Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics

Amalia Piro, Ilia Anna Serra, Antonia Spadafora, Monica Cardilio, Linda Bianco, Gaetano Perrotta, Rui Santos, Silvia Mazzuca

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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.
Original languageEnglish
Pages (from-to)4159 - 4174
Number of pages16
JournalProteomics
Volume15
Issue number23-24
DOIs
Publication statusPublished - 1 Dec 2015

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Piro, A., Serra, I. A., Spadafora, A., Cardilio, M., Bianco, L., Perrotta, G., ... Mazzuca, S. (2015). Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics. Proteomics, 15(23-24), 4159 - 4174. https://doi.org/10.1002/pmic.201500246