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

Ondřej Dlouhý,Janez Plavec,Győző Garab,Uroš Javornik,Václav Karlický,Vladimír Špunda,Ottó Zsiros,Primož Šket

doi:10.3390/cells10092354

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.

Highlights

In plants, the light reactions of photosynthesis occur in chloroplast thylakoid membranes (TMs), flattened lipid vesicles which separate the inner luminal and the outer, stroma-side aqueous phases
The 31P-NMR spectra of isolated granum and stroma thylakoid membranes recorded Cells 2021,a1t0,523°5C4 revealed that the signals originated from several different chemical environments5 of 16 of the phosphorous nucleus (Figure 1)
In this work we have shown that the granum and stroma TMs exhibit marked lipidphase polymorphisms, which are very similar to each other and to the intact TMs: in addition to the bilayer, they exhibit an HII phase and two isotropic phases

Summary

Introduction

The light reactions of photosynthesis occur in chloroplast thylakoid membranes (TMs), flattened lipid vesicles which separate the inner luminal and the outer, stroma-side aqueous phases These membranes embed the two photosystems (PSs) PSII and PSI, containing the photochemical reaction center core complexes and their lightharvesting antenna proteins (LHCII and LHCI, respectively), the cytochrome b6f complex and some additional components of the electron transport system, and the ATP-synthase. MGDG—similar to other non-bilayer lipid species in different biological membranes [6,7,8]—prefers to adopt nonlamellar or non-bilayer phases, such as the inverted hexagonal (HII), isotropic, and cubic phases [4,9] This holds true for total lipid mixtures of TMs, which are unable to maintain a stable bilayer phase under physiologically relevant conditions [4]; instead, they form stalks, non-bilayer molecular assemblies [10]. It has been shown that the addition of MGDG to loosely stacked lipid-LHCII membranes substantially increase their structural flexibility, and, their ability to undergo light-induced reversible reorganizations [14]—demonstrating that lipids with a high non-bilayer propensity lend the membranes additional structural plasticity

Methods

Results

Discussion

Conclusion