, the AtSnRK2.8 protein contained special domain I at the C-terminal catalytic domain. Real-time polymerase chain reaction (PCR) results showed that the AtSnRK2.8 gene was expressed in all tested tissues and was highly expressed in seeds. Our analyses proved that the AtSnRK2.8 gene expression was upregulated by salt and drought stresses. The recombinant AtSnRK2.8 protein was expressed using a pET-28a system and purified with HisTrap HP affinity columns. The recombinant AtSnRK2.8 protein has the properties of a protein kinase. The results of overexpressing AtSnRK2.8 in Nanlin895 poplar showed that the transgenic poplar plants had a better ability to resist drought and salt stress compared to the wild type, and adapted well to drought and a high-salt environment.


Arabidopsis thaliana Drought and salt Tolerance Heterologous Overexpression Populus SnRK 



This work was supported by the National Key Program on Transgenic Research (2018ZX08020002), the National Science Foundation of China (no. 31570650) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Author contributions

HW and AM designed the experiments and wrote the manuscript. CX, PW, WS did help to the HW and AM to do experiments. TY and QZ supervised this project.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

11816_2019_531_MOESM1_ESM.jpg (66 kb)
Supplementary material 1 Figure S1: Phosphorylation site analysis of the AtSnRK2.8 protein using the SignalP 4.1 Server ( online program (JPG 65 KB)
11816_2019_531_MOESM2_ESM.jpg (217 kb)
Supplementary material 2 Figure S2: 1 Expression analysis of the AtSRK2.8 gene in transgenic poplar plants. Total RNA was isolated from the leaves of wild-type and transgenic (numbers represent lines) poplar plants, and subjected to RT-PCR analysis. (A) Expression of AtSRK2.8 in AtSRK2.8 transgenic poplar plants. (B) Expression of Ptactin as the internal control in AtSRK2.8 transgenic plants. (C) Quantitative RT-PCR analysis of transcript levels of AtSRK2.8 in AtSRK2.8 transgenic poplar plants (JPG 217 KB)
11816_2019_531_MOESM3_ESM.jpg (772 kb)
Supplementary material 3 Figure S3: Transplanting process for the transgenic poplar after propagation (JPG 771 KB)
11816_2019_531_MOESM4_ESM.jpg (639 kb)
Supplementary material 4 Figure S4: Phenotypic analysis of the wild-type (WT) and AtSRK2.8 transgenic plants before and after the drought and salt treatments. (A) Phenotypic analysis of WT and AtSRK2.8 transgenic plants after a 2-week salt treatment. (B D) Phenotypic analysis of WT and AtSRK2.8 transgenic plants before stress treatment (under normal conditions). (C) Phenotypic analysis of WT and AtSRK2.8 transgenic plants after a 2-week drought treatment. Three independent experiments were performed (JPG 639 KB)


