Plant Molecular Biology

, Volume 99, Issue 1–2, pp 149–159 | Cite as

WRKY12 represses GSH1 expression to negatively regulate cadmium tolerance in Arabidopsis

  • Yangyang Han
  • Tingting Fan
  • Xiangyu Zhu
  • Xi Wu
  • Jian Ouyang
  • Li Jiang
  • Shuqing CaoEmail author


Key message

The WRKY transcription factor WRKY12 negatively regulates Cd tolerance in Arabidopsis via the glutathione-dependent phytochelatin synthesis pathway by directly targeting GSH1 and indirectly repressing phytochelatin synthesis-related gene expression.


Cadmium (Cd) is a widespread pollutant toxic to plants. The glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway plays key roles in Cd detoxification. However, its regulatory mechanism remains largely unknown. Here, we showed a previously unknown function of the WRKY transcription factor WRKY12 in the regulation of Cd tolerance by repressing the expression of PC synthesis-related genes. The expression of WRKY12 was inhibited by Cd stress. Enhanced Cd tolerance was observed in the WRKY12 loss-of-function mutants, whereas increased Cd sensitivity was found in the WRKY12-overexpressing plants. Overexpression and loss-of-function of WRKY12 were associated respectively with increased and decreased Cd accumulation by repressing or releasing the expression of the genes involved in the PC synthesis pathway. Transient expression assay showed that WRKY12 repressed the expression of GSH1, GSH2, PCS1, and PCS2. Further analysis indicated that WRKY12 could directly bind to the W-box of the promoter in GSH1 but not in GSH2, PCS1, and PCS2 in vivo. Together, our results suggest that WRKY12 directly targets GSH1 and indirectly represses PC synthesis-related gene expression to negatively regulate Cd accumulation and tolerance in Arabidopsis.


Arabidopsis Cd tolerance Glutathione Phytochelatins WRKY12 



We thank Wenjia Ma, Yun Meng, Xue Fang, Yuanyuan Wang and Jiena Xu for their technical assistance. This work was supported by the National Natural Science Foundation of China (31770284 and 31571250), and the Fundamental Research Funds for the Central Universities (JZ2018HGTB0248).

Author Contributions

SC conceived the original research plans; YH, XZ, XW and JO performed the experiments; SC, YH, and TF and LJ designed the experiments and analysed the data; TF and SC wrote the article with contributions of all the authors.

Supplementary material

11103_2018_809_MOESM1_ESM.docx (1.7 mb)
Supplementary material 1 (DOCX 1739 KB)


