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Monitoring of oxidative status in three native Australian species during cold acclimation and cryopreservation

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Abstract

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Three wild species exhibited a significant reduction in antioxidants throughout the cryopreservation protocol, whilst the half-cell reduction potential became more oxidised. Antioxidant content recuperated in recovering shoot tips.

Abstract

Cryopreservation is the most efficient and cost-effective long-term storage solution for the conservation of a wide range of plant species and material. Changes in the levels of antioxidants during the process of cryopreservation are known to reduce post-cryogenic survival due to oxidative stress. Low-molecular-weight thiols (cysteine, γ-glutamylcysteine, and glutathione) and ascorbic acid, which represent the two major water-soluble antioxidants in plants, were analysed at specific stages during cryopreservation of shoot tip material of three native Australian plant species [Anigozanthos viridis (Haemodoraceae), Lomandra sonderi (Asparagaceae), and Loxocarya cinerea (Restionaceae)] to quantify the oxidative stress experienced during cryopreservation. Post-cryogenic regeneration of shoot tips was greatest in A. viridis (78%) followed by L. sonderi (50%), whilst L. cinerea did not show any post-cryogenic regeneration. The application of a 3-week cold (5 °C) preconditioning regime, commonly used to increase post-cryogenic survival, resulted in significantly lower post-cryogenic regeneration for A. viridis (33%), but had little effect on the other two species. Total antioxidant concentration in shoot material decreased significantly with each step throughout the cryopreservation process, particularly in the cryoprotection and washing stages. Antioxidant levels in shoot tips then increased during the subsequent 7-day post-cryopreservation recovery period, with the greatest increase measured in A. viridis. Concentrations of thiols and their corresponding disulphides were used to calculate the corresponding half-cell reduction potentials, whereby the ability of these plant species to maintain a strong reducing environment in shoot tissues throughout the cryopreservation protocol was found to correlate with post-cryogenic survival.

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Fig. 1

Adapted from Kaczmarczyk et al. (2012); originally published under CC BY 3.0 license. Available from: doi:10.5772/32860

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Abbreviations

AsA:

Ascorbic acid

BM:

Basal medium

CPAs:

Cryoprotective agents

Cys:

Cysteine

DW:

Dry weight

FW:

Fresh weight

GSH:

Glutathione

GSSG:

Glutathione disulphide

γ-Glu-Cys:

Gamma-glutamylcysteine

LMW:

Low molecular weight

LN:

Liquid nitrogen

ROS:

Reactive oxygen species

References

  • Bachiri Y, Bajon C, Sauvanet A, Gazeau C, Morisset C (2000) Effect of osmotic stress on tolerance of air-drying and cryopreservation of Arabidopsis thaliana suspension cells. Protoplasma 214:227–243

    Article  CAS  Google Scholar 

  • 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, vol 32. Springer, New York, pp 1–18

    Google Scholar 

  • Barraco G, Chatelet P, Balsemin E, Decourcelle T, Sylvestre I, Engelmann F (2012) Cryopreservation of Prunus cerasus through vitrification and replacement of cold hardening with preculture on medium enriched with sucrose and/or glycerol. Sci Hortic 148:104–108

    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, Boca Raton, pp 206–241

    Google Scholar 

  • Berjak P, Pammenter N (2014) Cryostorage of germplasm of tropical recalcitrant-seeded species: approaches and problems. Int J Plant Sci 175:29–39

    Article  CAS  Google Scholar 

  • Berjak P, Varghese B, Pammenter N (2011) Cathodic amelioration of the adverse effects of oxidative stress accompanying procedures necessary for cryopreservation of embryonic axes of recalcitrant-seeded species. Seed Sci Res 21:187–203

    Article  CAS  Google Scholar 

  • Birtic S, Colville L, Pritchard HW, Pearce SR, Kranner I (2011) Mathematically combined half-cell reduction potentials of low-molecular-weight thiols as markers of seed ageing. Free Radic Res 45:1093–1102

    Article  PubMed  Google Scholar 

  • Canepa P, Panta A, Tay D (2011) The effect of antioxidants on the cryopreservation recovery of two potato cultivars following post-thawing culture. Acta Hort 908:101–105

    Article  CAS  Google Scholar 

  • Chang Y, Barker RE, Reed BM (2000) Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.). CryoLetters 21:107–116

