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Protoplasma

, Volume 256, Issue 5, pp 1333–1344 | Cite as

Functional domain analysis of LmSAP protein reveals the crucial role of the zinc-finger A20 domain in abiotic stress tolerance

  • Rania Ben Saad
  • Hela Safi
  • Anis Ben Hsouna
  • Faical Brini
  • Walid Ben RomdhaneEmail author
Original Article
  • 154 Downloads

Abstract

Stress-associated proteins (SAPs), such as A20/AN1 zinc-finger domain-containing proteins, have emerged as a novel class of proteins involved in abiotic stress signaling, and they are important candidates for preventing the loss of yield caused by exposure to environmental stresses. In a previous report, it was found that the ectopic-expression of Lobularia maritima stress-associated protein, LmSAP, conferred tolerance to abiotic and heavy metal stresses in transgenic tobacco plants. This study aimed to investigate the functions of the A20 and AN1 domains of LmSAP in salt and osmotic stress tolerance. To this end, in addition to the full-length LmSAP gene, we have generated three LmSAP-truncated forms (LmSAPΔA20, LmSAPΔAN1, and LmSAPΔA20-ΔAN1). Heterologous expression in Saccharomyces cerevisiae of different truncated forms of LmSAP revealed that the A20 domain is essential to increase cell tolerance to salt, ionic, and osmotic stresses. Transgenic tobacco plants overexpressing LmSAP and LmSAPΔAN1 constructs exhibited higher tolerance to salt and osmotic stresses in comparison to the non-transgenic plants (NT) and lines transformed with LmSAPΔA20 and LmSAPΔA20-ΔAN1 constructs. Similarly, transgenic plants overexpressing the full-length LmSAP gene and LmSAPΔAN1 truncated domain maintained higher superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) enzymatic activities due to the high expression levels of the genes encoding these key antioxidant enzymes, MnSOD, POD, and CAT1, as well as accumulated lower levels of malondialdehyde (MDA) under salt and osmotic stresses compared to NT and LmSAPΔA20 and LmSAPΔA20-ΔAN1 forms. These findings provide insights into the pivotal role of A20 and AN1 domains of LmSAP protein in salt and osmotic stress tolerance.

Keywords

Lobularia maritima Transgenic tobacco Domains deletion Abiotic stress tolerance 

Notes

Funding information

This work was financially supported in part by a grant from the Tunisian Ministry of Higher Education and Scientific Research (contract program 2015-2018, CBS-LBAP/code: LR15CBS03).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

709_2019_1390_MOESM1_ESM.docx (17 kb)
ESM 1 (DOCX 17 kb)

