Heat shock responses in Populus euphratica cell cultures: important role of crosstalk among hydrogen peroxide, calcium and potassium

Original Article


Populus euphratica is an important woody species in north-west China that can survive extremely high temperatures. In the present study, the defensive responses of P. euphratica cells to moderate heat shock (HS, 48 °C) were investigated using fluorescence imaging technique and a non-invasive vibrating ion-selective microelectrode. HS induced a hydrogen peroxide (H2O2)—and caspase-like protease activity-dependent programmed cell death (PCD, <25 %) in P. euphratica cells. After HS, the total activities of antioxidant enzymes, such as catalase, ascorbate peroxidase and glutathione reductase, as well as those of non-enzymatic antioxidants, were significantly enhanced, leading to a decline in H2O2 accumulation. However, the upregulated antioxidant system was dependent on the activation of plasma membrane (PM) Ca2+-ATPase in HS-treated P. euphratica cells. In addition, H2O2, which was inhibited by the DPI (an inhibitor of PM NADPH oxidase as well as mitochondrial flavin-containing enzymes), was required for the activation of PM Ca2+-ATPase under HS condition. Moreover, HS induced an early influx of Ca2+ and an early efflux of K+ in P. euphratica cells. However, the inhibition of this Ca2+ influx and K+ efflux by GdCl3 and Tetraethylammonium chloride significantly decreased the production of H2O2. These results suggest that P. euphratica cells adapt to heat stress via the improvement of the antioxidant system. The upregulation of the antioxidant system is regulated by PM Ca2+-ATPase, H2O2, and the early activation of Ca2+ and K+ channels in the PM. Finally, a model was postulated to reveal the HS responses in P. euphratica cells.


Heat shock responses Ca2+ K+ H2O2 PM Ca2+-ATPase Antioxidant system 



Heat shock


Heat shock response


Heat shock proteins


Heat stress transcription factors


Hydrogen peroxide


Programmed cell death




Ascorbate peroxidase


Glutathione reductase


Tetraethylammonium chloride


Cytosolic free Ca2+


Vacuolar free Ca2+


Dichlorodihydrofluorescein diacetate




Bis-(1,3-dibutylbarbituric acid)trimethine oxonol


Non-invasive micro-test technique


Reduced ascorbic acid


Dehydroascorbic acid


Reduced glutathione


Oxidized glutathione

Eosin Y

Eosin yellow


Membrane potential



This research was supported jointly by the National Science Foundation of China (Grant Nos. 31200470, 31270654), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Qing Lan Project, the Scientific Research Projects of Xuzhou City (No. XF13C056), the Research Project of Chinese Ministry of Education (Grant No. 113013A), the Key Project for Overseas Scholars by the Ministry of Human Resources and Social Security of PR China (Grant No. 2012001) and the Program of Introducing Talents of Discipline to Universities (111 Project, Grant No. B13007). The authors declare no conflict of interest.

Supplementary material

11240_2016_940_MOESM1_ESM.docx (32 kb)
Supplementary material 1 (DOCX 31 kb)


