Skip to main content
SpringerLink
Log in

Cell death patterns in Arabidopsis cells subjected to four physiological stressors indicate multiple signalling pathways and cell cycle phase specificity

  • Original Article
  • Published:
Protoplasma Aims and scope Submit manuscript

Abstract

Corpse morphology, nuclear DNA fragmentation, expression of senescence-associated genes (SAG) and cysteine protease profiles were investigated to understand cell death patterns in a cell cycle-synchronised Arabidopsis thaliana cell suspension culture treated with four physiological stressors in the late G2 phase. Within 4 h of treatment, polyethylene glycol (PEG, 20 %), mannose (100 mM) and hydrogen peroxide (2 mM) caused DNA fragmentation coinciding with cell permeability to Evans Blue (EB) and produced corpse morphology corresponding to apoptosis-like programmed cell death (AL-PCD) with cytoplasmic retraction from the cell wall. Ethylene (8 mL per 250-mL flask) caused permeability of cells to EB without concomitant nuclear DNA fragmentation and cytoplasmic retraction, suggesting necrotic cell death. Mannose inducing glycolysis block and PEG causing dehydration resulted in relatively similar patterns of upregulation of SAG suggesting similar cell death signalling pathways for these two stress factors, whereas hydrogen peroxide caused unique patterns indicating an alternate pathway for cell death induced by oxidative stress. Ethylene did not cause appreciable changes in SAG expression, confirming necrotic cell death. Expression of AtDAD, BoMT1 and AtSAG2 genes, previously shown to be associated with plant senescence, also changed rapidly during AL-PCD in cultured cells. The profiles of nine distinct cysteine protease-active bands ranging in size from ca. 21.5 to 38.5 kDa found in the control cultures were also altered after treatment with the four stressors, with mannose and PEG again producing similar patterns. Results also suggest that cysteine proteases may have a role in necrotic cell death.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Altschul S, Gish W, Miller W, Myers E, Lipman D (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Avci U, Petzold HE, Ismail IO, Beers EP, Haigler CH (2008) Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots. Plant J 56:303–315

    Article  CAS  PubMed  Google Scholar 

  • Baker J, Mock NM (1994) An improved method for monitoring cell death in cell suspension and leaf disc assays using Evans blue. Plant Cell Tissue Organ Cult 39:7–12

    Article  Google Scholar 

  • Bhalerao R, Keskitalo J, Sterky F, Erlandsson R, Bjorkbacka H, Birve SJ, Karlsson J, Gardestrom P, Gustafsson P, Lundeberg J, Jansson S (2003) Gene expression in autumn leaves. Plant Physiol 131:430–442

    Article  PubMed  PubMed Central  Google Scholar 

  • Buchannan-Wollaston V (1994) Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Identification of a gene encoding a senescence-specific metallothionein-like protein. Plant Physiol 105:839–846

    Article  Google Scholar 

  • Burbridge E, Diamond M, Dix PJ, McCabe PF (2007) Use of cell morphology to evaluate the effect of a peroxidase gene on the cell death induction thresholds in tobacco. Plant Sci 172:853–860

    Article  CAS  Google Scholar 

  • Carimi F, Zottini M, Formentin E, Terzi M, Schiavo FL (2003) Cytokinins: new apoptotic inducers in plants. Planta 216:413–421

    CAS  PubMed  Google Scholar 

  • Chakravarthy S, Park Y-J, Chodaparambil J, Edayathumangalam RS, Luger K (2005) Structure and dynamic properties of nucleosome core particles. FEBS Lett 579:895–898

    Article  CAS  PubMed  Google Scholar 

  • Coupe SA, Watson LM, Ryan DJ, Pinkney TT, Eason JR (2004) Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmityltransferase homologues. J Exp Bot 55:59–68

    Article  CAS  PubMed  Google Scholar 

  • Doyle SM, McCabe PF (2010) Type and cellular location of reactive oxygen species determine activation or suppression of programmed cell death in Arabidopsis suspension cultures. Plant Signal Behav 5:467–468

    Article  PubMed  Google Scholar 

  • Eason JR, Ryan DJ, Watson LM, Hedderley D, Christey MC, Braun RH, Coupe SA (2005) Suppression of the cysteine protease, aleurain, delays floret senescence in Brassica oleracea. Plant Mol Biol 57:645–657

    Article  CAS  PubMed  Google Scholar 

  • Eason JR, Patel D, Ryan D, Page B, Hedderley D, Watson LM, West DW (2007) Controlled atmosphere treatment of broccoli after harvest delays senescence and induces the expression of novel BoCAR genes. Plant Physiol Biochem 45:445–456