  1. Alderson A, Sabelli PA, Dickinson JR, Cole D, Richardson M, Kreis M et al (1991) Complementation of snf1, a mutation affecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA. Proc Natl Acad Sci 88(19):8602–8605Google Scholar
  2. Allen JA, Chambers JL, Stine M (1994) Prospects for increasing the salt tolerance of forest trees: a review. Tree Physiol 14(7_9):843Google Scholar
  3. Anderberg RJ, Walkersimmons MK (1992) Isolation of a wheat cdna clone for an abscisic acid-inducible transcript with homology to protein kinases. Proc Natl Acad Sci USA 89(21):10183–10187Google Scholar
  4. Boudsocq M, Barbierbrygoo H, Laurière C (2004) Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana. J Biol Chem 279(40):41758–41766Google Scholar
  5. Cheeke TE (2012) Effects of the cultivation of genetically modified Bt crops on nontarget soil organisms. In: Cheeke TE, Coleman DC, Wall DH (eds) Microbial ecology in sustainable agroecosystems. CRC Press, Boca Raton, pp 153–227Google Scholar
  6. Crompton T, Barrett J, Gliddon C, Kinderlerer J, Tzotzos G, Gates P (1998) Assessing risks of genetic engineering. Nature 392(6678):751Google Scholar
  7. Dale PJ (1997) Potential impacts from the release of transgenic plants into the environment. Acta Physiol Plant 19(4):595–600Google Scholar
  8. European Food Safety Authority (EFSA) (2013) International scientific workshop “Non‐Target Organisms and Genetically Modified Crops: Assessing the effects of Bt proteins” (29–30 November 2012, Amsterdam, The Netherlands). EFSA Support Publ 10(9):484EGoogle Scholar
  9. Gómezcadenas A, Verhey SD, Holappa LD, Shen Q, Ho TH, Walkersimmons MK (1999) An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers. Proc Natl Acad Sci USA 96(4):1767–1772Google Scholar
  10. Halford N, Hey S (2009) Snf1-related protein kinases (SNRKs) act within an intricate network that links metabolic and stress signaling in plants. Biochem J 419(2):247Google Scholar
  11. Halfter U, Ishitani M, Zhu JK (2000) The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3. Proc Natl Acad Sci USA 97(7):3735–3740Google Scholar
  12. Hey SJ, Byrne E, Halford NG (2010) The interface between metabolic and stress signalling. Ann Bot 105(2):197–203Google Scholar
  13. Holappa LD, Walkersimmons MK (1995) The wheat abscisic acid-responsive protein kinase mRNA, PKABA1, is up-regulated by dehydration, cold temperature, and osmotic stress. Plant Physiol 108(3):1203–1210PubMedCentralGoogle Scholar
  14. Hrabak EM, Chan CW, Gribskov M, Harper JF, Choi JH, Halford N et al (2003) The arabidopsis cdpk-snrk superfamily of protein kinases. Plant Physiol 132(2):666–680PubMedCentralGoogle Scholar
  15. Huai J, Wang M, He J, Zheng J, Dong Z, Lv H et al (2008) Cloning and characterization of the SnRK2 gene family from Zea mays. Plant Cell Rep 27(12):1861–1868Google Scholar
  16. Ikeda Y, Koizumi N, Kusano T, Sano H (1999) Sucrose and cytokinin modulation of WPK4, a gene encoding a SNF1-related protein kinase from wheat. Plant Physiol 121(3):813–820PubMedCentralGoogle Scholar
  17. Johnson RR, Wagner RL, Verhey SD, Walker-Simmons MK (2002) The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences. Plant Physiol 130(2):837–846PubMedCentralGoogle Scholar
  18. Jossier M, Bouly JP, Meimoun P, Arjmand A, Lessard P, Hawley S et al (2009) SnRK1 (SNF1-related kinase 1) has a central role in sugar and aba signalling in Arabidopsis thaliana. Plant J 59(2):316Google Scholar
  19. Kobayashi Y, Yamamoto S, Minami H, Kagaya Y, Hattori T (2004) Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid. Plant Cell 16(5):1163–1177PubMedCentralGoogle Scholar
  20. Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot 63(4):1593PubMedCentralGoogle Scholar
  21. Li J, Assmann SM (1996) An abscisic acid-activated and calcium-independent protein kinase from guard cells of fava bean. Plant Cell 8(12):2359–2368PubMedCentralGoogle Scholar
