Abstract
The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H+-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H+-ATPase and the V-H+-pyrophosphatase, also producing further information on vacuole purity.
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Acknowledgments
This work was supported by (1) Fundo Europeu de Desenvolvimento Regional funds through the Operational Competitiveness Programme COMPETE and by National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the projects FCOMP-01-0124-FEDER-037277 (PEst-C/SAU/LA0002/2013) and FCOMP-01-0124-FEDER-019664 (PTDC/BIA-BCM/119718/2010); (2) the FCT scholarships cosupported by FCT and POPH-QREN (European Social Fund), SFRH/BD/41907/2007 (I.C.) and SFRH/BD/74257/2010 (H.N.); (3) a Scientific Mecenate Grant from Grupo Jerónimo Martins.
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Carqueijeiro, I. et al. (2018). Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus . In: Pereira, C. (eds) Plant Vacuolar Trafficking. Methods in Molecular Biology, vol 1789. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7856-4_7
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DOI: https://doi.org/10.1007/978-1-4939-7856-4_7
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