    Article  CAS  PubMed  Google Scholar 

  • Errakhi R, Meimoun P, Lehner A, Vidal G, Briand J, Corbineau F, Rona J, Bouteau F (2008) Anion channel activity is necessary to induce ethylene synthesis and programmed cell death in response to oxalic acid. J Exp Bot 59:3121–3129

    Article  CAS  PubMed  Google Scholar 

  • Gallois P, Makishima T, Hecht V, Despres B, Laudié M, Nishimoto T, Cooke R (1997) An Arabidopsis thaliana cDNA complementing a hamster apoptosis suppressor mutant. Plant J 11:1325–1331

    Article  CAS  PubMed  Google Scholar 

  • Gao MG, Showalter AM (1999) Yariv reagent treatment induces programmed cell death in Arabidopsis cell cultures and implicates arabinogalactan protein involvement. Plant J 19:321–331

    Article  CAS  PubMed  Google Scholar 

  • Gapper N, Coupe S, McKenzie M, Sinclair B, Lill R, Jameson P (2005a) Regulation of harvest-induced senescence in broccoli (Brassica oleracea var. italica) by cytokinin, ethylene, and sucrose. J Plant Growth Regul 24:153–165

    Article  CAS  Google Scholar 

  • Gapper NE, Coupe SA, McKenzie MJ, Scott RW, Christey MC, Lill RE, McManus MT, Jameson PE (2005b) Senescence-associated down-regulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase delays harvest-induced senescence in broccoli. Funct Plant Biol 32:891–901

    Article  CAS  Google Scholar 

  • Garcia-Heredia JM, Hervas M, De la Rosa MA, Navarro JA (2008) Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures. Planta 228(1):89–97

    Article  CAS  PubMed  Google Scholar 

  • Gemperlova L, Cvikrova M, Fischerova L, Binarova P, Fischer L, Eder J (2009) Polyamine metabolism during the cell cycle of synchronized tobacco BY-2 cell line. Plant Physiol Biochem 47:584–591

    Article  CAS  PubMed  Google Scholar 

  • Gepstein S, Sabihi G, Carp M-J, Hajouj T, Falah M, Nesher O, Yariv I, Dor C, Bassani M (2003) Large scale identification of leaf senescence-associated genes. Plant J 36:629–642

    Article  CAS  PubMed  Google Scholar 

  • Goldsworthy A, Street HE (1965) The carbohydrate nutrition of tomato roots VIII: the mechanism of the inhibition by D-mannose of the respiration of excised roots. Ann Bot 29:45–58

    Article  CAS  Google Scholar 

  • Hensel LL, Grbic V, Baumgarten DA, Bleeker AB (1993) Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell 5:553–564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Herbert RJ, Vilhar B, Evett C, Orchard CB, Rogers HJ, Davies MS, Francis D (2001) Ethylene induces cell death at particular phases of the cell cycle in the tobacco TBY-2 cell line. J Exp Bot 52:1615–1623

    Article  CAS  PubMed  Google Scholar 

  • Higaki T, Kurusu T, Hasezawa S, Kuchitsu K (2011) Dynamic intracellular reorganization of cytoskeletons and the vacuole in defense responses and hypersensitive cell death in plants. J Plant Res 124(3):315–324

    Article  PubMed  Google Scholar 

  • Jones ML, Chaffin GS, Eason JR, Clark DG (2005) Ethylene-sensitivity regulates proteolytic activity and cysteine protease gene expression in petunia corollas. J Exp Bot 56:2733–2744

    Article  CAS  PubMed  Google Scholar 

  • Kadota Y, Watanabe T, Fujii S, Higashi K, Sano T, Nagata T, Hasezawa S, Kuschitsu K (2004) Crosstalk between elicitor-induced cell death and cell cycle regulation in tobacco BY-2 cells. Plant J 40:131–142

    Article  CAS  PubMed  Google Scholar 

  • Kadota Y, Furuichi T, Sano T, Kaya H, Gunji W, Murakami Y, Muto S, Hasezawa S, Kuchitsu K (2005a) Cell-cycle-dependent regulation of oxidative stress responses and Ca2+ permeable channels NtTPC1A/B in tobacco BY-2 cells. Biochem Biophys Res Commun 336(4):1259–1267

    Article  CAS  PubMed  Google Scholar 

  • Kadota Y, Watanabe T, Fujii S, Maeda Y, Ryoko O, Higashi K, Sano T, Muto S, Hasezawa S, Kuchitsu K (2005b) Cell cycle dependence of elicitor-induced signal transduction in tobacco BY-2 cells. Plant Cell Physiol 46:156–165

    Article  CAS  PubMed  Google Scholar 

  • Kuthanova A, Fischer L, Nick P, Opatrny Z (2008) Cell cycle phase-specific death response of tobacco BY-2 cell line to cadmium treatment. Plant Cell Environ 31:1634–1642

