• Publication type
• Editorial MeSH

The photosynthetic performance of crop plants under a variety of environmental factors and stress conditions, at the fundamental level, depends largely on the organization and structural flexibility of thylakoid membranes. These highly organized membranes accommodate virtually all protein complexes and additional compounds carrying out the light reactions of photosynthesis. Most regulatory mechanisms fine-tuning the photosynthetic functions affect the organization of thylakoid membranes at different levels of the structural complexity. In order to monitor these reorganizations, non-invasive techniques are of special value. On the mesoscopic scale, small-angle neutron scattering (SANS) has been shown to deliver statistically and spatially averaged information on the periodic organization of the thylakoid membranes in vivo and/or, in isolated thylakoids, under physiologically relevant conditions, without fixation or staining. More importantly, SANS investigations have revealed rapid reversible reorganizations on the timescale of several seconds and minutes. In this paper, we give a short introduction into the basics of SANS technique, advantages and limitations, and briefly overview recent advances and potential applications of this technique in the physiology and biotechnology of crop plants. We also discuss future perspectives of neutron crystallography and different neutron scattering techniques, which are anticipated to become more accessible and of more use in photosynthesis research at new facilities with higher fluxes and innovative instrumentation.

• Keywords
• Macro-organization, Neutron scattering, Regulatory mechanisms, Structural flexibility, Thylakoid membrane,
• MeSH
• Photosynthesis * MeSH
• Scattering, Small Angle MeSH
• Neutrons MeSH
• Thylakoids * metabolism   MeSH
• Publication type
• Journal Article MeSH

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 (HII) 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 HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII 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,
• MeSH
• Circular Dichroism methods   MeSH
• Microscopy, Electron methods   MeSH
• Photosynthesis genetics   MeSH
• Lipids genetics   MeSH
• Magnetic Resonance Spectroscopy methods   MeSH
• Thylakoids genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipids MeSH

Non-photochemical quenching (NPQ) is an important photoprotective mechanism in plants and algae. Although the process is extensively studied, little is known about its relationship with ultrastructural changes of the thylakoid membranes. In order to better understand this relationship, we studied the effects of illumination on the organization of thylakoid membranes in Monstera deliciosa leaves. This evergreen species is known to exhibit very large NPQ and to possess giant grana with dozens of stacked thylakoids. It is thus ideally suited for small-angle neutron scattering measurements (SANS)-a non-invasive technique, which is capable of providing spatially and statistically averaged information on the periodicity of the thylakoid membranes and their rapid reorganizations in vivo. We show that NPQ-inducing illumination causes a strong decrease in the periodic order of granum thylakoid membranes. Development of NPQ and light-induced ultrastructural changes, as well as the relaxation processes, follow similar kinetic patterns. Surprisingly, whereas NPQ is suppressed by diuron, it impedes only the relaxation of the structural changes and not its formation, suggesting that structural changes do not cause but enable NPQ. We also demonstrate that the diminishment of SANS peak does not originate from light-induced redistribution and reorientation of chloroplasts inside the cells.

• Keywords
• chloroplast thylakoid membranes, grana, lamellar repeat distance, non-photochemical quenching, small-angle neutron scattering,
• MeSH
• Araceae chemistry   MeSH
• X-Ray Diffraction * MeSH
• Intracellular Membranes chemistry   metabolism   MeSH
• Plant Leaves chemistry   MeSH
• Scattering, Small Angle * MeSH
• Neutrons * MeSH
• Light MeSH
• Thylakoids genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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.

• MeSH
• Spectrometry, Fluorescence * methods   MeSH
• Phosphorus Isotopes * MeSH
• Catalysis MeSH
• Hydrogen-Ion Concentration MeSH
• Lipids chemistry   MeSH
• Magnetic Resonance Spectroscopy * methods   MeSH
• Thylakoids chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphorus Isotopes * MeSH
• Lipids MeSH