title: A lower content of de-methylesterified homogalacturonan improves enzymatic cell separation and isolation of mesophyll protoplasts in Arabidopsis.
abstract: Cell adhesion occurs primarily at the level of middle lamella which is mainly composed by pectin polysaccharides. These can be degraded by cell wall degrading enzymes (CWDEs) during developmental processes to allow a controlled separation of plant cells. Extensive cell wall degradation by CWDEs with consequent cell separation is performed when protoplasts are isolated from plant tissues by using mixtures of CWDEs. We have evaluated whether modification of pectin affects cell separation and protoplast isolation. Arabidopsis plants overexpressing the pectin methylesterase inhibitors AtPMEI-1 or AtPMEI-2, and Arabidopsis pme3 plants, mutated in the gene encoding pectin methylesterase 3, showed an increased efficiency of isolation of viable mesophyll protoplasts as compared with Wild Type Columbia-0 plants. The release of protoplasts was correlated with the reduced level of long stretches of de-methylesterified homogalacturonan (HGA) present in these plants. Response to elicitation, cell wall regeneration and efficiency of transfection in protoplasts from transgenic plants was comparable to those of wild type protoplasts.
authors.affiliation: Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, Roma 00185, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Lionetti
authors.affiliation: Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, Roma 00185, Italy.
authors.forename: Felice
authors.initials: F
authors.lastname: Cervone
authors.affiliation: Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", "Sapienza" Università di Roma, Piazzale Aldo Moro 5, Roma 00185, Italy. Electronic address: giulia.delorenzo@uniroma1.it.
authors.forename: Giulia
authors.initials: G
authors.lastname: De Lorenzo
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000502:physiology; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D004951:Esterification; D058503:Mesophyll Cells / Q000166:cytology*; D010368:Pectins / Q000378:metabolism*; D011523:Protoplasts / Q000166:cytology* / Q000201:enzymology*; D013312:Stress, Physiological; D014162:Transfection
chemical_list: D010368:Pectins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; C003181:polygalacturonic acid
keywords: Arabidopsis thaliana; Cell adhesion; Enzymatic cell separation; Homogalacturonan; Pectin; Pectin methylesterase; Pectin methylesterase inhibitors; Protoplast isolation