    Article  CAS  PubMed  Google Scholar 

  • Lam E, Zhang Y (2012) Regulating the reapers: activating metacaspases for programmed cell death. Trends Plant Sci 17(8):487–494

    Article  CAS  PubMed  Google Scholar 

  • Lennon SV, Martin SJ, Cotter TG (1991) Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Prolif 24:203–214

    Article  CAS  PubMed  Google Scholar 

  • Luger K, Richmond TJ (1998) DNA binding within the nucleosome core. Curr Opin Struct Biol 8:33–40

    Article  CAS  PubMed  Google Scholar 

  • McCabe PF, Leaver CJ (2000) Programmed cell death in cell cultures. Plant Mol Biol 44:359–368

    Article  CAS  PubMed  Google Scholar 

  • McCabe PF, Levine A, Meijer PJ, Tapon NA, Pennell RI (1997) A programmed cell death pathway activated in carrot cells cultured at low cell density. Plant J 12:267–280

    Article  CAS  Google Scholar 

  • Mergemann H, Sauter M (2000) Ethylene induces epidermal cell death at the site of adventitious root emergence in rice. Plant Physiol 124:609–614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Meshi T, Taoka K-I, Iwabuchi M (2000) Regulation of histone gene expression during the cell cycle. Plant Mol Biol 43:643–657

    Article  CAS  PubMed  Google Scholar 

  • Nakashima T, Sekiguchi T, Kuraoka A, Sihibata Y, Komiyana S, Nishimoto T (1993) Molecular cloning of a human cDNA encoding a novel protein, DAD1, whose defect causes apoptotic cell death in hamster BHK21 cells. Mol Cell Biol 13:6367–6374

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pathirana R, Eason JR (2006) Establishment and characterization of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana L. suitable for cell cycle synchronisation. Plant Cell Tissue Organ Cult 85:125–136

    Article  Google Scholar 

  • Petrov V, Hille J, Mueller-Roeber B, Gechev TS (2015) ROS-mediated abiotic stress-induced programmed cell death in plants. Front Plant Sci 6:69

    Article  PubMed  PubMed Central  Google Scholar 

  • Petrovska B, Cenklova V, Pochylova Z, Kourova H, Doskocilova A, Plihal O, Binarova L, Binarova P (2012) Plant Aurora kinases play a role in maintenance of primary meristems and control of endoreduplication. New Phytol 193:590–604

    Article  CAS  PubMed  Google Scholar 

  • Pogson BJ, Downs CG, Davies KM (1995) Differential expression of two 1-aminocyclopropane-1-carboxylic acid oxidase genes in broccoli after harvest. Plant Physiol 108:651–657

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reape TJ, McCabe PF (2008) Apoptotic-like programmed cell death in plants. New Phytol 180:13–26

    Article  CAS  PubMed  Google Scholar 

  • Reape TJ, McCabe PF (2010) Apoptotic-like regulation of programmed cell death in plants. Apoptosis 15(3):249–256

    Article  CAS  PubMed  Google Scholar 

  • Reape TJ, Molony EM, McCabe PF (2008) Programmed cell death in plants: distinguishing between different modes. J Exp Bot 59:435–444

    Article  CAS  PubMed  Google Scholar 

  • Schiller M, Hegelund JN, Pedas P, Husted S, Schjoerring JK (2009) Plant metallothioneins and functional analysis of a barley metallothionein promoter. Proc Intl Plant Nutr Colloq XVI. Retrieved from: http://escholarship.cor/uc/item/6pf0s32h

  • Swidzinski JA, Sweetlove LJ, Leaver CJ (2002) A custom microarray analysis of gene expression during programmed cell death in Arabidopsis thaliana. Plant J 30:431–446

    Article  CAS  PubMed  Google Scholar 

  • Takahashi S, Seki M, Ishida J, Satou M, Sakurai T, Narusaka M, Kamiya A, Nakajima M, Enju A, Akiyama K, Yamaguchi-Shinozaki K, Shinozaki K (2004) Monitoring the expression profiles of genes induced by hyperosmotic, high salinity, and oxidative stress and abscisic acid treatment in Arabidopsis cell culture using a full-length cDNA microarray. Plant Mol Biol 56:29–55

    Article  CAS  PubMed  Google Scholar 

  • Tanaka Y, Makishima T, Sasabe M, Ichinose Y, Shiraishi T, Nishimoto T, Yamada T (1997) dad-1, a putative programmed cell death suppressor gene in rice. Plant Cell Physiol 38:379–383

