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ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox

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Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability.

Abstract

We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O ·−2 , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA–GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

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References

  • Bajaj YPS (1995) Cryopreservation of plant cell, tissue and organ culture for the conservation of germplasm and biodiversity. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry. Cryopreservation of plant germplasm I. Springer, USA, pp 3–18

    Chapter  Google Scholar 

  • Balk J, Leaver CJ, McCabe PF (1999) Translocation of cytochrome c from the mitochondria to the cytosol occurs during heat-induced programmed cell death in cucumber plants. FEBS Lett 463:151–154

    Article  CAS  PubMed  Google Scholar 

  • Baust JM (2002) Molecular mechanisms of cellular demise associated with cryopreservation failure. Cell Preserv Technol 1:17–31

    Article  CAS  Google Scholar 

  • Benelli C, De Carlo A, Engelmann F (2013) Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis. Biotechnol Adv 31:175–185

    Article  CAS  PubMed  Google Scholar 

  • Benson EE (1990) Free radical damage in stored plant germplasm. International Board for Plant Genetic Resources, Italy

    Google Scholar 

  • Benson EE (2008) Cryopreservation of phytodiversity: a critical appraisal of theory and practice. Crit Rev Plant Sci 27:141–219

    Article  CAS  Google Scholar 

  • Benson EE, Bremner D (2004) Oxidative stress in the frozen plant: a free radical point of view. In: Fuller BJ, Lane N, Benson EE (eds) Life in the frozen state. CRC Press, USA, pp 205–241

    Chapter  Google Scholar 

  • Bissoyi A, Nayak B, Pramanik K, Sarangi SK (2014) Targeting cryopreservation-induced cell death: a review. Biopreserv Biobank 12:23–34

    Article  CAS  PubMed  Google Scholar 

  • Buttke TM, Sandstrom PA (1994) Oxidative stress as a mediator of apoptosis. Immunol Today 15:7–10

    Article  CAS  PubMed  Google Scholar 

  • Chakraborti T, Das S, Mondal M, Roychoudhury S, Chakraborti S (1999) Oxidant, mitochondria and calcium: an overview. Cell Signal 11:77–85

    Article  CAS  PubMed  Google Scholar 

  • del Pozo O, Lam E (1998) Caspases and programmed cell death in the hypersensitive response of plants to pathogens. Curr Biol 8:1129–1132

    Article  PubMed  Google Scholar 

  • del Rio LA, Sandalio LM, Corpas FJ, Palma JM, Barroso JB (2006) Reactive oxygen species and reactive nitrogen species in peroxisomes. Production, scavenging, and role in cell signaling. Plant Physiol 141:330–335

    Article  PubMed Central  PubMed  Google Scholar 

  • Dowling DK, Simmons LW (2009) Reactive oxygen species as universal constraints in life-history evolution. Proc R Soc B 276:1737–1745

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • El-Maarouf-Bouteau H, Mazuy C, Corbineau F, Bailly C (2011) DNA alteration and programmed cell death during ageing of sunflower seed. J Exp Bot 62:5003–5011

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Engelmann F (2000) Importance of cryopreservation for the conservation of plant genetic resources. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm: current research progress and application. JIRCAS, Tsukuba/IPGRI, Rome, pp 8–20

    Google Scholar 

  • Engelmann F (2004) Cryopreservation: progress and prospects. In Vitro Cell Dev Plant 40:427–433

    Article  Google Scholar 

  • Erdei L, Tari I, Csiszár J, Pécsváradi A, Horváth F, Szabó M, Ördög M, Cseuz L, Zhiponova M, Szilák L, Györgyey J (2002) Osmotic stress responses of wheat species and cultivars differing in drought tolerance: some interesting genes (advices for gene hunting). Acta Biol Szeged 46:63–65

    Google Scholar 

  • Fang JY, Wetten A, Johnston J (2008) Headspace volatile markers for sensitivity of cocoa (Theobroma cacao L) somatic embryos to cryopreservation. Plant Cell Rep 27:453–461