  22. Li LB, Zhang YR, Liu KC, Ni ZF, Fang ZJ, Sun QX, Gao JW (2010) Identification and bioinformatics analysis of SnRK2 and CIPK family genes in sorghum. Agric Sci China 9(1):19–30Google Scholar
  23. Movahedi A, Zhang J, Gao P, Yang Y, Wang L, Yin T et al (2015) Expression of the chickpea CarNAC3, gene enhances salinity and drought tolerance in transgenic poplars. Plant Cell Tissue Org Cult 120(1):141–154Google Scholar
  24. Mustilli AC, Giraudat J (2002) Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14(12):3089–3099PubMedCentralGoogle Scholar
  25. Nishizawa A, Yabuta Y, Shigeoka S (2008) Galactinol and raffinose constitute a novel function to protect plants from oxidative damage. Plant Physiol 147(3):1251–1263PubMedCentralGoogle Scholar
  26. Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y et al (2009) Abscisic acid inhibits type 2c protein phosphatases via the PYR/PYL family of start proteins. Science 324(5930):1068–1071PubMedCentralGoogle Scholar
  27. Santiago J, Dupeux F, Betz K, Antoni R, Gonzalez-Guzman M, Rodriguez L et al (2012) Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs. Plant Sci 182(1):3–11Google Scholar
  28. Song X, Ohtani M, Hori C, Takebayasi A, Zhuge Q (2015) Physical interaction between snrk2 and pp2c is conserved in populus trichocarpa. Plant Biotechnol 32(4):359–364Google Scholar
  29. Song X, Yu X, Hori C, Demura T, Ohtani M, Zhuge Q (2016) Heterologous overexpression of poplar SnRK2 genes enhanced salt stress tolerance in Arabidopsis thaliana. Front Plant Sci 7:612PubMedCentralGoogle Scholar
  30. Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M et al (2010) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J 29(4):417–426Google Scholar
  31. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739PubMedCentralGoogle Scholar
  32. Tilman D, Fargione J, Wolff B, D’Antonio C, Dobson A, Howarth R et al (2001) Forecasting agriculturally driven global environmental change. Science 292(5515):281Google Scholar
  33. Ton J, Flors V, Mauch-Mani B (2009) The multifaceted role of ABA in disease resistance. Trends Plant Sci 14(6):310–317Google Scholar
  34. Umezawa T, Yoshida R, Maruyama K, Yamaguchishinozaki K, Shinozaki K (2004) SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana. Proc Natl Acad Sci USA 101(49):17306–17311Google Scholar
  35. Umezawa T, Sugiyama N, Takahashi F, Anderson JC, Ishihama Y, Peck SC et al (2013) Genetics and phosphoproteomics reveal a protein phosphorylation network in the abscisic acid signaling pathway in Arabidopsis thaliana. Sci Signal 6(270):rs8Google Scholar
  36. Watkinson AR, Freckleton RP, Robinson RA, Sutherland WJ (2000) Predictions of biodiversity response to genetically modified herbicide-tolerant crops. Science 289(5484):1554–1557Google Scholar
  37. Yoshida R, Umezawa T, Mizoguchi T, Takahashi S, Takahashi F, Shinozaki K (2006) The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis. J Biol Chem 281(8):5310–5318Google Scholar
  38. Yoshida T, Mogami J, Yamaguchi-Shinozaki K (2014) ABA-dependent and ABA-independent signaling in response to osmotic stress in plants. Curr Opion Plant Biol 21:133–139Google Scholar
  39. Yu X, Ohtani M, Kusano M, Nishikubo N, Uenoyama M, Umezawa T et al (2017) Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, atsrk2c, and atgols2. Mol Breed 37(5):57Google Scholar
  40. Zhou X, Hao H, Zhang Y, Bai Y, Zhu W, Qin Y et al (2015) SOS2-like protein kinase5, an SNF1-related protein kinase3-type protein kinase, is important for abscisic acid responses in Arabidopsis through phosphorylation of abscisic acid-insensitive5. Plant Physiol 168(2):659–676PubMedCentralGoogle Scholar

Copyright information

© Korean Society for Plant Biotechnology 2019

Authors and Affiliations

  1. 1.Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics and Biotechnology, Ministry of EductionNanjing Forestry UniversityNanjingChina
  2. 2.Jiangsu Provincial Key Construction Laboratory of Special Biomass Resource UtilizationNanjing Xiaozhuang UniversityNanjingChina

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