    CAS  PubMed  Google Scholar 

  • Chen H, Osuna D, Colville L, Lorenzo O, Graeber K, Küster H, Leubner-Metzger G, Kranner I (2013) Transcriptome-wide mapping of pea seed ageing reveals a pivotal role for genes related to oxidative stress and programmed cell death. PLoS One 8:e78471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen G, Ren L, Zhang J, Reed BM, Zhang D, Shen X (2015) Cryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings. Cryobiology 70:38–47

    Article  CAS  PubMed  Google Scholar 

  • Colville L, Kranner I (2010) Desiccation tolerant plants as model systems to study redox regulation of protein thiols. Plant Growth Regul 62:241–255

    Article  CAS  Google Scholar 

  • Department of Parks and Wildlife (2014) Wildlife Conservation (Rare Flora) Notice. https://www.dpaw.wa.gov.au/plants-and-animals/threatened-species-and-communities/threatened-plants

  • Dussert S, Chabrillange N, Montillet JL, Agnel JP, Engelmann F, Noirot M (2003) Basis of coffee seed sensitivity to liquid nitrogen exposure: oxidative stress or imbibitional damage? Physiol Plant 119:534–543

    Article  CAS  Google Scholar 

  • Fadzillah NM, Gill V, Finch RP, Burdon RH (1996) Chilling, oxidative stress and antioxidant responses in shoot cultures of rice. Planta 199:552–556

    Article  CAS  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 

  • Folgado R, Panis B, Sergeant K, Renaut J, Swennen R, Hausman J-F (2015) Unravelling the effect of sucrose and cold pretreatment on cryopreservation of potato through sugar analysis and proteomics. Cryobiology 71:432–441

    Article  CAS  PubMed  Google Scholar 

  • Foyer CH, Noctor G (2011) Ascorbate and glutathione: the heart of the redox hub. Plant Physiol 155:2–18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fujikawa S, Jitsuyama Y, Kuroda K (1999) Determination of the role of cold acclimation-induced diverse changes in plant cells from the viewpoint of avoidance of freezing injury. J Plant Res 112:237–244

    Article  Google Scholar 

  • Funnekotter B, Kaczmarczyk A, Turner SR, Bunn E, Zhou W, Smith S, Flematti G, Mancera RL (2013) Acclimation-induced changes in cell membrane composition and influence on cryotolerance of in vitro shoots of native plant species. Plant Cell Tissue Organ Cult 114:83–96

    Article  CAS  Google Scholar 

  • Funnekotter B, Sortey A, Bunn E, Turner S, Mancera R (2016) Influence of abiotic stress preconditioning on antioxidant enzymes in shoot tips of Lomandra sonderi (Asparagaceae) prior to cryostorage. Aust J Bot 64:260–268

    CAS  Google Scholar 

  • Gupta S, Reed BM (2006) Cryopreservation of shoot tips of blackberry and raspberry by encapsulation–dehydration and vitrification. CryoLetters 27:29–42

    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 

  • Hincha D, Zuther E (2014) Introduction: plant cold acclimation and freezing tolerance. In: Hincha DK, Zuther E (eds) Plant cold acclimation. Springer, New York, pp 1–6

    Chapter  Google Scholar 

  • Hopper SD, Gioia P (2004) The southwest Australian floristic region: evolution and conservation of a global hot spot of biodiversity. Annu Rev Ecol Evol Syst 35:623–650

    Article  Google Scholar 

  • Hughes ZE, Mancera RL (2013) Molecular dynamics simulations of mixed DOPC–β-sitosterol bilayers and their interactions with DMSO. Soft Matter 9:2920–2935

    Article  CAS  Google Scholar 

  • Hughes ZE, Mancera RL (2014) Molecular mechanism of the synergistic effects of vitrification solutions on the stability of phospholipid bilayers. Biophys J 106:2617–2624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hughes ZE, Mark AE, Mancera RL (2012) Molecular dynamics simulations of the interactions of DMSO with DPPC and DOPC phospholipid membranes. J Phys Chem B 116:11911–11923

    Article  CAS  PubMed  Google Scholar 

  • Johnston JW, Harding K, Benson EE (2007) Antioxidant status and genotypic tolerance of Ribes in vitro cultures to cryopreservation. Plant Sci 172:524–534