  1. Alonso JM, Stepanova AN, Leisse TJ et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657CrossRefGoogle Scholar
  2. Brunetti P, Zanella L, Proia A, De Paolis A, Falasca G, Altamura MM, Sanità di Toppi L, Costantino P, Cardarelli M (2011) Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1. J Exp Bot 62:5509–5519CrossRefGoogle Scholar
  3. Cazalé AC, Clemens S (2001) Arabidopsis thaliana expresses a second functional phytochelatin synthase. FEBS Lett 507:215–219CrossRefGoogle Scholar
  4. Chen YF, Li LQ, Xu Q, Kong YH, Wang H, Wu WH (2009) The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis. Plant Cell 21:3554–3566CrossRefGoogle Scholar
  5. Chen L, Song Y, Li S, Zhang L, Zou C, Yu D (2012) The role of WRKY transcription factors in plant abiotic stresses. Biochem Biophys Acta 1819:120–128Google Scholar
  6. Chen J, Yang L, Gu J, Bai X, Ren Y, Fan T, Han Y, Jiang L, Xiao F, Liu Y, Cao S (2015) MAN3 gene regulates cadmium tolerance through the glutathione-dependent pathway in Arabidopsis thaliana. New Phytol 205:570–582CrossRefGoogle Scholar
  7. Chen J, Yang L, Yan X, Liu Y, Wang R, Fan T, Ren Y, Tang X, Xiao F, Liu Y, Cao S (2016) Zinc-finger transcription factor ZAT6 positively regulates cadmium tolerance through the glutathione-dependent pathway in Arabidopsis. Plant Physiol 171:707–719CrossRefGoogle Scholar
  8. Clemens S (2006) Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 88:1707–1719CrossRefGoogle Scholar
  9. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefGoogle Scholar
  10. Cobbett C, Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53:159–182CrossRefGoogle Scholar
  11. Cobbett CS, May MJ, Howden R, Rolls B (1998) The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in γ-glutamylcysteine synthetase. Plant J 16:73–78CrossRefGoogle Scholar
  12. Devaiah BN, Karthikeyan AS, Raghothama KG (2007) WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol 143:1789–1801CrossRefGoogle Scholar
  13. Ding ZJ, Yan JY, Xu XY, Li GX, Zheng SJ (2013) WRKY46 functions as a transcriptional repressor of ALMT1, regulating aluminum-induced malate secretion in Arabidopsis. Plant J 76:825–835CrossRefGoogle Scholar
  14. Dong J, Chen C, Chen Z (2003) Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol 51:21–37CrossRefGoogle Scholar
  15. Eulgem T, Somssich IE (2007) Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol 10:366–371CrossRefGoogle Scholar
  16. Farinati S, DalCorso G, Varotto S, Furini A (2010) The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants. New Phytol 185:964–978CrossRefGoogle Scholar
  17. Gasic K, Korban SS (2007) Transgenic Indian mustard (Brassica juncea) plants expressing an Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced As and Cd tolerance. Plant Mol Biol 64:361–369CrossRefGoogle Scholar
  18. Guo J, Dai X, Xu W, Ma M (2008) Overexpressing GSH1 and AsPCS1 simultaneously increases the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana. Chemosphere 72:1020–1026CrossRefGoogle Scholar
  19. Hong C, Cheng D, Zhang G, Zhu D, Chen Y, Tan M (2017) The role of ZmWRKY4 in regulating maize antioxidant defense under cadmium stress. Biochem Biophys Res Commun 482:1504–1510CrossRefGoogle Scholar
  20. Jiang Y, Liang G, Yang S, Yu D (2014) Arabidopsis WRKY57 functions as a node of convergence for jasmonic acid- and auxin-mediated signaling in jasmonic acid-induced leaf senescence. Plant Cell 26:230–245CrossRefGoogle Scholar
  21. Jozefczak M, Remans T, Vangronsveld J, Cuypers A (2012) Glutathione is a key player in metal-induced oxidative stress defenses. Int J Mol Sci 13:3145–3175CrossRefGoogle Scholar
  22. Kalde M, Barth M, Somssich IE, Lippok B (2003) Members of the Arabidopsis WRKY group III transcription factors are part of different plant defense signaling pathways. Mol Plant Microbe Interact 16:295–305CrossRefGoogle Scholar
  23. Kaufmann K, Muiño JM, Østerås M, Farinelli L, Krajewski P, Angenent GC (2010) Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nat Protoc 5:457–472CrossRefGoogle Scholar
  24. Kim DY, Bovet L, Kushnir S, Noh EW, Martinoia E, Lee Y (2006) AtATM3 is involved in heavy metal resistance in Arabidopsis. Plant Physiol 140:922–932CrossRefGoogle Scholar
  25. Kim HS, Park YH, Nam H, Lee YM, Song K, Choi C, Ahn I, Park SR, Lee YH, Hwang DJ (2013) Overexpression of the Brassica rapa transcription factor WRKY12 results in reduced soft rot symptoms caused by Pectobacterium carotovorum in Arabidopsis and Chinese cabbage. Plant Biol 16:973–981CrossRefGoogle Scholar
  26. Kühnlenz T, Schmidt H, Uraguchi S, Clemens S (2014) Arabidopsis thaliana phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the PCS1-deficient cad1-3 mutant on Cd-contaminated soil. J Exp Bot 65:4241–4253CrossRefGoogle Scholar
  27. Li W, Wang H, Yu D (2016) Arabidopsis WRKY transcription factors WRKY12 and WRKY13 oppositely regulate flowering under short-day conditions. Mol Plant 9:1492–1503CrossRefGoogle Scholar
  28. Lin YF, Aarts MG (2012) The molecular mechanism of zinc and cadmium stress response in plants. Cell Mol Life Sci 69:3187–3206CrossRefGoogle Scholar
  29. Liu B, Hong YB, Zhang YF, Li XH, Huang L, Zhang HJ, Li DY, Song FM (2014) Tomato WRKY transcriptional factor SlDRW1 is required for disease resistance against Botrytis cinerea and tolerance to oxidative stress. Plant Sci 227:145–156CrossRefGoogle Scholar
  30. Luo M, Dennis ES, Berger F, Peacock WJ, Chaudhury A (2005) MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis. Proc Natl Acad Sci USA 102:17531–17536CrossRefGoogle Scholar
