Lipid polymorphism in chloroplast thylakoid membranes \u2013 as revealed by 31P-NMR and time-resolved merocyanine fluorescence spectroscopy

Győző Garab,Henk Van As,Pieter De Waard,Parveen Akhtar,Janez Plavec,Vladimír Špunda,Václav Karlický,Christos Kotakis,Primož Šket,Petar H Lambrev,Zuzana Materová,László Vígh,Herbert Van Amerongen,Bettina Ughy,Uroš Javornik

doi:10.1038/s41598-017-13574-y

Abstract

Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.

Highlights

For the homogenous catalytic hydrogenation, thylakoid membranes were resuspended in 150 ml 50 mM HEPES buffer and supplemented with 0.8 M sorbitol
By using 31P-NMR and time-resolved MC540 fluorescence spectroscopy we provided additional experimental evidence on the polymorphic phase behaviour of the lipids in isolated spinach thylakoid membranes
With short lifetimes and small red shifts compared to the dye in the reaction medium, appear to originate from domains closer to the aqueous phase – one of them might originate from HII phase

Summary

Introduction

For the homogenous catalytic hydrogenation, thylakoid membranes were resuspended in 150 ml 50 mM HEPES buffer (pH 7.0) and supplemented with 0.8 M sorbitol. VDE, the key XC enzyme is activated at low pH58 and its functioning depends on the presence of non-bilayer lipid phase[73].

Results

Conclusion