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Optimized conditions for the isolation of mesophyll protoplasts along the growing season from Arbutus unedo and their use in single cell gel electrophoresis

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Abstract

Global warming is affecting Mediterranean ecosystems, in which Arbutus unedo represents an important species. Mesophyll protoplasts are convenient material to study plant DNA integrity, nevertheless their release from mature plants along the year has not been reported in sclerophyll species. In this respect, in the present study the chance to isolate protoplasts from leaf tissues of A. unedo in different seasons was investigated. The digestion was obtained using Macerozyme R10 (1%) and Cellulase Onozuka RS (2%), with 0.6 M of mannitol, incubated for 4 h at 25 °C in the dark. In spring, soft leaves of different ages were studied to identify the most suitable material and protoplast yields were significantly influenced, with the still expanding 4th leaf characterized by the highest amount. Protoplast release decreased during the growing season when leaves become partially hardened, while their obtainment was quite impossible in summer and winter, due to leaf hardening. In November, an increase of incubation time (16 h) and of Cellulase RS (4%) or mannitol concentration (0.8 M) was needed with chilled leaves from the field; conversely, a good source for protoplasts were the green soft leaves coming from detached and pruned branches placed in controlled environment at spring temperatures. The employment of polyvinylpyrrolidone-40 during isolation procedure is discussed. The isolated protoplasts were employed to assess the DNA integrity, by using single cell gel electrophoresis (SCGE). The X-irradiated nucleoids were significantly damaged confirming that the present approach could be used in the selection of elite material of A. unedo devoted to fruit plantation.

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Abbreviations

CPW:

Cell and protoplast washing

SCGE:

Single cell gel electrophoresis

DTT:

1,4-Dithiothreitol

PVP-40:

Polyvinylpyrrolidone

References

  • Abbruzzese G, Beritognolo I, Muleo R, Piazzai M, Sabatti M, Scarascia Mugnozza G et al (2009) Leaf morphological plasticity and stomatal conductance in three Populus alba L. genotypes subjected to salt stress. Environ Exp Bot 66:349–522. https://doi.org/10.1016/j.envexpbot.2009.04.008

    Article  Google Scholar 

  • Collins AR (2004) The Comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26:249–261. https://doi.org/10.1385/MB:26:3:249

    Article  CAS  PubMed  Google Scholar 

  • Davey MR, Anthony P, Powera JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23:131–171. https://doi.org/10.1016/j.biotechadv.2004.09.008

    Article  CAS  PubMed  Google Scholar 

  • Dentener F, Drevet J, Lamarque JF, Bey I, Eickhout B, Fiore AM, Hauglustaine D, Horowitz LW, Krol M, Kulshrestha UC, Lawrence M, Galy-Lacaux C, Rast S, Shindell D, Stevenson D, Van Noije T, Atherton C, Bell N, Bergman D, Butler T, Cofala J, Collins B, Doherty R, Ellingsen K, Galloway J, Gauss M, Montanaro V, Müller JF, Pitari G, Rodriguez J, Sanderson M, Solmon F, Strahan S, Schultz M, Sudo K, Szopa S, Wild O (2006) Nitrogen and sulfur deposition on regional and global scales: a multimodel evaluation. Glob Biogeochem Cycles 20:GB4003. https://doi.org/10.1029/2005GB002672

    Article  Google Scholar 

  • Doblas-Miranda E, Alonso R, Arnan X, Bermejo V, Brotons L, de las Heras J, Estiarte M, Hódar JA, Llorens P, Lloret F, López-Serrano FR, Martínez-Vilalta J, Moya D, Peñuelas JP, Rodrigo A, Roura-Pascual N, Valladares F, Vilà M, Zamora R, Retana J (2017) A review of the combination among global change factors in forests, shrublands and pastures of the Mediterranean Region: beyond drought effects. Global Planet Change 148:42–54