References

  1. Aebi H (1984) Catalase in vitro. In: Methods Enzymol., vol 105. Elsevier, pp 121–126Google Scholar
  2. Atanassov II, Atanassov II, Etchells JP, Turner SR (2009) A simple, flexible and efficient PCR-fusion/gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps. Plant Methods 5(1):14PubMedPubMedCentralCrossRefGoogle Scholar
  3. Ben Saad R, Zouari N, Ben Ramdhan W, Azaza J, Meynard D, Guiderdoni E, Hassairi A (2010) Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-finger “AlSAP” gene isolated from the halophyte grass Aeluropus littoralis. Plant Mol Biol 72(1–2):171–190PubMedCrossRefGoogle Scholar
  4. Ben Saad R, Fabre D, Mieulet D, Meynard D, Dingkuhn M, Al-Doss A, Guiderdoni E, Hassairi A (2012) Expression of the Aeluropus littoralis AlSAP gene in rice confers broad tolerance to abiotic stresses through maintenance of photosynthesis. Plant Cell Environ 35(3):626–643PubMedCrossRefGoogle Scholar
  5. Ben Saad R, Farhat-Khemekhem A, Ben Halima N, Ben Hamed K, Brini F, Saibi W (2017) The LmSAP gene isolated from the halotolerant Lobularia maritima improves salt and ionic tolerance in transgenic tobacco lines. Funct Plant Biol 45(3):378–391CrossRefGoogle Scholar
  6. Ben Saad R, Hsouna AB, Saibi W, Hamed KB, Brini F, Ghneim-Herrera T (2018) A stress-associated protein, LmSAP, from the halophyte Lobularia maritima provides tolerance to heavy metals in tobacco through increased ROS scavenging and metal detoxification processes. J Plant Physiol 231:234–243CrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedPubMedCentralCrossRefGoogle Scholar
  8. Catrysse L, Vereecke L, Beyaert R, van Loo G (2014) A20 in inflammation and autoimmunity. Trends Immunol 35(1):22–31PubMedCrossRefGoogle Scholar
  9. Chang EJ, Ha J, Kang SS, Lee ZH, Kim HH (2011) AWP1 binds to tumor necrosis factor receptor-associated factor 2 (TRAF2) and is involved in TRAF2-mediated nuclear factor-kappaB signaling. Int J Biochem Cell Biol 43(11):1612–1620PubMedCrossRefGoogle Scholar
  10. Chang L, Chang HH, Chang JC, Lu HC, Wang TT, Hsu DW, Tzean Y, Cheng AP, Chiu YS, Yeh HH (2018) Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity. PLoS Pathog 14(9):e1007288PubMedPubMedCentralCrossRefGoogle Scholar
  11. Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. BioTechniques 16 (4):664–668, 670Google Scholar
  12. Dansana PK, Kothari KS, Vij S, Tyagi AK (2014) OsiSAP1 overexpression improves water-deficit stress tolerance in transgenic rice by affecting expression of endogenous stress-related genes. Plant Cell Rep 33(9):1425–1440PubMedCrossRefGoogle Scholar
  13. Dixit VM, Green S, Sarma V, Holzman LB, Wolf FW, O’Rourke K, Ward PA, Prochownik EV, Marks RM (1990) Tumor necrosis factor-alpha induction of novel gene products in human endothelial cells including a macrophage-specific chemotaxin. J Biol Chem 265(5):2973–2978PubMedGoogle Scholar
  14. Dixit A, Tomar P, Vaine E, Abdullah H, Hazen S, Dhankher OP (2018) A stress-associated protein, AtSAP13, from Arabidopsis thaliana provides tolerance to multiple abiotic stresses. Plant Cell Environ 41(5):1171–1185PubMedCrossRefGoogle Scholar
  15. Dong QL, Duan DY, Zhao S, Xu BY, Luo JW, Wang Q, Huang D, Liu CH, Li C, Gong XQ, Mao K, Ma FW (2018) Genome-wide analysis and cloning of the apple stress-associated protein gene family reveals MdSAP15, which confers tolerance to drought and osmotic stresses in transgenic Arabidopsis. Int J Mol Sci 19(9):2478PubMedCentralCrossRefGoogle Scholar
  16. Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M (1993) A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med 15(4):353–363PubMedCrossRefPubMedCentralGoogle Scholar
  17. Duan W, Sun BG, Li TW, Tan BJ, Lee MK, Teo TS (2000) Cloning and characterization of AWP1, a novel protein that associates with serine/threonine kinase PRK1 in vivo. Gene 256(1–2):113–121PubMedCrossRefGoogle Scholar
  18. Elble R (1992) A simple and efficient procedure for transformation of yeasts. BioTechniques 13(1):18–20PubMedGoogle Scholar
  19. Evans PC, Ovaa H, Hamon M, Kilshaw PJ, Hamm S, Bauer S, Ploegh HL, Smith TS (2004) Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity. Biochem J 378 (Pt 3:727–734PubMedPubMedCentralCrossRefGoogle Scholar
