From chloroplasts to photosystems: in situ scanning force microscopy on intact thylakoid membranes

Abstract

Envelope-free chloroplasts were imaged in situ by contact and tapping mode scanning force microscopy at a lateral resolution of 3-5 nm and vertical resolution of approximately 0.3 nm. The images of the intact thylakoids revealed detailed structural features of their surface, including individual protein complexes over stroma, grana margin and grana-end membrane domains. Structural and immunogold-assisted assignment of two of these complexes, photosystem I (PS I) and ATP synthase, allowed direct determination of their surface density, which, for both, was found to be highest in grana margins. Surface rearrangements and pigment- protein complex redistribution associated with salt-induced membrane unstacking were followed on native, hydrated specimens. Unstacking was accompanied by a substantial increase in grana diameter and, eventually, led to their merging with the stroma lamellae. Concomitantly, PS IIalpha effective antenna size decreased by 21% and the mean size of membrane particles increased substantially, consistent with attachment of mobile light-harvesting complex II to PS I. The ability to image intact photosynthetic membranes at molecular resolution, as demonstrated here, opens up new vistas to investigate thylakoid structure and function.

Contrasting effect of dark-chilling on chloroplast structure and arrangement of chlorophyll–protein complexes in pea and tomato: plants with a different susceptibility to non-freezing temperature

“…6c vs. d). These data may be related to those obtained by the use of atomic force microscopy (Kaftan et al 2002) indicating that due to Mg 2+ -depletion the unstacking of thylakoids starts at the stromal-exposed edges of the appressed grana domains and leads to their merging with the stroma lamellae. Grana diameter may increase due to the unstacking process, however, the grana height remains relatively constant (Kaftan et al 2002).…”

“…These data may be related to those obtained by the use of atomic force microscopy (Kaftan et al 2002) indicating that due to Mg 2+ -depletion the unstacking of thylakoids starts at the stromal-exposed edges of the appressed grana domains and leads to their merging with the stroma lamellae. Grana diameter may increase due to the unstacking process, however, the grana height remains relatively constant (Kaftan et al 2002). Thus, merging of the edges of grana membranes to stroma lamellae and vice versa could proceed independently of vertical interactions between membranes in central parts of grana domains, as observed in darkchilled pea and tomato leaves (Fig.…”

“…At low Mg 2+ concentrations isolated chloroplasts do not exhibit the characteristic stacked thylakoids and PSII and PSI are homogeneously distributed within thylakoid membranes, whereas at higher cation concentrations the segregation of the photosystems and restacking of grana are observed (KirchhoV et al 2000(KirchhoV et al , 2004 and references therein; Kaftan et al 2002). Therefore, in order to conWrm that the main source of Chl Xuorescence within thylakoid regions comes from PSII/LHCII, a control experiment concerning the Mg 2+ -eVect on the distribution of Chl Xuorescence in isolated intact pea chloroplasts was performed (Fig.…”

Three-Dimensional Organization of Higher-Plant Chloroplast Thylakoid Membranes Revealed by Electron Tomography

“…One may also use electron microscopy techniques to determine the organization of photosynthetic complexes and supercomplexes in the thylakoid membranes under different conditions of illumination (Boekema et al, 2000;Ford et al, 2002;Kirchhoff et al, 2004). The information obtained from such analyses can be combined with data derived from other high-resolution imaging methods, such as atomic force microscopy (Kaftan et al, 2002), as well as from x-ray crystallography to provide a detailed picture of the macroscopic and microscopic makeup of the photosynthetic apparatus, which can then be correlated to its activity and light adaptability.…”

The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains

“…Interestingly, these dimensions are within the order of magnitude reported for lipid rafts in eukaryotic systems, ranging from <70 to 300 nm (Munro, 2003;Eggeling et al, 2009). However, they are significantly smaller than grana membranes in higher plant thylakoids with diameters of 300 to 500 nm (Mehta et al, 1999;Kaftan et al, 2002;Staehelin, 2003;Shimoni et al, 2005).…”