    Article  CAS  Google Scholar 

  • Kaczmarczyk A, Shvachko N, Lupysheva Y, Hajirezaei MR, Keller ER (2008) Influence of alternating temperature preculture on cryopreservation results for potato shoot tips. Plant Cell Rep 27:1551–1558

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kaczmarczyk A, Turner SR, Bunn E, Mancera RL, Dixon KW (2011) Cryopreservation of threatened native Australian species—what have we learned and where to from here? In Vitro Cell Dev Biol Plant 47:17–25

    Article  Google Scholar 

  • Kaczmarczyk A, Funnekotter B, Menon A, Phang PY, Al-Hanbali A, Bunn E, Mancera RL (2012) Current issues in plant cryopreservation. In: Katkov II (ed) Current frontiers in cryobiology. InTech, Croatia, pp 417–438

    Google Scholar 

  • Kaczmarczyk A, Funnekotter B, Turner SR, Bunn E, Bryant G, Hunt TE, Mancera RL (2013) Development of cryopreservation for Loxocarya cinerea—an endemic Australian plant species important for post-mining restoration. CryoLetters 34:508–519

    CAS  PubMed  Google Scholar 

  • Kamata T, Uemura M (2004) Solute accumulation in wheat seedlings during cold acclimation: contribution to increased freezing tolerance. CryoLetters 25:311–322

    CAS  PubMed  Google Scholar 

  • Kocsy G, Szalai G, Vágújfalvi A, Stéhli L, Orosz G, Galiba G (2000) Genetic study of glutathione accumulation during cold hardening in wheat. Planta 210:295–301

    Article  CAS  PubMed  Google Scholar 

  • Kocsy G, Galiba G, Brunold C (2001) Role of glutathione in adaptation and signalling during chilling and cold acclimation in plants. Physiol Plant 113:158–164

    Article  CAS  PubMed  Google Scholar 

  • Kranner I (1998) Determination of glutathione, glutathione disulphide and two related enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase, in fungal and plant cells. In: Varma A (ed) Mycorrhiza manual. Springer, Berlin, pp 227–241

    Chapter  Google Scholar 

  • Kranner I, Birtić 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 

  • Leunufna S, Keller ER (2005) Cryopreservation of yams using vitrification modified by including droplet method: effects of cold acclimation and sucrose. CryoLetters 26:93–102

    CAS  PubMed  Google Scholar 

  • Lynch PT, Siddika A, Johnston JW, Trigwell SM, Mehra A, Benelli C, Lambardi M, Benson EE (2011) Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos. Plant Sci 181:47–56

    Article  CAS  PubMed  Google Scholar 

  • Martinez-Montero M, Harding K (2015) Cryobionomics: evaluating the concept in plant cryopreservation. In: Barh D, Khan MS, Davies E (eds) PlantOmics: the omics of plant science. Springer India, New Delhi, pp 655–682

    Google Scholar 

  • Menon A, Funnekotter B, Kaczmarczyk A, Bunn E, Turner S, Mancera RL (2012) Cryopreservation of Lomandra sonderi (Asparagaceae) shoot tips using droplet-vitrification. CryoLetters 33:259–270

    CAS  PubMed  Google Scholar 

  • Menon A, Funnekotter B, Kaczmarczyk A, Bunn E, Turner S, Mancera RL (2014) Cold-induced changes affect survival after exposure to vitrification solution during cryopreservation in the south-west Australian Mediterranean climate species Lomandra sonderi (Asparagaceae). Plant Cell Tissue Organ Cult 119:347–358

    Article  CAS  Google Scholar 

  • Nagel M, Kranner I, Neumann K, Rolletschek H, Seal CE, Colville L, Fernández-Marín B, Börner A (2014) Genome-wide association mapping and biochemical markers reveal that seed ageing and longevity are intricately affected by genetic background and developmental and environmental conditions in barley. Plant Cell Environ. doi:10.1111/pce.12474

    Google Scholar 

  • Prasad TK (1996) Mechanisms of chilling-induced oxidative stress injury and tolerance in developing maize seedlings: changes in antioxidant system, oxidation of proteins and lipids, and protease activities. Plant J 10:1017–1026