  20. Gimeno-Gilles C, Gervais ML, Planchet E, Satour P, Limami AM, Lelievre E (2011) A stress-associated protein containing A20/AN1 zing-finger domains expressed in Medicago truncatula seeds. Plant Physiol Biochem 49(3):303–310PubMedCrossRefGoogle Scholar
  21. Giri J, Vij S, Dansana PK, Tyagi AK (2011) Rice A20/AN1 zinc-finger containing stress-associated proteins (SAP1/11) and a receptor-like cytoplasmic kinase (OsRLCK253) interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants. New Phytol 191(3):721–732PubMedCrossRefGoogle Scholar
  22. Giri J, Dansana PK, Kothari KS, Sharma G, Vij S, Tyagi AK (2013) SAPs as novel regulators of abiotic stress response in plants. BioEssays : News Rev Mol, Cell Dev Biol 35(7):639–648CrossRefGoogle Scholar
  23. Gururani MA, Mohanta TK, Bae H (2015) Current understanding of the interplay between phytohormones and photosynthesis under environmental stress. Int J Mol Sci 16(8):19055–19085PubMedPubMedCentralCrossRefGoogle Scholar
  24. Hishiya A, Iemura S, Natsume T, Takayama S, Ikeda K, Watanabe K (2006) A novel ubiquitin-binding protein ZNF216 functioning in muscle atrophy. EMBO J 25(3):554–564PubMedPubMedCentralCrossRefGoogle Scholar
  25. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180CrossRefGoogle Scholar
  26. Hozain M, Abdelmageed H, Lee J, Kang M, Fokar M, Allen RD, Holaday AS (2012) Expression of AtSAP5 in cotton up-regulates putative stress-responsive genes and improves the tolerance to rapidly developing water deficit and moderate heat stress. J Plant Physiol 169(13):1261–1270PubMedCrossRefGoogle Scholar
  27. Huang J, Teng L, Li L, Liu T, Li L, Chen D, Xu LG, Zhai Z, Shu HB (2004) ZNF216 is an A20-like and IkB kinase gamma-interacting inhibitor of NFkB activation. J Biol Chem 279(16):16847–16853PubMedCrossRefGoogle Scholar
  28. Huang XS, Luo T, Fu XZ, Fan QJ, Liu JH (2011) Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobacco. J Exp Bot 62:5191–5206PubMedPubMedCentralCrossRefGoogle Scholar
  29. Jia HX, Li JB, Zhang J, Ren YQ, Hu JJ, Lu MZ (2016) Genome-wide survey and expression analysis of the stress-associated protein gene family in desert poplar, Populus euphratica. Tree Genet Genomes 12(4):78CrossRefGoogle Scholar
  30. Jin Y, Wang M, Fu J, Xuan N, Zhu Y, Lian Y, Jia Z, Zheng J, Wang G (2007) Phylogenetic and expression analysis of ZnF-AN1 genes in plants. Genomics 90(2):265–275PubMedCrossRefGoogle Scholar
  31. Kang M, Fokar M, Abdelmageed H, Allen RD (2011) Arabidopsis SAP5 functions as a positive regulator of stress responses and exhibits E3 ubiquitin ligase activity. Plant Mol Biol 75(4–5):451–466PubMedCrossRefGoogle Scholar
  32. Kanneganti V, Gupta AK (2008) Overexpression of OsiSAP8, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice. Plant Mol Biol 66(5):445–462PubMedCrossRefGoogle Scholar
  33. Kothari KS, Dansana PK, Giri J, Tyagi AK (2016) Rice stress associated protein 1 (OsSAP1) interacts with aminotransferase (OsAMTR1) and pathogenesis-related 1a protein (OsSCP) and regulates abiotic stress responses. Front Plant Sci 7:1057PubMedPubMedCentralCrossRefGoogle Scholar
  34. Lee D, Takayama S, Goldberg AL (2018) ZFAND5/ZNF216 is an activator of the 26S proteasome that stimulates overall protein degradation. Proc Natl Acad Sci U S A 115(41):E9550–E9559PubMedPubMedCentralCrossRefGoogle Scholar
  35. Linnen JM, Bailey CP, Weeks DL (1993) Two related localized mRNAs from Xenopus laevis encode ubiquitin-like fusion proteins. Gene 128(2):181–188PubMedCrossRefGoogle Scholar
  36. Liu YJ, Xu YY, Xiao J, Ma QB, Li D, Xue Z, Chong K (2011) OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice. J Plant Physiol 168(10):1098–1105PubMedCrossRefGoogle Scholar
  37. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔC T method. methods 25(4):402–408PubMedCrossRefGoogle Scholar
  38. Loriaux M (2016) R.R. Sokal and F.J. Rohlf Biometry. The principles and practice of statistics in biological research. San Francisco, W.H. Freeman and Company, 1969, XXI p. 776 p., 126/-. - F.J. Rohlf and R.R. Sokal Statistical Tables. San Francisco, W.H. Freeman and Company, 1969, XI p. 253 p., . Louvain Economic Review 37 (4):461–462Google Scholar
  39. Ma A, Malynn BA (2012) A20: linking a complex regulator of ubiquitylation to immunity and human disease. Nat Rev Immunol 12(11):774–785PubMedPubMedCentralCrossRefGoogle Scholar
  40. Maehly A, Chance B (1954) Methods of biochemical analysis. by Glick D, Interscience, New York:454Google Scholar