  31. May MJ, Vernoux T, Leaver C, Van Montagu M, Inzé D (1998) Glutathione homeostasis in plants: Implications for environmental sensing and plant development. J Exp Bot 49:649–667Google Scholar
  32. Mills RF, Francini A, Ferreira da Rocha PSC, Baccarini PJ, Aylett M, Krijger GC, Williams LE (2005) The plant P1B-type ATPase AtHMA4 transports Zn and Cd plays a role in detoxification of transition metals supplied at elevated levels. FEBS Lett 579:783–791CrossRefGoogle Scholar
  33. Morel M, Crouzet J, Gravot A, Auroy P, Leonhardt N, Vavasseur A, Richaud P (2009) AtHMA3, a P1B-ATPase allowing Cd/Zn/Co/Pb vacuolar storage in Arabidopsis. Plant Physiol 149:894–904CrossRefGoogle Scholar
  34. Moreno I, Norambuena L, Maturana D, Toro M, Vergara C, Orellana A, Zurita-Silva A, Ordenes VR (2008) AtHMA1 is a thapsigargin-sensitive Ca2+ /heavy metal pump. J Biol Chem 283:9633–9641CrossRefGoogle Scholar
  35. Mucha S, Walther D, Müller TM, Hincha DK, Glawischnig E (2015) Substantial reprogramming of the Eutrema salsugineum (Thellungiella salsuginea) transcriptome in response to UV and silver nitrate challenge. BMC Plant Biol 15:137CrossRefGoogle Scholar
  36. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  37. Noctor G, Gomez L, Vanacker H, Foyer CH (2002) Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling. J Exp Bot 53:1283–1304CrossRefGoogle Scholar
  38. Noctor G, Mhamdi A, Chaouch S, Han Y, Neukermans J, Marquez-Garcia B, Queval G, Foyer CH (2012) Glutathione in plants: an integrated overview. Plant Cell Environ 35:454–484CrossRefGoogle Scholar
  39. Rauser WE (1995) Phytochelatins and related peptides. Structure, biosynthesis, and function. Plant Physiol 109:1141–1149CrossRefGoogle Scholar
  40. Seth CS, Remans T, Keunen E, Jozefczak M, Gielen H, Opdenakker K, Weyens N, Vangronsveld J, Cuypers A (2012) Phytoextraction of toxic metals: a central role for glutathione. Plant Cell Environ 35:334–346CrossRefGoogle Scholar
  41. Shanmugam V, Tsednee M, Yeh KC (2012) ZINC TOLERANCE INDUCED BY IRON 1 reveals the importance of glutathione in the cross-homeostasis between zinc and iron in Arabidopsis thaliana. Plant J 69:1006–1017CrossRefGoogle Scholar
  42. Sharma SS, Dietz KJ (2009) The relationship between metal toxicity and cellular redox imbalance. Trends Plant Sci 14:43–50CrossRefGoogle Scholar
  43. Sheng YB, Yan XX, Huang Y, Han YY, Zhang C, Ren YB, Fan TT, Xiao FM, Liu YS, Cao SQ (2018) The WRKY transcription factor, WRKY13, activates PDR8 expression to positively regulate cadmium tolerance in Arabidopsis. Plant Cell Environ. Google Scholar
  44. Shim D, Hwang JU, Lee J, Lee S, Choi Y, An G, Martinoia E, Lee Y (2009) Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice. Plant Cell 21:4031–4043CrossRefGoogle Scholar
  45. Smeets K, Ruytinx J, Semane B, Van Belleghem F, Remans T, Van Sanden S, Vangronsveld J, Cuypers A (2008) Cadmium-induced transcriptional and enzymatic alterations related to oxidative stress. Environ Exp Bot 63:1–8CrossRefGoogle Scholar
  46. Tang W, Charles TM, Newton RJ (2005) Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine (Pinus virginiana Mill.) confers multiple stress tolerance and enhances organ growth. Plant Mol Biol 59:603–617CrossRefGoogle Scholar
  47. Ulker B, Somssich IE (2004) WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol 7:491–498CrossRefGoogle Scholar
  48. Verbruggen N, Hermans C, Schat H (2009) Molecular mechanisms of metal hyperaccumulation in plants. New Phytol 181:759–776CrossRefGoogle Scholar
  49. Wang H, Avci U, Nakashima J, Hahn MG, Chen F, Dixon RA (2010) Mutation of WRKY transcription factors initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants. Proc Natl Acad Sci USA 107:22338–22343CrossRefGoogle Scholar
  50. Wei W, Zhang Y, Han L, Guan Z, Chai T (2008) A novel WRKY transcriptional factor from Thlaspi caerulescens negatively regulates the osmotic stress tolerance of transgenic tobacco. Plant Cell Rep 27:795–803CrossRefGoogle Scholar
  51. Wu Z, Zhao X, Sun X, Tan Q, Tang Y, Nie Z, Qu C, Chen Z, Hu C (2015) Antioxidant enzyme systems and the ascorbate–glutathione cycle as contributing factors to cadmium accumulation and tolerance in two oilseed rape cultivars (Brassica napus L.) under moderate cadmium stress. Chemosphere 138:526–536CrossRefGoogle Scholar
  52. Xu J, Li HD, Chen LQ, Wang Y, Liu LL, He L, Wu WH (2006) A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell 125:1347–1360CrossRefGoogle Scholar
  53. Yang G, Wang C, Wang Y, Guo Y, Zhao Y, Yang C, Gao C (2016) Overexpression of ThVHAc1 and its potential upstream regulator, ThWRKY7, improved plant tolerance of Cadmium stress. Sci Rep 6:18752CrossRefGoogle Scholar
  54. Zhu YL, Pilon-Smits EA, Jouanin L, Terry N (1999a) Overexpression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance. Plant Physiol 119:73–80CrossRefGoogle Scholar
  55. Zhu YL, Pilon-Smits EA, Tarun AS, Weber SU, Jouanin L, Terry N (1999b) Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing γ-glutamylcysteine synthetase. Plant Physiol 121:1169–1178CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Yangyang Han
    • 1
  • Tingting Fan
    • 1
  • Xiangyu Zhu
    • 1
  • Xi Wu
    • 1
  • Jian Ouyang
    • 1
  • Li Jiang
    • 1
  • Shuqing Cao
    • 1
    Email author
  1. 1.School of Food and Biological EngineeringHefei University of TechnologyHefeiChina

Personalised recommendations