3-D modelling of chloroplast structure under (Mg2+) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking

Abstract

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.

Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid-Light-Harvesting Complex II Model Membranes

“…The formation of such stable lamellar membrane stacks has been confirmed by the results of the microscopy analyses, which include CLSM (Figure 9) and transmission electron microscopy ( Figure 10). Microscopy techniques, and in particular CLSM, show close similarities in the organization of natural chloroplast membranes and the model systems studied in this work (Rumak et al, 2010(Rumak et al, , 2012. In particular, the process of grana stack formation by the thylakoid membranes appeared to have its analog in the model system composed of the mixture of chloroplast lipids (MGDG with DGDG, 2:1) and nonphosphorylated and zeaxanthin-lacking LHCII isolated from dark-adapted leaves.…”

“…These merge only at the edge, but some ovals are not connected with the others ( Figure 9F). It might be suggested that the adjacent membranes in the L-LHCII-HL are weakly connected in a stack due to electrostatic and hydration repulsion between membranes (Rumak et al, 2010). The general view of the model revealed that relatively small L-LHCII-HL stacks are evenly arranged in all axes of three-dimensional space, forming a wide three-dimensional cluster ( Figure 9F).…”

“…Visualization of chlorophyll a fluorescence by confocal laser scanning microscopy (CLSM) enabled observation of the natural lipid membranes containing the chlorophyll-protein complexes (e.g., inside intact chloroplasts or leaves) without any fixation procedure and in situ (Mehta et al, 1999;Garstka et al, 2005Garstka et al, , 2007Hasegawa et al, 2010;Rumak et al, 2010Rumak et al, , 2012. Red fluorescence was attributed mainly to chlorophyll a species emitted at around 680 nm, because at room temperature the long-wavelength emission, above 700 nm, was rather weak (Mehta et al, 1999;Hasegawa et al, 2010).…”

“…Large numbers of fluorescence images were taken in different focal depths (Garstka et al, 2005(Garstka et al, , 2007Rumak et al, 2010Rumak et al, , 2012. This allowed the creation of computer-generated threedimensional structures in which the spatial layout of chlorophyll a fluorescence intensity was shown.…”

“…This indicates that these bilayers formed the continuous surface, where neighboring membranes fit tightly on a long distance. Since the stack of two adjacent thylakoid membranes, mostly containing LHCII as a protein component, is ;17 nm high Rumak et al, 2010), the stacks of L-LHCII should be composed from ;20 membrane pairs ( Figure 9E). A similar conclusion can be reached based on the diffractometrically determined periodicity parameter of the multibilayer, taking into account the relatively broad diffraction maxima observed in the case of the L-LHCII samples (Figure 2).…”