  41. Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819(2):86–96PubMedCrossRefGoogle Scholar
  42. Mukhopadhyay A, Vij S, Tyagi AK (2004) Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco. Proc Natl Acad Sci U S A 101(16):6309–6314PubMedPubMedCentralCrossRefGoogle Scholar
  43. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321–4325PubMedPubMedCentralCrossRefGoogle Scholar
  44. Nakashima K, Takasaki H, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) NAC transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819(2):97–103PubMedCrossRefGoogle Scholar
  45. Opipari AW Jr, Boguski MS, Dixit VM (1990) The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein. J Biol Chem 265(25):14705–14708PubMedGoogle Scholar
  46. Rogers S, Horsch R, Fraley R (1986) Gene transfer in plants: production of transformed plants using Ti plasmid vectors. In: Methods in Enzymol, vol 118. Academic Press, pp 627–640Google Scholar
  47. Scebba F, Sebastiani L, Vitagliano C (1999) Protective enzymes against activated oxygen species in wheat (Triticum aestivum L.) seedlings: responses to cold acclimation. J Plant Physiol 155(6):762–768CrossRefGoogle Scholar
  48. Scharf KD, Berberich T, Ebersberger I, Nover L (2012) The plant heat stress transcription factor (Hsf) family: structure, function and evolution. Biochim Biophys Acta 1819(2):104–119PubMedCrossRefGoogle Scholar
  49. Soni R, Carmichael JP, Murray JA (1993) Parameters affecting lithium acetate-mediated transformation of Saccharomyces cerevisiae and development of a rapid and simplified procedure. Curr Genet 24(5):455–459PubMedCrossRefGoogle Scholar
  50. Sreedharan S, Shekhawat UK, Ganapathi TR (2012) MusaSAP1, a A20/AN1 zinc finger gene from banana functions as a positive regulator in different stress responses. Plant Mol Biol 80(4–5):503–517PubMedCrossRefGoogle Scholar
  51. Stroher E, Wang XJ, Roloff N, Klein P, Husemann A, Dietz KJ (2009) Redox-dependent regulation of the stress-induced zinc-finger protein SAP12 in Arabidopsis thaliana. Mol Plant 2(2):357–367PubMedCrossRefGoogle Scholar
  52. Vij S, Tyagi AK (2006) Genome-wide analysis of the stress associated protein (SAP) gene family containing A20/AN1 zinc-finger(s) in rice and their phylogenetic relationship with Arabidopsis. Mol Gen Genomics 276(6):565–575CrossRefGoogle Scholar
  53. Vij S, Tyagi AK (2008) A20/AN1 zinc-finger domain-containing proteins in plants and animals represent common elements in stress response. Funct Integr Genomics 8(3):301–307PubMedCrossRefGoogle Scholar
  54. Wang YH, Zhang LR, Zhang LL, Xing T, Peng JZ, Sun SL, Chen G, Wang XJ (2013) A novel stress-associated protein SbSAP14 from Sorghum bicolor confers tolerance to salt stress in transgenic rice. Mol Breed 32(2):437–449CrossRefGoogle Scholar
  55. Wani SH, Kumar V, Shriram V, Sah SK (2016) Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants. Crop J 4(3):162–176CrossRefGoogle Scholar
  56. Xu QF, Mao XG, Wang YX, Wang JY, Xi YJ, Jing RL (2018) A wheat gene TaSAP17-D encoding an AN1/AN1 zinc finger protein improves salt stress tolerance in transgenic Arabidopsis. J Integr Agric 17(3):507–516CrossRefGoogle Scholar
  57. Xuan N, Jin Y, Zhang HW, Xie YH, Liu YJ, Wang GY (2011) A putative maize zinc-finger protein gene, ZmAN13, participates in abiotic stress response. Plant Cell Tiss Org 107(1):101–112CrossRefGoogle Scholar
  58. Yoon S-K, Bae E-K, Lee H, Choi Y-I, Han M, Choi H, Kang K-S, Park E-J (2018) Down regulation of stress-associated protein 1 (PagSAP1) increases salt stress tolerance in poplar (Populus alba× P. glandulosa). Trees:1–11Google Scholar
  59. Zhang Y, Lan H, Shao Q, Wang R, Chen H, Tang H, Zhang H, Huang J (2016) An A20/AN1-type zinc finger protein modulates gibberellins and abscisic acid contents and increases sensitivity to abiotic stress in rice (Oryza sativa). J Exp Bot 67(1):315–326PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Biotechnology and Plant Improvement Laboratory, Centre of Biotechnology of SfaxUniversity of SfaxSfaxTunisia
  2. 2.Department of Life SciencesFaculty of Sciences of GafsaGafsaTunisia
  3. 3.Plant Production Department, College of Food and Agricultural SciencesKing Saud UniversityRiyadhSaudi Arabia

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