    Article  CAS  PubMed  Google Scholar 

  • Fleck RA, Benson EE, Bremner DH, Day JG (2003) A comparative study of antioxidant protection in cryopreserved unicellular algae Euglena gracilis and Haematococcus pluvialis. CryoLett 24:213–228

    CAS  Google Scholar 

  • Fowler WE, Aebi U (1983) Preparation of single molecules and supramolecular complexes for high-resolution metal shadowing. J Ultrastruct Res 83:319–334

    Article  CAS  PubMed  Google Scholar 

  • Foyer CH, Halliwell B (1976) The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133:21–25

    Article  CAS  PubMed  Google Scholar 

  • Gallois P, Makishima T, Hecht V, Despres B, LaudiéM 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 C, Xing D, Li L, Zhang L (2008) Implication of reactive oxygen species and mitochondrial dysfunction in the early stages of plant programmed cell death induced by ultraviolet-C overexposure. Planta 227:755–767

    Article  CAS  PubMed  Google Scholar 

  • Gechev TS, van Breusegem F, Stone JM, Denev I, Laloi C (2006) Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. BioEssays 28:1091–1101

    Article  CAS  PubMed  Google Scholar 

  • Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930

    Article  CAS  PubMed  Google Scholar 

  • Hansen G (2000) Evidence for Agrobacterium-induced apoptosis in maize cells. Mol Plant Microbe Interact 13:649–657

    Article  CAS  PubMed  Google Scholar 

  • Harding K, Johnston JW, Benson EE (2009) Exploring the physiological basis of cryopreservation success and failure in clonally propagated in vitro crop plant germplasm. Agric Food Sci 18:103–116

    Article  CAS  Google Scholar 

  • Hoeberichts FA, Woltering EJ (2002) Multiple mediators of plant programmed cell death: interplay of conserved cell death mechanisms and plant-specific regulators. BioEssays 25:47–57

    Article  CAS  Google Scholar 

  • Hu D, Ma G, Wang Q, Yao J, Wang Y, Pritchard HW, Wang X (2012) Spatial and temporal nature of reactive oxygen species production and programmed cell death in elm (Ulmus pumila L.) seeds during controlled deterioration. Plant Cell Environ 35:2045–2059

    Article  CAS  PubMed  Google Scholar 

  • Johnston JW, Benson EE, Harding K (2009) Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm. Plant Physiol Biochem 47:123–131

    Article  CAS  PubMed  Google Scholar 

  • Jones A (2000) Does the plant mitochondrion integrate cellular stress and regulate programmed cell death? Trends Plant Sci 5:225–230

    Article  CAS  PubMed  Google Scholar 

  • Korthout HA, Berecki G, Bruin W, Van Duijn B, Wang M (2000) The presence and subcellular localization of caspase 3-like proteinases in plant cells. FEBS Lett 475:139–144

    Article  CAS  PubMed  Google Scholar 

  • Kranner I, Birtic S, Anderson KM, Pritchard HW (2006) Glutathione half-cell reduction potential: a universal stress marker and modulator of programmed cell death? Free Radic Biol Med 40:2155–2165

    Article  CAS  PubMed  Google Scholar 

  • Kulus D, Zalewska M (2014) Cryopreservation as a tool used in long-term storage of ornamental species—a review. Sci Hortic 168:88–107

    Article  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    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 

  • Meryman HT (2007) Cryopreservation of living cells: principles and practice. Transfusion 47:935–945

    Article  CAS  PubMed  Google Scholar 

  • Meyer AJ (2008) The integration of glutathione homeostasis and redox signaling. J Plant Physiol 165:1390–1403

    Article  CAS  PubMed  Google Scholar 

  • Navrot N, Rouhier N, Gelhaye E, Jaquot JP (2007) Reactive oxygen species generation and antioxidant systems in plant mitochondria. Physiol Plant 129:185–195

    Article  CAS  Google Scholar 

  • Peng J, Wang D, Xu C, Chen L, Deng F, Zhang X (2009) Ammonium molybdate method for detecting the activities of rice catalase. Chin Agric Sci Bull 25:61–64