  1. Ahuja I, de Vos RC, Bones AM, Hall RD (2010) Plant molecular stress responses face climate change. Trends Plant Sci 15:664–674CrossRefPubMedGoogle Scholar
  2. Anderson ME (1985) Determination of glutathione and glutathione disulphide in biological samples. Methods Enzymol 113:548–555CrossRefPubMedGoogle Scholar
  3. Anil VS, Rajkumar P, Kumar P, Mathew M (2008) A plant Ca2+ pump, ACA2, relieves salt hypersensitivity in yeast. J Biol Chem 283:3497–3506CrossRefPubMedGoogle Scholar
  4. Ara N, Nakkanong K, Lv W, Yang J, Hu Z, Zhang M (2013) Antioxidant enzymatic activities and gene expression associated with heat tolerance in the stems and roots of two cucurbit species (“Cucurbita maxima” and “Cucurbita moschata”) and their interspecific inbred line “Maxchata”. Int J Mol Sci 14:24008–24028CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bhardwaj AR, Joshi G, Kukreja B et al (2015) Global insights into high temperature and drought stress regulated genes by RNA-Seq in economically important oilseed crop Brassica juncea. BMC Plant Biol 15:9CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bita CE, Zenoni S, Vriezen WH, Mariani C, Pezzotti M, Gerats T (2011) Temperature stress differentially modulates transcription in meiotic anthers of heat-tolerant and heat-sensitive tomato plants. BMC Genom 12:384CrossRefGoogle Scholar
  7. Bose J, Pottosin II, Shabala SS, Palmgren MG, Shabala S (2011) Calcium efflux systems in stress signaling and adaptation in plants. Front Plant Sci 2:85CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen J, Yin W, Xia X (2014a) Transcriptome profiles of Populus euphratica upon heat shock stress. Curr Genom 15:326–340CrossRefGoogle Scholar
  9. Chen S, Hawighorst P, Sun J, Polle A (2014b) Salt tolerance in Populus: significance of stress signaling networks, mycorrhization, and soil amendments for cellular and whole-plant nutrition. Environ Exp Bot 107:113–124CrossRefGoogle Scholar
  10. Chen S, Liu A, Zhang S, Li C, Chang R, Liu D, Ahammed GJ, Lin X (2013) Overexpression of mitochondrial uncoupling protein conferred resistance to heat stress and Botrytis cinerea infection in tomato. Plant Physiol Biochem 73:245–253CrossRefPubMedGoogle Scholar
  11. Dalla Costa L, Piazza S, Campa M, Flachowsky H, Hanke MV, Malnoy M (2016) Efficient heat-shock removal of the selectable marker gene in genetically modified grapevine. DOI, Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-015-0907-z Google Scholar
  12. de Pinto MC, Paradiso A, Leonetti P, De Gara L (2006) Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death. Plant J 48:784–795CrossRefPubMedGoogle Scholar
  13. Demidchik V, Shabala SN, Davies JM (2007) Spatial variation in H2O2 response of Arabidopsis thaliana root epidermal Ca2+ flux and plasma membrane Ca2+ channels. Plant J 49:377–386CrossRefPubMedGoogle Scholar
  14. Demidchik V, Cuin TA, Svistunenko D, Smith SJ, Miller AJ, Shabala S, Sokolik A, Yurin V (2010) Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death. J Cell Sci 123:1468–1479CrossRefPubMedGoogle Scholar
  15. Ferreira S, Hjernø K, Larsen M, Wingsle G, Larsen P, Fey S, Roepstorff P, Salomé Pais M (2006) Proteome profiling of Populus euphratica Oliv. upon heat stress. Ann Bot 98:361–377CrossRefPubMedPubMedCentralGoogle Scholar
  16. Finka A, Cuendet AF, Maathuis FJ, Saidi Y, Goloubinoff P (2012) Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance. Plant Cell 24:3333–3348CrossRefPubMedPubMedCentralGoogle Scholar
  17. Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866–1875CrossRefPubMedPubMedCentralGoogle Scholar
  18. Geisler M, Frangne N, Gomes E, Martinoia E, Palmgren MG (2000) The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast. Plant Physiol 124:1814–1827CrossRefPubMedPubMedCentralGoogle Scholar
