An efficient protocol for extracting thylakoid membranes and total leaf proteins from Posidonia oceanica and other polyphenol-rich plants

• Published in: Plant methods Vol. 20; no. 1; p. 38
• Main Authors: Charras, Quentin, Rey, Pascal, Guillemain, Dorian, Dourguin, Fabian, Laganier, Hugo, Peschoux, Sacha, Molinié, Roland, Ismaël, Marwa, Caffarri, Stefano, Rayon, Catherine, Jungas, Colette
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 11.03.2024; BioMed Central; BMC
• Subjects: Analysis; Arabidopsis thaliana; Ascorbic acid; Ascorbic acid (vitamin C); Cell membranes; Chemical compounds; Chlorophyll; Controlled conditions; Crosslinking; Environmental stress; Extraction (Chemistry); Flowers & plants; Grapes; Identification and classification; Leaves; Life Sciences; Marine plants; Membranes; Metabolism; Methodology; Methods; Microscopy; Native-PAGE; Oxidation; Photosynthesis; Photosystem II; Photosystems; Plant extracts; Plant species; Plants; Polyethylene glycol; Polyols; Polyphenols; Posidonia oceanica; Proteins; SDS-PAGE; Seagrasses; Thylakoid membranes; Thylakoids; Vitamin C; Wild plants; France; SDS-PAGE; Polyethylene glycol; Photosystems; Polyphenols; Ascorbic acid (vitamin C); Wild plants; Native-PAGE; Seagrasses; Thylakoid membranes
• ISSN: 1746-4811; 1746-4811
• DOI: 10.1186/s13007-024-01166-7

The extraction of thylakoids is an essential step in studying the structure of photosynthetic complexes and several other aspects of the photosynthetic process in plants. Conventional protocols have been developed for selected land plants grown in controlled conditions. Plants accumulate defensive chemical compounds such as polyphenols to cope with environmental stresses. When the polyphenol levels are high, their oxidation and cross-linking properties prevent thylakoid extraction. In this study, we developed a method to counteract the hindering effects of polyphenols by modifying the grinding buffer with the addition of both vitamin C (VitC) and polyethylene glycol (PEG4000). This protocol was first applied to the marine plant Posidonia oceanica and then extended to other plants synthesizing substantial amounts of polyphenols, such as Quercus pubescens (oak) and Vitis vinifera (grapevine). Native gel analysis showed that photosynthetic complexes (PSII, PSI, and LHCII) can be extracted from purified membranes and fractionated comparably to those extracted from the model plant Arabidopsis thaliana. Moreover, total protein extraction from frozen P. oceanica leaves was also efficiently carried out using a denaturing buffer containing PEG and VitC. Our work shows that the use of PEG and VitC significantly improves the isolation of native thylakoids, native photosynthetic complexes, and total proteins from plants containing high amounts of polyphenols and thus enables studies on photosynthesis in various plant species grown in natural conditions.