    Article  CAS  PubMed  Google Scholar 

  • Tomastikova E, Demidov D, Jerabkova H, Binarova P, Houben A, Dolezel J, Petrovska B (2016) TPX2 protein of Arabidopsis activates Aurora Kinase 1, but not Aurora Kinase 3 in vitro. Plant Mol Biol Report 33:1988–1995

    Article  Google Scholar 

  • Trobacher CP, Senatore A, Holley C, Greenwood JS (2013) Induction of a ricinosomal-protease and programmed cell death in tomato endosperm by gibberellic acid. Planta 237(3):665–679

    Article  CAS  PubMed  Google Scholar 

  • van der Hoorn RAL, Leeuwenburgh A, Bogyo M, Joosten MHAJ, Peck SC (2004) Activity profiling of papain-like cysteine proteases in plants. Plant Physiol 135:1170–1178

    Article  PubMed  PubMed Central  Google Scholar 

  • Van Hautegem T, Waters AJ, Goodrich J, Nowack MK (2015) Only in dying, life: programmed cell death during plant development. Trends Plant Sci 20(2):102–113. doi:10.1016/j.tplants.2014.10.003

    Article  PubMed  Google Scholar 

  • Watanabe N, Lam E (2009) Bax inhibitor-1, a conserved cell death suppressor, is key metabolic switch downstream from a variety of biotic and abiotic signals in plants. Int J Mol Sci 10:3149–3167

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weaver LM, Gan S, Quirino B, Amasino RM (1998) A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment. Plant Mol Biol 37:455–469

    Article  CAS  PubMed  Google Scholar 

  • Williams B, Dickman M (2008) Plant programmed cell death: can’t live with it; can’t live without it. Mol Plant Pathol 9:531–544

    Article  CAS  PubMed  Google Scholar 

  • Woltering EJ (2010) Death proteases: alive and kicking. Trends Plant Sci 15:185–188

    Article  CAS  PubMed  Google Scholar 

  • Woltering EJ, Yakimova ET, Michaeli R, Kapchina-Toteva VM (2007) Chemical-induced programmed cell death in tomato suspension cells is mediated through ethylene and lipid signalling. In: Ramina A, Chang C, Giovannoni J, Klee H, Perata P, Woltering E (eds) Advances in Plant Ethylene Research: Proc 7th Int Symp on the Plant Hormone Ethylene, pp 341–351

  • Yakimova ET, Kapchina-Toteva VM, Laarhoven L-J, Harren FM, Waltering EJ (2006) Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells. Plant Physiol Biochem 44:581–589

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Dr. Renier van der Hoorn for supplying the biotin-labelled protease inhibitor (DCG-04), Dr. Murray Grant (Imperial College, London Wye campus, Kent, UK) for supplying the AtBI-1 cDNA, Dr. Jeff Dangl (University of North Carolina, Chapel Hill, NC, USA) for supplying the LSD1 cDNA, Dr. Paul Birch (SCRI, Invergowrie, UK) for supplying AtSTLB1 and the ABRC (Columbus, OH, USA) for supplying the AtSPT1 EST. We wish to thank Dr. Ross Bicknell and Sylvia Erasmuson for the use of their equipment and technical expertise in flow cytometry. We thank Andrew Mullen and Sriya Pathirana for culture media preparation. Valuable comments by Drs. Dave Brummell and Donald Hunter on the manuscript are also gratefully appreciated. This work was supported by the New Zealand Foundation for Research, Science and Technology (now the Ministry of Business, Innovation and Employment) under C02X0701 ‘Future Vegetables’ programme.

Author contribution

JE, RP and DH designed the experiments; RP, PW and JE conducted the research; DH conducted the statistical analysis; RP, JE and DH wrote the manuscript.

Author information

Author notes

  1. Phillip West

    Present address: NZ Avocado, Level 5 Harrington House, 32 Harington Street, Tauranga, 3110, New Zealand

Authors and Affiliations

  1. The New Zealand Institute for Plant & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand

    Ranjith Pathirana, Phillip West, Duncan Hedderley & Jocelyn Eason

Authors
  1. Ranjith Pathirana
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Phillip West
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Duncan Hedderley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jocelyn Eason
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ranjith Pathirana.

Additional information

Handling Editor: Bhumi Nath Tripathi

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 174 kb)

ESM 2

(PDF 174 kb)

ESM 3

(PDF 175 kb)

ESM 4

(PDF 193 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pathirana, R., West, P., Hedderley, D. et al. Cell death patterns in Arabidopsis cells subjected to four physiological stressors indicate multiple signalling pathways and cell cycle phase specificity. Protoplasma 254, 635–647 (2017). https://doi.org/10.1007/s00709-016-0977-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00709-016-0977-8

Keywords

Search

Navigation