    Google Scholar 

  • Poobathy R, Sinniah UR, Xavier R, Subramaniam S (2013) Catalase and superoxide dismutase activities and the total protein content of protocorm-like bodies of Dendrobium Sonia-28 subjected to vitrification. Appl Biochem Biotechnol 170:1066–1079

    Article  CAS  PubMed  Google Scholar 

  • Quan LJ, Zhang B, Shi WW, Li HY (2008) Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. J Integr Plant Biol 50:2–18

    Article  CAS  PubMed  Google Scholar 

  • Queval G, Issakidis-Bourguet E, Hoeberichts FA, Vandorpe M, Gakière B, Vanacker H, Miginiac-Maslow M, Breusegem FV, Noctor G (2007) Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cell death. Plant J 52:640–657

    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 

  • Reed BM (2008) Cryopreservation—practical considerations. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, USA, pp 3–13

    Chapter  Google Scholar 

  • Ren L, Zhang D, Jiang XN, Gai Y, Wang WM, Reed BM, Shen XH (2013) Peroxidation due to cryoprotectant step is a vital factor for cell survival in Arabidopsis cryopreservation. Plant Sci 212:37–47

    Article  CAS  PubMed  Google Scholar 

  • Sakai A, Hirai D, Niino T (2008) Development of PVS-based vitrification and encapsulation-vitrification protocols. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, USA, pp 33–57

    Chapter  Google Scholar 

  • Scandalios JG (1997) Molecular genetics of SOD in plants. In: Scandalios JG (ed) Oxidative stress and the molecular biology of antioxidant defense. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 527–568

    Google Scholar 

  • Skyba M, Petijová L, Košuth J, Koleva DP, Ganeva TG, Kapchina-Toteva VM, Cellárová E (2012) Oxidative stress and antioxidant response in Hypericum perforatum L. plants subjected to low temperature step. J Plant Physiol 169:955–964

    Article  CAS  PubMed  Google Scholar 

  • Sun YL, Zhao Y, Hong X, Zhai ZH (1999) Cytochrome c release and caspase activation during menadione-induced apoptosis in plants. FEBS Lett 462:317–321

    Article  CAS  PubMed  Google Scholar 

  • Tatone C, Emidio GD, Ventol M, Ciriminna R, Artini PG (2010) Cryopreservation and oxidative stress in reproductive cells. Gynecol Endocrinol 26:563–567

    Article  PubMed  Google Scholar 

  • Uchendu EE, Leonard SW, Traber MG, Reed BM (2010a) Vitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation. Plant Cell Rep 29:25–35

    Article  CAS  PubMed  Google Scholar 

  • Uchendu EE, Muminova M, Gupta S, Reed BM (2010b) Antioxidant and anti-stress compounds improve regrowth of cryopreserved Rubus shoot tips. In Vitro Cell Dev-Pl 46:386–393

    Article  CAS  Google Scholar 

  • Vacca RA, de Pinto MC, Valenti D, Passarella S, Marra E, De Gara L (2004) Production of reactive oxygen species, alteration of cytosolic ascorbate peroxidase, and impairment of mitochondrial metabolism are early events in heat shock-induced programmed cell death in tobacco Bright-Yellow 2 cells. Plant Physiol 134:1100–1112

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Vacca RA, Valenti D, Bobba A, MeraWna 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 en route to heat shock-induced cell death. Plant Physiol 141:208–219

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Van Breusegem F, Vranová E, Dat JF, Inzé D (2001) The role of active oxygen species in plant signal transduction. Plant Sci 161:405–414

    Article  Google Scholar 

  • Volk GM, Henk A, Basu C (2011) Gene expression in response to cryoprotectant and liquid nitrogen exposure in Arabidopsis shoot tips. Acta Hortic 908:55–66

    CAS  Google Scholar 

  • Vranová E, Atichartpongkul S, Villarroel R, Van Montagu M, Inzé D, Van Camp W (2002) Comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress. Proc Natl Acad Sci USA 99:870–875

