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The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM-OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM-OEM complexes in various physiological contexts and using virtually any other purified membrane organelle. [ABSTRACT FROM AUTHOR]

Chlorophyll biosynthesis requires a metabolic dialog between the chloroplast envelope and thylakoids where biosynthetic activities are localized. Here, we report the first plant S -adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase (MgPIX MT) sequence identified in the Arabidopsis genome owing to its similarity with the Synechocystis sp. MgP IX MT gene. After expression in Escherichia coli, the recombinant Arabidopsis thaliana cDNA was shown to encode a protein having MgPIX MT activity. The full-length polypeptide exhibits a chloroplast transit peptide that is processed during import into the chloroplast. The mature protein contains two functional regions. The C-terminal part aligns with the Synechocystis full-length protein. The corresponding truncated region binds to Ado-met, as assayed by UV crosslinking, and is shown to harbor the MgPIX MT activity. Downstream of the cleaved transit peptide, the 40 N-terminal amino acids of the mature protein are very hydrophobic and enhance the association of the protein with the membrane. In A. thaliana and spinach, the MgPIX MT protein has a dual localization in chloroplast envelope membranes as well as in thylakoids. The protein is active in each membrane and has the same apparent size corresponding to the processed mature protein. The protein is very likely a monotopic membrane protein embedded within one leaflet of the membrane as indicated by ionic and alkaline extraction of each membrane. The rationale for a dual localization of the protein in the chloroplast is discussed. [ABSTRACT FROM AUTHOR]

Plants and algae have to adapt to environmental changes and face various stresses that negatively affect their growth and development. One common stress is phosphate (Pi) deficiency, which is often present in the environment at limiting levels. In response to Pi deficiency, these organisms increase Pi uptake and remobilize intracellular Pi. Phospholipids are degraded to provide Pi and replaced by non-phosphorus lipids, such as glycolipids or betaine lipids. During evolution, seed plants lost the ability to synthesize betaine lipids. By expressing BTA1 genes, which are involved in the synthesis of diacylglyceryl-N,N,N-trimethyl-homoserine (DGTS), from different species, our work shows that DGTS can be produced in seed plants. In Arabidopsis, expression of BTA1 under a phosphate starvation-inducible promoter resulted in limited DGTS production without having any impact on plant growth or lipid remodeling. In transient expression systems in Nicotiana benthamiana, leaves were able to accumulate DGTS up to 30 % of their glycerolipid content at a slight expense of galactolipid and phospholipid production. At the subcellular level, we showed that DGTS is absent from the plastids and seems to be enriched in the endomembranes, driving an ER membrane proliferation. Finally, the DGTS synthesis pathway seems to compete with PC synthesis via the Kennedy pathway but does not seem to be derived from the PC diacylglycerol backbone and therefore does not interfere with the eukaryotic pathway involved in galactolipid synthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Plant cell chloroplasts are bounded by a two-membrane envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are distinct from the chloroplast envelope, and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three compartments. A protocol is detailed for leaves of spinach, Arabidopsis or pea., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Chloroplasts are specific organelles of plant cells dedicated to photosynthesis and delimited by a two-membrane chloroplast envelope. Their photosynthetic function is based on the development of an operational large internal membrane network, called the thylakoids, and on enzymatic processes present in the chloroplast matrix, called the stroma. Thylakoid membranes are clearly different from the chloroplast envelope and their biogenesis is dependent on biosynthetic and transport activities specific of the chloroplast envelope. Starting with the isolation of intact chloroplasts, the method presents the separation by differential centrifugation of the three main compartments of the chloroplast: the stroma, the thylakoids, and the chloroplast envelope. Three different protocols are provided, adapted for starting leaves of spinach, Arabidopsis, and pea.