Optimized protoplast isolation and establishment of transient gene expression system for the Antarctic flowering plant Colobanthus quitensis (Kunth) Bartl.

  • Ok-Kyoung Cha
  • Jungeun Lee
  • Hyoung Seok Lee
  • Horim LeeEmail author
Research Note


Colobanthus quitensis is one of two terrestrial plants that grow in the maritime Antarctic. Despite its important ecological niche in extreme environments, the molecular mechanisms of its adaptation and tolerance have not been elucidated due to difficulties with genetic or transgenic approaches. For this reason, in many other plant species mesophyll protoplasts as a versatile cell-based system have been developed and used to analyze the biological functions of genes of interest. Here we report an optimized method of protoplast isolation from C. quitensis leaves. The main parameters evaluated to reach the highest protoplast yield were the use of a cell wall-degrading enzyme, an osmotic stabilizer, and different pH conditions. Moreover, transient expression and subcellular localization of proteins were validated by an immunoblot assay and a confocal microscopy, respectively, using C. quitensis protoplasts. Therefore, these results suggest that protoplasts can provide a useful cell-based system to facilitate the molecular, biochemical, and cellular characterizations of C. quitensis genes.

Key message

C. quitensis protoplasts can provide a physiologically relevant cell system to facilitate the molecular, biochemical, and cellular characterization of C. quitensis genes.


Cell-based assay Low temperature Molecular adaptation PEG–CaCl2-mediated transfection Subcellular localization 



This research was supported by a grant from the Korea Polar Research Institute (PE18290).

Author contributions

JL and HL developed concept and supplied plant materials. JL, HSL, and HL designed the research. HL wrote the manuscript and OKC performed all experiments.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

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Supplementary material 1 (PDF 464 kb)
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Supplementary material 2 (PDF 584 kb)
11240_2019_1651_MOESM3_ESM.pdf (534 kb)
Supplementary material 3 (PDF 533 kb)