  19. Gillespie KM, Ainsworth EA (2007) Measurement of reduced, oxidized and total ascorbate content in plants. Nat Protoc 2:871–874CrossRefPubMedGoogle Scholar
  20. Gong M, van der Luit AH, Knight MR, Trewaves AJ (1998) Heat-shock-induced changes in intracellular Ca2+ level in tobacco seedlings in relation to thermotolerance. Plant Physiol 116:429–437CrossRefPubMedCentralGoogle Scholar
  21. Gong B, Wang X, Wei M, Yang F, Li Y, Shi Q (2016) Overexpression of S-adenosylmethionine synthetase 1 enhances tomato callus tolerance to alkali stress through polyamine and hydrogen peroxide cross-linked networks. DOI, Plant Cell Tiss Organ Cult. doi: 10.1007/s11240-015-0901-5 Google Scholar
  22. Hasanuzzaman M, Nahar K, Alam MM, Roychowdhury R, Fujita M (2013) Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. Int J Mol Sci 14:9643–9684CrossRefPubMedPubMedCentralGoogle Scholar
  23. Hernandez M, Fernandez-Garcia N, Garcia-Garma J, Rubio-Asensio JS, Rubio F, Olmos E (2012) Potassium starvation induces oxidative stress in Solanum lycopersicum L. roots. J Plant Physiol 169:1366–1374CrossRefPubMedGoogle Scholar
  24. Hosy E, Vavasseur A, Mouline K, Dreyer I, Gaymard F, Poree F, BoucherezJ LebaudyA, Bouchez D, Very AA, Simonneau T, Thibaud JB, Sentenac H (2003) The Arabidopsis outward K+ channel GORK is involved in regulation of stomatal movements and plant transpiration. Proc Natl Acad Sci USA 100:5549–5554CrossRefPubMedPubMedCentralGoogle Scholar
  25. Huda KM, Yadav S, Akhter Banu MS, Trivedi DK, Tuteja N (2013a) Genome-wide analysis of plant-type II Ca2+ ATPases gene family from rice and Arabidopsis: potential role in abiotic stresses. Plant Physiol Biochem 65:32–47CrossRefGoogle Scholar
  26. Huda KM, Banu MS, Tuteja R, Tuteja N (2013b) Global calcium transducer P-type Ca2+-ATPases open new avenues for agriculture by regulating stress signalling. J Exp Bot 64:3099–3109CrossRefPubMedGoogle Scholar
  27. Huda KM, Banu MS, Garg B, Tula S, Tuteja R, Tuteja N (2013c) OsACA6, a P-type IIB Ca2+ ATPase promotes salinity and drought stress tolerance in tobacco by ROS scavenging and enhancing the expression of stress-responsive genes. Plant J 76:997–1015CrossRefPubMedGoogle Scholar
  28. Jia L, Chu H, Wu D, Feng M, Zhao L (2014) Role of calmodulin in thermotolerance. Plant Signal Behav 9:e28887CrossRefPubMedPubMedCentralGoogle Scholar
  29. Jiang M, Zhang J (2003) Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings. Plant Cell Environ 26:929–939CrossRefPubMedGoogle Scholar
  30. Larkindale J, Hall JD, Knight MR, Vierling E (2005) Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol 138:882–897CrossRefPubMedPubMedCentralGoogle Scholar
  31. Li S, Yu J, Zhu M, Zhao F, Luan S (2012) Cadmium impairs ion homeostasis by altering K+ and Ca2+ channel activities in rice root hair cells. Plant Cell Environ 35:1998–2013CrossRefPubMedGoogle Scholar
  32. Li YF, Wang Y, Tang Y, Kakani VG, Mahalingam R (2013) Transcriptome analysis of heat stress response in switchgrass (Panicum virgatum L.). BMC Plant Biol 13:153CrossRefPubMedPubMedCentralGoogle Scholar
  33. Liu HT, Sun DY, Zhou RG (2005) Ca2+ and AtCaM3 are involved in the expression of heat shock protein gene in Arabidopsis. Plant Cell Environ 28:1276–1284CrossRefGoogle Scholar
  34. Locato V, Gadaleta C, De Gara L, De Pinto MC (2008) Production of reactive species and modulation of antioxidant network in response to heat shock: a critical balance for cell fate. Plant Cell Environ 31:1606–1619CrossRefPubMedGoogle Scholar
  35. Lopreiato R, Giacomello M, Carafoli E (2014) The plasma membrane calcium pump: new ways to look at an old enzyme. J Biol Chem 289:10261–10268CrossRefPubMedPubMedCentralGoogle Scholar
  36. Ma T, Wang J, Zhou G et al (2013) Genomic insights into salt adaptation in a desert poplar. Nat Commun 4:2797PubMedGoogle Scholar
