Abstract
We have developed an optimized protocol for isolating protoplasts from chlorenchyma cells of the single-cell C4 species Bienertia sinuspersici. The isolated protoplasts maintained the integrity of the unique single-cell C4 intracellular compartmentation of organelles as observed in chlorenchyma cells after cell wall digestion. Approximately over 80% of isolated protoplasts expressed the fusion reporter gene following the polyethylene glycol-mediated transfection procedures. Overall, fluorescent protein fusion tagged with various intraorganellular sorting signals validated the potential use of the transient gene expression system in subcellular localization and organelle dynamics studies.
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References
Voznesenskaya EV, Koteyeva NK, Chuong SDX, Edwards GE, Akhani H, Franceschi VR (2005) Differentiation of cellular and biochemical features of the single cell C4 syndrome during leaf development in Bienertia cycloptera (Chenopodiaceae). Am J Bot 92:1784–1795
Chuong SDX, Franceschi VR, Edwards GE (2006) The cytoskeleton maintains organelle partitioning required for single-cell C4 photosynthesis in Chenopodiaceae species. Plant Cell 18:2207–2223
Park J, Knoblauch M, Okita TW, Edwards GE (2009) Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C4 photosynthesis in Bienertia sinuspersici. Planta 229:369–382
Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475
Cocking EC (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187:962–963
Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23:131–171
Lung SC, Yanagisawa M, Chuong SDX (2011) Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici. Plant Cell Rep 30:473–484
Lung SC, Chuong SDX (2012) A transit peptide-like sorting signal at the C-terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the chloroplast outer membrane. Plant Cell 24:1560–1578
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572
Tzfira T, Tian GW, Lacroix B, Vyas S, Li J, Leitner-Dagan Y, Krichevsky A, Taylor T, Vainstein A, Citovsky V (2005) pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants. Plant Mol Biol 57:503–516
Acknowledgments
This research was supported by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2017-04416 to SDXC.
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Lung, SC., Yogadasan, N., Yanagisawa, M., Chuong, S.D.X. (2022). Protoplast Isolation and Transfection in the Single-Cell C4 Species Bienertia sinuspersici . In: Wang, K., Zhang, F. (eds) Protoplast Technology. Methods in Molecular Biology, vol 2464. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2164-6_2
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DOI: https://doi.org/10.1007/978-1-0716-2164-6_2
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