Lipid Polymorphism of the Subchloroplast—Granum and Stroma Thylakoid Membrane—Particles. I. 31P-NMR Spectroscopy

Abstract

Build-up of the energized state of thylakoid membranes and the synthesis of ATP are warranted by organizing their bulk lipids into a bilayer. However, the major lipid species of these membranes, monogalactosyldiacylglycerol, is a non-bilayer lipid. It has also been documented that fully functional thylakoid membranes, in addition to the bilayer, contain an inverted hexagonal (HII) phase and two isotropic phases. To shed light on the origin of these non-lamellar phases, we performed 31P-NMR spectroscopy experiments on sub-chloroplast particles of spinach: stacked, granum and unstacked, stroma thylakoid membranes. These membranes exhibited similar lipid polymorphism as the whole thylakoids. Saturation transfer experiments, applying saturating pulses at characteristic frequencies at 5 °C, provided evidence for distinct lipid phases—with component spectra very similar to those derived from mathematical deconvolution of the 31P-NMR spectra. Wheat-germ lipase treatment of samples selectively eliminated the phases exhibiting sharp isotropic peaks, suggesting easier accessibility of these lipids compared to the bilayer and the HII phases. Gradually increasing lipid exchanges were observed between the bilayer and the two isotropic phases upon gradually elevating the temperature from 5 to 35 °C, suggesting close connections between these lipid phases. Data concerning the identity and structural and functional roles of different lipid phases will be presented in the accompanying paper.

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes—Selective Susceptibilities to Different Lipases and Proteases

“…The addition of 20 U mL −1 WGL further suppressed the isotropic phases and diminished the L and H II phases; at 100 U mL −1 of WGL, all phases were eliminated ( Figure 6 ). These data are in good agreement with our previous results on isolated granum and stroma sub-chloroplast TM particles [ 38 , 39 ]. It is important to note that 5 U mL −1 WGL, even over long incubation times, did not disturb the structure and function of TMs; it did not increase the permeability of the membranes and exerted no effects on the excitation-energy distribution in the TMs, on the activity of PSII, nor the pattern and macro-organization of PPCs ( Figure S4 ).…”

“…The large amplitude of the 31 P-NMR band arising from the lipid phase with inverted hexagonal geometry showed that this phase, in intact TMs, contained large amounts of lipids (~40% of the detectable bulk lipids—see Figure S1b ). However, as previously tested on granum and stroma TM fractions [ 38 ] and on intact TMs (unpublished data), they did not adopt long-range order structures.…”

“…31 P-NMR spectrometry was performed similarly as in our previous study [ 38 ]. 31 P-NMR spectra were acquired with an Avance Neo 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA) with BBFO SmartProbe tuned at the phosphorus frequency.…”

“…In our earlier studies, we established correlations among the variations in the temperature, pH, and osmotic and ionic strengths of the medium as well as the activity of VDE and the phase behavior of the bulk lipid molecules [ 31 , 36 , 37 ]; we also clarified that isolated grana and stroma lamellae contain all the three non-bilayer phases [ 38 ]. Analyses of our structural and functional data strongly suggested that the isotropic phases originate from membrane fusions and junctions [ 39 ] and from VDE: lipid assemblies [ 31 ].…”

Overexpression the BnLACS9 could increase the chlorophyll and oil content in Brassica napus

“…Previous studies showed that the main lipid component of the thylakoid layer structure such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG) [ 2 , 35 , 36 ]. Therefore, we used thin-layer chromatography to analyze the lipid content in leaves.…”