Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. II. Structure and Functions

Abstract

In Part I, by using ³¹P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (H_II) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our ³¹P-NMR spectroscopy. Signatures of H_II-phase could not be discerned with small-angle X-ray scattering-but traces of H_II structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

Keywords: SAXS; bilayer; chlorophyll fluorescence; cryo-electron-tomography; electron microscopy; membrane energization; membrane networks; non-bilayer lipid phases; violaxanthin de-epoxidase.

Figure 1

Circular dichroism (CD) of lipase-treated (a,b) and thermally treated (c,d) granum (a,c) and stroma (b,d) thylakoid membranes (TMs). The activity of the wheat germ lipase (a,b) was 0 U (blue curves) and 10 U for granum and 5 U for stroma TMs (red curves). The effect of temperature (c,d) was recorded at 5 °C (blue curves) and 25 °C (red curves). Four individual batches of granum TMs were measured and averaged. We used two different batches of stroma TMs for measuring the effects of thermal- and lipase treatments.

Figure 2

The 77 K fluorescence emission spectra of lipase-treated granum (a) and stroma (b) thylakoid membranes. Excitation at 435 nm; spectra are normalized at 685 nm ((a); emission of PSII) and at 730 nm ((b); emission of PSI). All experiments were performed at 5 °C on dark-adapted samples. Four individual batches of granum TMs were measured and averaged. A single batch of stroma TMs was used. (For further details, see Methods.).

Figure 3

Immunoblotting of a recombinant VDE (positive control, with a MW slightly affected because of the His-tag) and VDE in intact TMs and in granum and stroma membrane particles. Proteins were separated and VDE detected using SDS-PAGE/western blotting. Total Chl contents loaded in the wells were 2 and 20 µg for intact TMs subchloroplast particles, respectively. MW of the bands, 49.5 kDa (upper) and 44.0 kDa (lower). Images of two equally treated blots were processed for granum and stroma TMs. Exposure times: 38 s (intact and granum TMs) and 30 s (stroma TMs).

Figure 4

Small angle X-ray scattering (SAXS) of granum (blue) and stroma (red) TMs. Monochromatized and collimated Cu Kα radiation, with a 0.1542 nm wavelength was used. Scattering pattern was recorded in the range of 0.02–5 nm⁻¹. Total measurement time was 12 h for each sample at three geometries. For an easier comparison, the SAXS signal of granum TMs was multiplied by a factor of 10. (For further details, see Methods.).

Figure 5

Freeze-fracture electron microscopy images of granum (a,b) and stroma (c,d) TMs; images of different regions with different magnifications; insets in (a,d), protein rich regions; P, W, and NL in (b) stand for regions dominated by proteins, water and non-bilayer lipid phase.

Figure 6

Scanning electron microscopy (SEM) images of granum (a) and stroma (b) TMs, captured using accelerating voltages of 2 and 15 kV, respectively, of the EM. Inset in (a), an example of two apparently fused grana.

Figure 7

Surface views of reconstructed tomograms of granum and stroma thylakoid membranes. Panels (a,c) represent the top views of the granum and stroma thylakoid membranes, respectively. Panels (b,d) represent their corresponding tilted views. Cross-sections of the membranes of individual thylakoids, which are perpendicular to the X-Y image plane, are marked in cyan, magenta, and blue, small membrane vesicles are depicted in yellow. Membrane vesicles, which are inside the thylakoids, are shown in red. A tubular assembly in the stromal thylakoid membrane (c,d, in salmon) is supposedly formed by lipids in the H_II phase. Scale bars, 50 nm.