    Article  CAS  Google Scholar 

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

    Chapter  Google Scholar 

  • Ren L, Zhang D, Jiang X-N, Gai Y, Wang W-M, Reed BM, Shen X-H (2013) Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in Arabidopsis cryopreservation. Plant Sci 212:37–47

    Article  CAS  PubMed  Google Scholar 

  • Ren L, Zhang D, G-q Chen, Reed BM, X-h Shen, H-y Chen (2015) Transcriptomic profiling revealed the regulatory mechanism of Arabidopsis seedlings response to oxidative stress from cryopreservation. Plant Cell Rep 34:2161–2178

    Article  CAS  PubMed  Google Scholar 

  • Roach T, Ivanova M, Beckett RP, Minibayeva FV, Green I, Pritchard HW, Kranner I (2008) An oxidative burst of superoxide in embryonic axes of recalcitrant sweet chestnut seeds as induced by excision and desiccation. Physiol Plant 133:131–139

    Article  CAS  PubMed  Google Scholar 

  • Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33

    Article  CAS  PubMed  Google Scholar 

  • Sánchez-Fernández R, Fricker M, Corben LB, White NS, Sheard N, Leaver CJ, Van Montagu M, Inzé D, May MJ (1997) Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms of redox control. Proc Natl Acad Sci 94:2745–2750

    Article  PubMed  PubMed Central  Google Scholar 

  • Sasaki H, Ichimura K, Oda M (1996) Changes in sugar content during cold acclimation and deacclimation of cabbage seedlings. Ann Bot 78:365–369

    Article  CAS  Google Scholar 

  • Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30:1191–1212

    Article  CAS  PubMed  Google Scholar 

  • Skyba M, Urbanová M, Kapchina-Toteva V, Košuth J, Harding K, Čellárová E (2010) Physiological, biochemical and molecular characteristics of cryopreserved Hypericum perforatum L. shoot tips. CryoLetters 31:249–260

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Steponkus PL (1984) Role of the plasma membrane in freezing injury and cold acclimation. Ann Rev Plant Physiol 35:543–584

    Article  CAS  Google Scholar 

  • Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 118:1–7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Turner S, Touchell D, Dixon K, Tan B (2000) Cryopreservation of Anigozanthos viridis ssp. viridis and related taxa from the south-west of Western Australia. Aust J Bot 48:739–744

    Article  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 Biol Plant 46:386–393

    Article  CAS  Google Scholar 

  • Wang ZC, Deng XX (2004) Cryopreservation of shoot-tips of citrus using vitrification: effect of reduced form of glutathione. CryoLetters 25:43–50

    PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  • Western Australian Herbarium (1998) FloraBase—the Western Australian flora. Department of Environment and Conservation, Kensington

    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. South Afr J Bot 76:112–118

    Article  CAS  Google Scholar 

  • Zhang D, Ren L, G-q Chen, Zhang J, Reed BM, X-h Shen (2015) 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

    Article  PubMed  Google Scholar 

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Acknowledgements

The authors acknowledge the Australian Research Council (Grants LP0884027 and LP140100993) and generous support from Alcoa of Australia Ltd. The Royal Botanic Gardens, Kew receives grant-in-aid from Defra, UK. BF was recipient of an Australian Postgraduate Award and MERIWA scholarship.

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Authors and Affiliations

  1. School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, GPO Box U1987, Perth, WA, 6845, Australia

    Bryn Funnekotter & Ricardo L. Mancera

  2. Botanic Gardens and Parks Authority, 1 Kattidj Close, Kings Park, WA, 6005, Australia

    Bryn Funnekotter, Anja Kaczmarczyk, Shane R. Turner & Eric Bunn

  3. Department of Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex, RH17 6TN, UK

    Louise Colville

  4. School of Biological Sciences, University of Western Australia, Crawley, WA, 6009, Australia

    Shane R. Turner & Eric Bunn

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  1. Bryn Funnekotter
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  2. Louise Colville
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  3. Anja Kaczmarczyk
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  4. Shane R. Turner
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Correspondence to Ricardo L. Mancera.

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Communicated by Emmanuel Guiderdoni.

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Funnekotter, B., Colville, L., Kaczmarczyk, A. et al. Monitoring of oxidative status in three native Australian species during cold acclimation and cryopreservation. Plant Cell Rep 36, 1903–1916 (2017). https://doi.org/10.1007/s00299-017-2204-2

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