title: Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics.
abstract: Posidonia oceanica is a marine angiosperm, or seagrass, adapted to grow to the underwater life from shallow waters to 50 m depth. This raises questions of how their photosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolism of the chloroplast are the basis of adaptive capacity. In the present study, we described a protocol that was adapted from those optimized for terrestrial plants, to extract chloroplasts from as minimal tissue as possible. We obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant. After isopynic separations, the chloroplasts purity and integrity were evaluated by biochemical assay and using a proteomic approach. Chloroplast proteins were extracted from highly purified organelles and resolved by 1DE SDS-PAGE. Proteins were sequenced by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against NCBInr green plant databases, Dr. Zompo database for seagrasses in a local customized dataset. The curated localization of proteins in sub-plastidial compartments (i.e. envelope, stroma and thylakoids) was retrieved in the AT_CHLORO database. This purification protocol and the validation of compartment markers may serve as basis for sub-cellular proteomics in P. oceanica and other seagrasses.
authors.affiliation: Laboratory of Plant Biology and Plant Proteomics (Lab.Bio.Pro.Ve), Department of Chemistry and Chemical Technologies, Università della Calabria, Rende, Italy.
authors.forename: Amalia
authors.initials: A
authors.lastname: Piro
authors.affiliation: Laboratory of Plant Biology and Plant Proteomics (Lab.Bio.Pro.Ve), Department of Chemistry and Chemical Technologies, Università della Calabria, Rende, Italy.
authors.forename: Ilia Anna
authors.initials: IA
authors.lastname: Serra
authors.affiliation: Laboratory of Plant Biology and Plant Proteomics (Lab.Bio.Pro.Ve), Department of Chemistry and Chemical Technologies, Università della Calabria, Rende, Italy.
authors.forename: Antonia
authors.initials: A
authors.lastname: Spadafora
authors.affiliation: A.S. Diving Center Paolano, Lungomare Nord, Paola, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Cardilio
authors.affiliation: Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), TRISAIA Research Center, Rotondella (Matera), Italy.
authors.forename: Linda
authors.initials: L
authors.lastname: Bianco
authors.affiliation: Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), TRISAIA Research Center, Rotondella (Matera), Italy.
authors.forename: Gaetano
authors.initials: G
authors.lastname: Perrotta
authors.affiliation: ALGAE - Marine Plant Ecology, Center of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal.
authors.forename: Rui
authors.initials: R
authors.lastname: Santos
authors.affiliation: Laboratory of Plant Biology and Plant Proteomics (Lab.Bio.Pro.Ve), Department of Chemistry and Chemical Technologies, Università della Calabria, Rende, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Mazzuca
mesh_terms: D035961:Alismatales / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
keywords: Chloroplast purification; Marine proteomics; Plant proteomics; Seagrasses