    Article  Google Scholar 

  • Facchini PJ, Hagel J, Zulak KG (2002) Hydroxycinnamic acid amide metabolism: physiology and biochemistry. Can J Bot 80:577–589. https://doi.org/10.1139/b02-065

    Article  CAS  Google Scholar 

  • García-Alonso M, Pascual-Teresa S, Santos-Buelga C, Rivas-Gonzalo JC (2004) Evaluation of the antioxidant properties of fruits. Food Chem 84:13–18

    Article  Google Scholar 

  • Guillet G, De Luca V (2005) Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines. Plant Phys 137:692–699. https://doi.org/10.1104/pp.104.050294

    Article  CAS  Google Scholar 

  • Hughes NM (2011) Winter leaf reddening in ‘evergreen’ species. New Phytol 190(3):573–581. https://doi.org/10.1111/j.1469-8137.2011.03662.x

    Article  PubMed  Google Scholar 

  • Jones AMP, Shukla MR, Biswas GCG, Saxena PK (2015) Protoplast-to-plant regeneration of American elm (Ulmus americana). Protoplasma 52:925–931. https://doi.org/10.1007/s00709-014-0724-y

    Article  Google Scholar 

  • Kästner U, Klocke E, Abel S (2017) Regeneration of protoplasts after somatic hybridisation of Hydrangea. Plant Cell Tissue Organ Cult 129:359–373. https://doi.org/10.1007/s11240-017-1183-x

    Article  Google Scholar 

  • Kuzminsky E, Meschini R, Terzoli S, Pavani L, Silvestri C, Choury Z, Scarascia-Mugnozza G (2016) Isolation of mesophyll protoplasts from mediterranean woody plants for the study of DNA integrity under abiotic stress. Front Plant Sci 7:1168. https://doi.org/10.3389/fpls.2016.01168

    Article  PubMed  PubMed Central  Google Scholar 

  • Liqing Z, Bochu W, Jing Z, Lingxi C, Chuanyun D, Chuanren D (2005) Protoplast isolation of callus in Echinacea augustofolia. Colloids Surf B 44:1–5. https://doi.org/10.1016/j.colsurfb.2005.05.002

    Article  Google Scholar 

  • Lopez-Arellano M, Dhir S, Albino NC, Santiago A, Morris T, Dhir SK (2015) Somatic embryogenesis and plantlet regeneration from protoplast culture of Stevia rebaudiana. Br Biotechnol J 5(1):1–12

    Article  CAS  Google Scholar 

  • Malcolm JR, Liu C, Neilson RP, Hansen L, Hannah L (2006) Global warming and extinctions of endemic species from biodiversity hotspots. Conserv Biol 20:538–548

    Article  PubMed  Google Scholar 

  • Matsumoto K, Oka S (1997) Plant regeneration from protoplasts of a brazilian dessert banana (Musa spp., AAB GROUP). Acta Hortic 490:455–462

    Google Scholar 

  • Mills D, Hammerschlag FA (1994) Isolation of cells and protoplasts from leaves of in vitro propagated peach plants. Plant Cell Tissue Org Cult 36:99–105

    Article  CAS  Google Scholar 

  • Moreno-Jiménez E, Peñalosa J, Carpena-Ruiz R, Esteban E (2008) Comparison of arsenic resistance in Mediterranean woody shrubs used in restoration activities. Chemosphere 71:466–473

    Article  PubMed  Google Scholar 

  • Mukhtar I, Bajwa R, Nasim G (2012) Isolation of Mesophyll Protoplasts from Leaves of Dalbergia sissoo Roxb. J Appl Sci Environ Manage 16:11–15

    Google Scholar 

  • Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent, J (2000) Biodiversity hotspots for conservation priorities. Nature 403:853–858

    Article  CAS  PubMed  Google Scholar 

  • Nali C, Paoletti E, Marabottini R, Della Rocca G, Lorenzini G, Paolacci AR, Ciaffi M, Badiani M (2004) Ecophysiological and biochemical strategies of response to ozone in Mediterranean evergreen broadleaf species. Atmos Environ 38:2247–2257

