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 (H_II) 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 H_II 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.

Keywords: 31P-NMR; DEM—dynamic exchange model; HII phase; bilayer membrane; grana; isotropic phase; non-bilayer lipids; non-lamellar lipid phases; structural flexibility; thylakoid membranes.

Figure 1

³¹P-NMR spectra (a,c) and relative intensities (b,d) of isolated spinach granum (a,b) and stroma (c,d) thylakoid membranes at 5 °C. Average of (a) five spectra from three batches and (c) six spectra from five batches. Integrated areas of the component spectra (b,d) associated with the different lipid phases, relative to the overall integrated area; mean values ± SD.

Figure 2

³¹P-NMR spectra of granum thylakoid membranes in the absence (Control, blue curves) and presence (SP, red curves) of saturation pulses applied at different frequencies, as indicated by the arrows, at or close to the peak position of different phases: L, −12 ppm (a); H_II, 20 ppm (b); I₁, 2.8 ppm (c); I₂, 4.3 ppm (d). Each panel shows the deconvoluted component spectra and, in inset, the measured spectra. The measurements were performed on different batches; the spectra represent averages from two independent batches with similar polymorphisms; temperature, 5 °C.

Figure 3

³¹P-NMR spectra of stroma thylakoid membranes in the absence (Control, blue curves) and presence (SP, red curves) of saturation pulses applied at different frequencies, as indicated by the arrows, at or close to the peak position of different phases: L, −12 ppm (a); H_II, 20 ppm (b); I₁, 2.8 ppm (c) and I₂, 4.3 ppm (d). Each panel shows the deconvoluted component spectra and, in inset, the measured spectra, averages from two independent batches with similar spectral features; temperature, 5 °C.

Figure 4

³¹P-NMR spectra of granum thylakoid membranes at different temperatures (a) and their component spectra showing variations in L, the lamellar phase (b), H_II, the inverted hexagonal phase (c), and the two isotropic phases, I₁ (d) and I₂ (e). The experiments were performed by gradually increasing the temperature from 5 to 35 °C, with data acquisition times between 1 and 2 h. Four experiments on three independent batches, with similar polymorphic features, were averaged to improve the signal to noise ratio. The inset in panel a shows the isotropic region.

Figure 5

³¹P-NMR spectra of stroma thylakoid membranes at different temperatures (a) and their component spectra showing variations in L, the lamellar phase (b), H_II, the inverted hexagonal phase (c), and the two isotropic phases, I₁ (d) and I₂ (e). The experiments were performed by gradually increasing the temperature from 5 to 35 °C, with data acquisition times between 1 and 2 h. Two experiments on two independent batches, possessing similar spectra, were averaged. The inset in Panel a shows the isotropic region.

Figure 6

Effects of wheat-germ lipase treatments on the ³¹P-NMR spectra (a) of granum thylakoid membranes and on their component spectra: L, the lamellar phase (b), H_II, the inverted hexagonal phase (c), and the two isotropic phases, I₁ (d) and I₂ (e). The spectra represent averages of two measurements from two batches exhibiting similar spectra; recorded at 5 °C. The inset in panel a shows the isotropic region.

Figure 7

Effects of wheat-germ lipase treatments on the ³¹P-NMR spectra of stroma thylakoid membranes (a) and on their component spectra: L, the lamellar phase (b), H_II, the inverted hexagonal phase (c), and the two isotropic phases, I₁ (d) and I₂ (e). The spectra were recorded at 5 °C. The inset in panel a shows the isotropic region.