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Using Arabidopsis Protoplasts to Study Cellular Responses to Environmental Stress

  • Ana Confraria
  • Elena Baena-GonzálezEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)

Abstract

Arabidopsis mesophyll protoplasts can be readily isolated and transfected in order to transiently express proteins of interest. As freshly isolated mesophyll protoplasts maintain essentially the same physiological characteristics of whole leaves, this cell-based transient expression system can be used to molecularly dissect the responses to various stress conditions. The response of stress-responsive promoters to specific stimuli can be accessed via reporter gene assays. Additionally, reporter systems can be easily engineered to address other levels of regulation, such as transcript and/or protein stability. Here we present a detailed protocol for using the Arabidopsis mesophyll protoplast system to study responses to environmental stress, including preparation of reporter and effector constructs, large scale DNA purification, protoplast isolation, transfection, treatment, and quantification of luciferase-based reporter gene activities.

Key words

Protoplasts Arabidopsis thaliana Mesophyll Transient expression system Stress Luciferase-based reporters Promoter activity 

Notes

Acknowledgements

We thank Vera Nunes for excellent plant care and all the members of the Baena-González lab for helping to establish various reporter assays in Arabidopsis mesophyll protoplasts. We also thank Jen Sheen (Harvard Medical School/Massachusetts General Hospital) for all the guidance and training regarding this technique and cell signaling. Ana Confraria is supported by a fellowship from Fundação para a Ciência e Tecnologia (FCT, SFRH/BPD/47280/2008). The Baena-González lab is supported by grants from the EMBO Installation program, the Marie Curie ITN program (“MERIT”, PITN-GA-2010-264474) and FCT (PTDC/BIA-PLA/3937/2012; Research unit GREEN-it "Bioresources for Sustainability” UID/Multi/04551/2013).

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© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Plant Stress Signaling, Instituto Gulbenkian de CiênciaOeirasPortugal

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