    Article  CAS  Google Scholar 

  • Nenadis N, Llorens L, Koufogianni A, Díaz L, Font J, Gonzalez JA, Verdaguer D (2015) Interactive effects of UV radiation and reduced precipitation on the seasonal leaf phenolic content/composition and the antioxidant activity of naturally growing Arbutus unedo plants. J Photochem Photobiol B 153:435–444. https://doi.org/10.1016/j.jphotobiol.2015.10.016

    Article  CAS  PubMed  Google Scholar 

  • Olive PL, Banath JP, Durand RE (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the “Comet” assay. Radiat Res 122:86–94. https://doi.org/10.2307/3577587

    Article  CAS  PubMed  Google Scholar 

  • Paoletti E (2005) Ozone slows stomatal response to light and leaf wounding in a Mediterranean evergreen broadleaf, Arbutus unedo. Environ Pollut 134:439–445

    Article  CAS  PubMed  Google Scholar 

  • Paoletti E (2006) Impact of ozone on Mediterranean forests: a review. Environ Pollut 144(2):463–474

    Article  CAS  PubMed  Google Scholar 

  • Papadakis AK, Roubelakis-Angelakis KA (2002) Oxidative stress could be responsible for the recalcitrance of plant protoplasts. Plant Physiol Biochem 40:549–559. https://doi.org/10.1016/S0981-9428(02)01423-7

    Article  CAS  Google Scholar 

  • Reustle G, Natter I (1994) Effect of polyvinylpyrrolidone and activated-charcoal on formation of microcallus from grapevine protoplasts (Vitis sp.). Vitis 33:117–121

    CAS  Google Scholar 

  • Revilla MA, Ochatt SJ, Doughty S, Power JB (1987) A general strategy for the isolation of mesophyll protoplasts from deciduous fruit and nut tree species. Plant Sci 50:133–137

    Article  CAS  Google Scholar 

  • Ribeiro MM, Piotti A, Ricardo A, Gaspar D, Costa R, Parducci L, Vendramin GG (2017) Genetic diversity and divergence at the Arbutus unedo L. (Ericaceae) westernmost distribution limit. PLoS ONE 12(4):e0175239

    Article  PubMed  PubMed Central  Google Scholar 

  • Rivas-Ubach A, Gargallo-Garriga A, Sardans J, Oravec M, Mateu-Castell L, Pérez-Trujillo M et al (2014) Drought enhances folivory by shifting foliar metabolomes in Quercus ilex trees. New Phytol 202:874–885. https://doi.org/10.1111/nph.12687

    Article  CAS  PubMed  Google Scholar 

  • Sala OE, Chapin FS III, Armesto JJ, Berlow E, Bloomfield J, Dirzo R, Huber-Sanwald E, Huenneke LF, Jackson RB, Kinzig A, Leemans R, Lodge DM, Mooney HA, Oesterheld M, Poff NL, Sykes MT, Walker BH, Walker M, Wall DH (2000) Global biodiversity scenarios for the year 2100. Science 287:1770–1774. https://doi.org/10.1126/science.287.5459.1770

    Article  CAS  PubMed  Google Scholar 

  • Santiso X, López L, Gilbert KJ, Barreiro R, Whitlock MC, Retuerto R (2015) Patterns of genetic variation within and among populations in Arbutus unedo and its relation with selection and evolvability. Perspect Plant Ecol Evol Syst 17(3):185–192

    Article  Google Scholar 

  • Santos CLV, Pourrut B, Ferreira de Oliveira JMP (2015) The use of comet assay in plant toxicology: recent advances. Front Genet 6:216. https://doi.org/10.3389/fgene.2015.00216

    Article  PubMed  PubMed Central  Google Scholar 

  • Sinha A, Wetten AC, Caligari PDS (2003) Effect of biotic factors on the isolation of Lupinus albus protoplasts. Aust J Bot 51:103–109