  1. Androsiuk P, Jastrzębski JP, Paukszto Ł, Okorski A, Pszczółkowska A, Chwedorzewska KJ, Koc J, Górecki R, Giełwanowska I (2018) The complete chloroplast genome of Colobanthus apetalus (Labill.) Druce: genome organization and comparison with related species. PeerJ 6:e4723CrossRefGoogle Scholar
  2. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983CrossRefGoogle Scholar
  3. Burris KP, Dlugosz EM, Collins AG, Stewart CN Jr, Lenaghan SC (2016) Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.). Plant Cell Rep 35:693–704CrossRefGoogle Scholar
  4. Cavieres LA, Sáez P, Sanhueza C, Sierra-almeida A, Rabert C, Corcuera LJ, Alberdi M, Bravo LA (2016) Ecophysiological traits of Antarctic vascular plants: their importance in the responses to climate change. Plant Ecol 217:343–358CrossRefGoogle Scholar
  5. Cheng MC, Liao PM, Kuo WW, Lin TP (2013) The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals. Plant Physiol 162:1566–1582CrossRefGoogle Scholar
  6. Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330CrossRefGoogle Scholar
  7. Cho SM, Lee H, Jo H, Lee H, Kang Y, Park H, Lee J (2018) Comparative transcriptome analysis of field- and chamber-grown samples of Colobanthus quitensis (Kunth) Bartl, an Antarctic flowering plant. Sci Rep 8:11049CrossRefGoogle Scholar
  8. Cuba-Díaz M, Klagges M, Fuentes-Lillo E, Cordero C, Acuña D, Opazo G, Troncoso-Castro JM (2017) Phenotypic variability and genetic differentiation in continental and island populations of Colobanthus quitensis (Caryophyllaceae: Antarctic pearlwort). Polar Biol 40:2397–2409CrossRefGoogle Scholar
  9. Grundt HH, Kjølner S, Borgen L, Rieseberg LH, Brochmann C (2006) High biological species diversity in the arctic flora. Proc Natl Acad Sci USA 103:972–975CrossRefGoogle Scholar
  10. Guo WJ, Ho THD (2008) An abscisic acid-induced protein, HVA22, inhibits gibberellin-mediated programmed cell death in cereal aleurone cells. Plant Physiol 147:1710–1722CrossRefGoogle Scholar
  11. Hachez C, Veljanovski V, Reinhardt H, Guillaumot D, Vanhee C, Chaumont F, Batoko H (2014) The Arabidopsis abiotic stress-induced TSPO-related protein reduces cell-surface expression of the aquaporin PIP2;7 through protein-protein interactions and autophagic degradation. Plant Cell 26:4974–4990CrossRefGoogle Scholar
  12. Huo A, Chen Z, Wang P, Yang L, Wang G, Wang D, Liao S, Cheng T, Chen J, Shi J (2017) Establishment of transient gene expression systems in protoplasts from Liriodendron hybrid mesophyll cells. PLoS ONE 12:e0172475CrossRefGoogle Scholar
  13. Kang Y, Lee H, Kim MK, Shin SC, Park H, Lee J (2016) The complete chloroplast genome of Antarctic pearlwort Colobanthus quitensis (Kunth) Bartl. Mitochondrial DNA Part A 27:4677–4678CrossRefGoogle Scholar
  14. Liu S, Liu C, Huang X, Chai Y, Cong B (2006) Optimization of parameters for isolation of protoplasts from the Antarctic sea ice alga Chlamydomonas sp. ICE-L. J Appl Phycol 18:783–786CrossRefGoogle Scholar
  15. Lung S-C, Yanagisawa M, Chuong SDX (2011) Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici. Plant Cell Rep 30:473–484CrossRefGoogle Scholar
  16. Pérez-Torres E, Dinamarca J, Bravo LA, Corcuera LJ (2004) Responses of Colobanthus quitensis (Kunth) Bartl. to high light and low temperature. Polar Biol 27:183–189CrossRefGoogle Scholar
  17. Sáez PL, Bravo LA, Cavieres LA, Vallejos V, Sanhueza C, Font-Carrascosa M, Gil-Pelegrín E, Peguero-Pina JJ, Galmés J (2017) Photosynthetic limitations in two Antarctic vascular plants: importance of leaf anatomical traits and Rubisco kinetic parameters. J Exp Bot 68:2871–2883CrossRefGoogle Scholar
  18. Sanhueza C, Vallejos V, Cavieres LA, Saez P, Bravo LA, Corcuera LJ (2017) Growing temperature affects seed germination of the antarctic plant Colobanthus quitensis (Kunth) Bartl (Caryophyllaceae). Polar Biol 40:449–455CrossRefGoogle Scholar
  19. Singh J, Singh RP, Khare R (2018) Influence of climate change on Antarctic flora. Polar Sci 18:94–101CrossRefGoogle Scholar
  20. Smith RIL (2003) The enigma of Colobanthus quitensis and Deschampsia antarctica in Antarctica. In: Huiskes AHL, Gieskes WWC, Rocema J, Schorno RML, Van Der Vies SM, Wolff WJ (eds) Antarctic biology in a global context. Backhuys, Leiden, pp 234–239Google Scholar
  21. Wu JZ, Liu Q, Geng XS, Li KM, Luo LJ, Liu JP (2017) Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz). BMC Biotechnol 17:29CrossRefGoogle Scholar
  22. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572CrossRefGoogle Scholar
  23. Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J, Wang H (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7:30CrossRefGoogle Scholar
  24. Zúñiga G, Zamora P, Ortega M, Obrecht A (2009) Micropropagation of Antarctic Colobanthus quitensis. Antarct Sci 21:149–150CrossRefGoogle Scholar
  25. Zúñiga-Feest A, Bascuñán-Godoy L, Reyes-Diaz M, Bravo LA, Corcuera LJ (2009) Is survival after ice encasement related with sugar distribution in organs of the Antarctic plants Deschampsia Antarctica Desv. (Poaceae) and Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae)? Polar Biol 32:583–591CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyDuksung Women’s UniversitySeoulRepublic of Korea
  2. 2.Unit of Polar GenomicsKorea Polar Research Institute (KOPRI)IncheonRepublic of Korea
  3. 3.Polar ScienceUniversity of Science & TechnologyDaejeonRepublic of Korea

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