    Article  Google Scholar 

  • Wang Y, Fan X, Zhang D, Shen X (2012) Regeneration of Agapanthus praecox ssp. orientalis ‘big blue’ via somatic embryogenesis. Propag Ornam Plants 12:148–154

    CAS  Google Scholar 

  • Wen B, Wang RL, Cheng HY, Song SQ (2010) Cytological and physiological changes in orthodox maize embryos during cryopreservation. Protoplasma 239:57–67

    Article  PubMed  Google Scholar 

  • Wen B, Cai C, Wang R, Song S, Song J (2012) Cytological and physiological changes in recalcitrant Chinese fan palm (Livistona chinensis) embryos during cryopreservation. Protoplasma 249:323–335

    Article  CAS  PubMed  Google Scholar 

  • Whitaker C, Beckett RP, Minibayeva FV, Kranner I (2010) Production of reactive oxygen species in excised, desiccated and cryopreserved explants of Trichilia dregeana Sond. S Afr J Bot 76:112–118

    Article  CAS  Google Scholar 

  • Wu N, Ren F, Wu X (2001) Assay of hydroxyl radical produced in Co2+-H2O2 reaction system by spectrophotometry with bromopyrogallol red. J Instrum Anal 20:52–54

    Google Scholar 

  • Xu J, Liu Q, Jia M, Liu Y, Li B, Shi Y (2014) Generation of reactive oxygen species during cryopreservation may improve Lilium × siberia pollen viability. In Vitro Cell Dev Plant 50:369–375

    Article  CAS  Google Scholar 

  • Zhang LR, Xing D (2008) Methyl jasmonate induces production of reactive oxygen species and alterations in mitochondrial dynamics that precede photosynthetic dysfunction and subsequent cell death. Plant Cell Physiol 49:1092–1111

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Wang J, Tang J, Guo L, Yang J, Huang Y, Tan Y, Fu S, Kong X, Zheng F (2009) In vivo protein transduction: delivery of PEP-1-SOD1 fusion protein into Myocardium efficiently protects against Ischemic Insult. Mol Cells 27:159–166

    Article  PubMed  Google Scholar 

  • Zhang D, Ren L, Yue JH, Wang L, Zhuo LH, Shen XH (2013) A comprehensive analysis of flowering transition in Agapanthus praecox ssp. orientalis (Leighton) Leighton by using transcriptomic and proteomic techniques. J Proteomics 80:1–25

    Article  CAS  PubMed  Google Scholar 

  • Zhang D, Ren L, Yue J, Wang L, Zhuo L, Shen X (2014) GA4 and IAA were involved in the morphogenesis and development of flowers in Agapanthus praecox ssp. orientalis. J Plant Physiol 171:966–976

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study received support from the National Natural Science Foundation of China (No. 31300580 and No. 31170655), Special Financial Grant from the China Postdoctoral Science Foundation (2013T60451), and Shanghai Graduate Education and Innovation Program (Horticulture).

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The authors declare that they have no conflict of interest.

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    Authors and Affiliations

    1. Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, No. 800, Rd. Dong Chuan, Shanghai, People’s Republic of China

      Di Zhang, Li Ren, Guan-qun Chen, Jie Zhang & Xiao-hui Shen

    2. United States Department of Agriculture-Agricultural Research Service, National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR, 97333-2521, USA

      Barbara M. Reed

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    1. Di Zhang
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    2. Li Ren
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    Correspondence to Xiao-hui Shen.

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    Communicated by H. S. Judelson.

    D. Zhang and L. Ren contributed equally to this work.

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    299_2015_1802_MOESM1_ESM.xlsx

    Supplementary material 1 (XLSX 10 kb) Supporting Information Table S1 the sequence and annealing temperature of all primers for qRT-PCR

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    Zhang, D., Ren, L., Chen, Gq. et al. ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox . Plant Cell Rep 34, 1499–1513 (2015). https://doi.org/10.1007/s00299-015-1802-0

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