title: Using Arabidopsis Protoplasts to Study Cellular Responses to Environmental Stress.
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.
authors: Plant Stress Signaling, Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156, Oeiras, Portugal.
authors: Ana
authors: A
authors: Confraria
authors: Plant Stress Signaling, Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156, Oeiras, Portugal. ebaena@igc.gulbenkian.pt.
authors: Elena
authors: E
authors: Baena-González
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins
keywords: Arabidopsis thaliana; Luciferase-based reporters; Mesophyll; Promoter activity; Protoplasts; Stress; Transient expression system