  37. Mittler R, Finka A, Goloubinoff P (2012) How do plants feel the heat? Trends Biochem Sci 37:118–125CrossRefPubMedGoogle Scholar
  38. Pei ZM, Murata Y, Benning G, Thomine S, Klüsener B, Allen GJ, Grill E, Schroeder JI (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406:731–734CrossRefPubMedGoogle Scholar
  39. Polle A, Chen S (2014) On the salty side of life: molecular, physiological and anatomical adaptation and acclimation of trees to extreme habitats. Plant Cell Environ 38:1794–1816CrossRefPubMedGoogle Scholar
  40. Pottosin I, Velarde-Buendía AM, Bose J, Zepeda-Jazo I, Shabala S, Dobrovinskaya O (2014) Cross-talk between reactive oxygen species and polyamines in regulation of ion transport across the plasma membrane: implications for plant adaptive responses. J Exp Bot 65:1271–1283CrossRefPubMedGoogle Scholar
  41. Pucciariello C, Banti V, Perata P (2012) ROS signaling as common element in low oxygen and heat stresses. Plant Physiol Biochem 59:3–10CrossRefPubMedGoogle Scholar
  42. Qudeimat E, Faltusz AMC, Wheeler G, Lang D, Brownlee C, Reski R, Frank W (2008) A PIIB-type Ca2+-ATPase is essential for stress adaptation in Physcomitrella patens. Proc Natl Acad Sci USA 105:19555–19560CrossRefPubMedPubMedCentralGoogle Scholar
  43. Richards SL, Laohavisit A, Mortimer JC, Shabala L, Swarbreck SM, Shabala S, Davies JM (2014) Annexin 1 regulates the H2O2-induced calcium signature in Arabidopsis thaliana roots. Plant J 77:136–145CrossRefPubMedGoogle Scholar
  44. Saidi Y, Finka A, Goloubinoff P (2011) Heat perception and signalling in plants: a tortuous path to thermotolerance. New Phytol 190:556–565CrossRefPubMedGoogle Scholar
  45. Saidi Y, Finka A, Muriset M, Bromberg Z, Weiss YG, Maathuis FJ, Goloubinoff P (2009) The heat shock response in moss plants is regulated by specific calcium-permeable channels in the plasma membrane. Plant Cell 21:2829–2843CrossRefPubMedPubMedCentralGoogle Scholar
  46. Schiøtt M, Palmgren MG (2005) Two plant Ca2+ pumps expressed in stomatal guard cells show opposite expression patterns during cold stress. Physiol Plant 124:278–283CrossRefGoogle Scholar
  47. Shabala S, Cuin TA (2008) Potassium transport and plant salt tolerance. Physiol Plant 133:651–669CrossRefPubMedGoogle Scholar
  48. Shabala S (2009) Salinity and programmed cell death: unravelling mechanisms for ion specific signalling. J Exp Bot 60:709–712CrossRefPubMedGoogle Scholar
  49. Shabala S, Pottosin I (2014) Regulation of potassium transport in plants under hostile conditions: implications for abiotic and biotic stress tolerance. Physiol Plant 151:257–279CrossRefPubMedGoogle Scholar
  50. Shabala S, Shabala L, Barcelo J, Poschenrieder C (2014) Membrane transporters mediating root signalling and adaptive responses to oxygen deprivation and soil flooding. Plant Cell Environ 37:2216–2233PubMedGoogle Scholar
  51. Shin R, Schachtman DP (2004) Hydrogen peroxide mediates plant root cell response to nutrient deprivation. Proc Natl Acad Sci USA 101:8827–8832CrossRefPubMedPubMedCentralGoogle Scholar
  52. Shukla D, Huda KM, Banu MS, Gill SS, Tuteja R, Tuteja N (2014) OsACA6, a P-type 2B Ca2+ ATPase functions in cadmium stress tolerance in tobacco by reducing the oxidative stress load. Planta 240:809–824CrossRefPubMedGoogle Scholar
  53. Song Y, Chen Q, Ci D, Shao X, Zhang D (2014) Effects of high temperature on photosynthesis and related gene expression in poplar. BMC Plant Biol 14:111CrossRefPubMedPubMedCentralGoogle Scholar
  54. Sun J, Dai S, Wang R et al (2009a) Calcium mediates root K+/Na+ homeostasis in poplar species differing in salt tolerance. Tree Physiol 29:1175–1186CrossRefPubMedGoogle Scholar