    Article  Google Scholar 

  • Tan B, Xu M, Chen Y. Huang M (2013) Transient expression for functional gene analysis using Populus protoplasts. Plant Cell Tissue Organ Cult 114:11. https://doi.org/10.1007/s11240-013-0299-x

    Article  CAS  Google Scholar 

  • Tice RR, Strauss GH (1995) The single cell gel electrophoresis/comet assay: a potential tool for detecting radiation-induced DNA damage in humans. Stem Cells 13(Suppl. 1):207–214

    PubMed  Google Scholar 

  • Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221

    Article  CAS  PubMed  Google Scholar 

  • von Rӧpenack E, Parr A, Schulze-Lefert P (1998) Structural analyses and dynamics of soluble and cell wall-bound phenolics in a broad spectrum resistance to the powdery mildew fungus in barley. Biol Chem 273:9013–9022. https://doi.org/10.1074/jbc.273.15.9013

    Article  Google Scholar 

  • Wang T, McFarlane HE, Persson S (2016) The impact of abiotic factors on cellulose synthesis. J Exp Bot 67:543–552. https://doi.org/10.1093/jxb/erv488

    Article  CAS  PubMed  Google Scholar 

  • Ximenes E, Kim Y, Mosier N, Dien B, Ladisch M (2010) Inhibition of cellulases by phenols. Enzyme Microb Technol 46:170–176. https://doi.org/10.1016/j.enzmictec.2009.11.001

    Article  CAS  Google Scholar 

  • Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7):1565–1572

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The research was funded by the Italian “Progetti di Ricerca di Interesse Nazionale” (PRIN). Project TreeCity (prot. 20109E8F95_002). Thanks are due to Mrs. Matilde Tamantini for her technical assistance, and to Dr. Liliana Pavani for her kind assistance in the handling of the images.

Author information

Authors and Affiliations

  1. Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Viterbo, Italy

    Zineb Choury, Most Jannatul Fardusi, Giuseppe Scarascia Mugnozza & Elena Kuzminsky

  2. Department of Ecological and Biological Science, University of Tuscia, Viterbo, Italy

    Roberta Meschini & Ambra Dell’Orso

  3. Hawkesbury Institute for the Environment, University of Western Sydney, Penrith, NSW, 2751, Australia

    Zineb Choury

  4. Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria – Forestry Research Centre, Viale S. Margherita 80, 52100, Arezzo, Italy

    Most Jannatul Fardusi

Authors
  1. Zineb Choury
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  2. Roberta Meschini
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  3. Ambra Dell’Orso
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  4. Most Jannatul Fardusi
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  5. Giuseppe Scarascia Mugnozza
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  6. Elena Kuzminsky
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Contributions

EK is the scientist responsible for the funding project, expert in forest biotechnology and a substantial contributor to the conception and design of the work, and to the acquisition and interpretation of the data for the work. RM is an expert in SCGE and a substantial contributor to the conception and design of the work, and to the acquisition and interpretation of the data for the work. ZC is a substantial contributor to the conception and design of the work, and to the acquisition and interpretation of the data for the work, as well as the statistical analyses of the data. She provided for the drafting of the manuscript of the work. AD and MJF are involved in the acquisition, analyses and interpretation of the data. GS-M critically revised the work. All the authors approved the final version of the work to be published and are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Corresponding author

Correspondence to Elena Kuzminsky.

Additional information

Communicated by Wenwu Guo.

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Choury, Z., Meschini, R., Dell’Orso, A. et al. Optimized conditions for the isolation of mesophyll protoplasts along the growing season from Arbutus unedo and their use in single cell gel electrophoresis. Plant Cell Tiss Organ Cult 132, 535–543 (2018). https://doi.org/10.1007/s11240-017-1349-6

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  • DOI: https://doi.org/10.1007/s11240-017-1349-6

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