  55. Sun J, Chen SL, Dai SX et al (2009b) NaCl-induced alternations of cellular and tissue ion fluxes in roots of salt-resistant and salt-sensitive poplar species. Plant Physiol 149:1141–1153CrossRefPubMedPubMedCentralGoogle Scholar
  56. Sun J, Wang M, Ding M et al (2010a) H2O2 and cytosolic Ca2+ signals triggered by the PM H+-coupled transport system mediate K+/Na+ homeostasis in NaCl-stressed Populus euphratica cells. Plant Cell Environ 33:943–958CrossRefPubMedGoogle Scholar
  57. Sun J, Li LS, Liu MQ et al (2010b) Hydrogen peroxide and nitric oxide mediate K+/Na+ homeostasis and antioxidant defense in NaCl-stressed callus cells of two contrasting poplars. Plant Cell Tissue Organ Cult 103:205–215CrossRefGoogle Scholar
  58. Sun J, Zhang X, Deng S et al (2012a) Extracellular ATP signaling is mediated by H2O2 and cytosolic Ca2+ in the salt response of Populus euphratica cells. PLoS One 7:e53136CrossRefPubMedPubMedCentralGoogle Scholar
  59. Sun J, Zhang C, Deng S, Lu C, Shen X, Zheng X, Chen S (2012b) An ATP signaling pathway in plant cells: extracellular ATP triggers programmed cell death in Populus euphratica. Plant Cell Environ 35:893–916CrossRefPubMedGoogle Scholar
  60. Sun J, Wang R, Zhang X, Yu Y, Zhao R, Li Z, Chen S (2013) Hydrogen sulfide alleviates cadmium toxicity through regulations of cadmium transport across the plasma and vacuolar membranes in Populus euphratica cells. Plant Physiol Biochem 65:67–74CrossRefPubMedGoogle Scholar
  61. Tunc-Ozdemir M, Tang C, Ishka MR, Brown E, Groves NR, Myers CT, Rato C, Poulsen LR, McDowell S, Miller G, Mittler R, Harper JF (2013) A cyclic nucleotide-gated channel (CNGC16) in pollen is critical for stress tolerance in pollen reproductive development. Plant Physiol 161:1010–1020CrossRefPubMedPubMedCentralGoogle Scholar
  62. Vacca RA, Valenti D, Bobba A, Merafina RS, Passarella S, Marra E (2006) Cytochrome c is released in a reactive oxygen species-dependent manner and is degraded via caspase-like proteases in tobacco Bright-Yellow 2 cells enroute to heat shock-induced cell death. Plant Physiol 141:208–219CrossRefPubMedPubMedCentralGoogle Scholar
  63. Volkov RA, Panchuk II, Mullineaux PM, Schöffl F (2006) Heat stress-induced H2O2 is required for effective expression of heat shock genes in Arabidopsis. Plant Mol Biol 61:733–746CrossRefPubMedGoogle Scholar
  64. Wang RG, Chen SL, Zhou XY et al (2008) Ionic homeostasis and reactive oxygen species control in leaves and xylem sap of two poplars subjected to NaCl stress. Tree Physiol 28:947–957CrossRefPubMedGoogle Scholar
  65. Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9:244–252CrossRefPubMedGoogle Scholar
  66. Wang X, Cai J, Liu F, Dai T, Cao W, Wollenweber B, Jiang D (2014) Multiple heat priming enhances thermo-tolerance to a later high temperature stress via improving subcellular antioxidant activities in wheat seedlings. Plant Physiol Biochem 74:185–192CrossRefPubMedGoogle Scholar
  67. Wilson RA, Sangha MK, Banga SS, Atwal AK, Gupta S (2014) Heat stress tolerance in relation to oxidative stress and antioxidants in Brassica juncea. J Environ Biol 35:383–387PubMedGoogle Scholar
  68. Zhang W, Jeon BW, Assmann SM (2011) Heterotrimeric G-protein regulation of ROS signaling and calcium currents in Arabidopsis guard cells. J Exp Bot 62:2371–2379CrossRefPubMedGoogle Scholar
  69. Zhang X, Shen Z, Sun J, Yu Y, Deng S, Li Z, Sun C, Zhang J, Zhao R, Shen X, Chen S (2015) NaCl-elicited, vacuolar Ca2+ release facilitates prolonged cytosolic Ca2+ signaling in the salt response of Populus euphratica cells. Cell Calcium 57:348–365CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Institute of Integrative Plant Biology, School of Life ScienceJiangsu Normal UniversityXuzhouPeople’s Republic of China
  2. 2.College of Biological Sciences and TechnologyBeijing Forestry UniversityBeijingPeople’s Republic of China

Personalised recommendations