Differential Polarization Imaging of Plant Cells. Mapping the Anisotropy of Cell Walls and Chloroplasts

Modern light microscopy imaging techniques have substantially advanced our knowledge about the ultrastructure of plant cells and their organelles. Laser-scanning microscopy and digital light microscopy imaging techniques, in general-in addition to their high sensitivity, fast data acquisition, and great versatility of 2D-4D image analyses-also opened the technical possibilities to combine microscopy imaging with spectroscopic measurements. In this review, we focus our attention on differential polarization (DP) imaging techniques and on their applications on plant cell walls and chloroplasts, and show how these techniques provided unique and quantitative information on the anisotropic molecular organization of plant cell constituents: (i) We briefly describe how laser-scanning microscopes (LSMs) and the enhanced-resolution Re-scan Confocal Microscope (RCM of Confocal.nl Ltd. Amsterdam, Netherlands) can be equipped with DP attachments-making them capable of measuring different polarization spectroscopy parameters, parallel with the 'conventional' intensity imaging. (ii) We show examples of different faces of the strong anisotropic molecular organization of chloroplast thylakoid membranes. (iii) We illustrate the use of DP imaging of cell walls from a variety of wood samples and demonstrate the use of quantitative analysis. (iv) Finally, we outline the perspectives of further technical developments of micro-spectropolarimetry imaging and its use in plant cell studies

Far-red fluorescence:A direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching

AbstractTime-resolved fluorescence on oligomers of the main light-harvesting complex from higher plants indicate that in vitro oligomerization leads to the formation of a weakly coupled inter-trimer chlorophyll–chlorophyll (Chl) exciton state which converts in tens of ps into a state which is spectrally broad and has a strongly far-red enhanced fluorescence spectrum. Both its lifetime and spectrum show striking similarity with a 400ps fluorescence component appearing in intact leaves of Arabidopsis when non-photochemical quenching (NPQ) is induced. The fluorescence components with high far-red/red ratio are thus a characteristic marker for NPQ conditions in vivo. The far-red emitting state is shown to be an emissive Chl–Chl charge transfer state which plays a crucial part in the quenching

Fabrication, characterization and photoelectrochemical behavior of Fe2TiO5 screen printed thick films

Pseudobrookite paste was composed of a mixture of starting nanopowders of hematite (α-Fe2O3) and anatase (TiO2) in the molar ratio 1:1.5, organic vehicle and glass frit. The paste was screen printed on on fluorine-doped tin oxide (FTO) glass substrate using screen printing technology. Structural, morphological and optical studies have been carried out using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and transmission electron microscopy (TEM). The photo-electrochemical performance of Fe2TiO5 screen printed thick film was examined under xenon lamp illumination in 1 M NaOH electrolyte

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023 Å− 1, corresponding to spatially and statistically averaged repeat distance values of about 275–330 Å. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D2O in dim laboratory light or transpired with D2O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 Å, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy

Modifications in parenchyma cell wall structure related to stem twining in monocotyledonous liana Dioscorea balcanica Košanin

Anatomical adaptation of liana plants includes structural changes in cell walls of different tissues: fibers, vessel elements and tracheids. The contribution of parenchyma cells to stem twining is mostly unknown. Plants control the orientation and alignment of cellulose fibrils during the deposition in the cell walls with high precision, creating required anisotropy of the cell wall. Our aim was to determine possible changes in cellulose fibrils orientation and structural order in stem parenchyma cell walls related to stem twinning in liana plants. We applied different microscopy techniques: light microscopy, scanning electron microscopy and differential polarization laser scanning microscopy (DP-LSM) for fluorescence detected linear dichroism imaging (FDLD), on stem cross sections of straight and twisted internodes of monocotyledonous liana Dioscorea balcanica. Histochemical analysis showed no difference in parenchyma cell wall structure between straight and twisted internodes. Also, no difference in ‘‘cellulose fiber order’’ in parenchyma cell walls related to stem twining was found by FDLD microscopy. However, SEM micrographs suggested the difference in cellulose microfibril orientation in secondary cell walls of parenchyma cells related to stem twining. Our results indicate that adaptations to stem twining in liana plants involve modifications in cellulose microfibril orientation in parenchyma cell walls. Although the orientation of cellulose microfibrils dictates, among other properties, cell shape, living stem parenchyma cells in D. balcanica retain their shape regardless of stem twining, which is possibly enabled by retaining ‘‘cellulose fibril order’’

Fluorescence-Detected Linear Dichroism of Wood Cell Walls in Juvenile Serbian Spruce: Estimation of Compression Wood Severity.

Fluorescence-detected linear dichroism (FDLD) microscopy provides observation of structural order in a microscopic sample and its expression in numerical terms, enabling both quantitative and qualitative comparison among different samples. We applied FDLD microscopy to compare the distribution and alignment of cellulose fibrils in cell walls of compression wood (CW) and normal wood (NW) on stem cross-sections of juvenile Picea omorika trees. Our data indicate a decrease in cellulose fibril order in CW compared with NW. Radial and tangential walls differ considerably in both NW and CW. In radial walls, cellulose fibril order shows a gradual decrease from NW to severe CW, in line with the increase in CW severity. This indicates that FDLD analysis of cellulose fibril order in radial cell walls is a valuable method for estimation of CW severity

Ultrafast excitation quenching by the oxidized photosystem II reaction center

Photosystem II (PSII) is the pigment-protein complex driving the photoinduced oxidation of water and reduction of plastoquinone in all oxygenic photosynthetic organisms. Excitations in the antenna chlorophylls are photochemically trapped in the reaction center (RC) producing the chlorophyll-pheophytin radical ion pair P+ Pheo(-). When electron donation from water is inhibited, the oxidized RC chlorophyll P+ acts as an excitation quencher, but knowledge on the kinetics of quenching is limited. Here, we used femtosecond transient absorption spectroscopy to compare the excitation dynamics of PSII with neutral and oxidized RC (P+). We find that equilibration in the core antenna has a major lifetime of about 300 fs, irrespective of the RC redox state. Two-dimensional electronic spectroscopy revealed additional slower energy equilibration occurring on timescales of 3-5 ps, concurrent with excitation trapping. The kinetics of PSII with open RC can be described well with previously proposed models according to which the radical pair P+ Pheo(-) is populated with a main lifetime of about 40 ps, which is primarily determined by energy transfer between the core antenna and the RC chlorophylls. Yet, in PSII with oxidized RC (P+), fast excitation quenching was observed with decay lifetimes as short as 3 ps and an average decay lifetime of about 90 ps, which is shorter than the excited-state lifetime of PSII with open RC. The underlying mechanism of this extremely fast quenching prompts further investigation. Published under an exclusive license by AIP Publishing

Carotenoids are essential for the assembly of cyanobacterial photosynthetic complexes.

In photosynthetic organisms, carotenoids (carotenes and xanthophylls) are important for light harvesting, photoprotection and structural stability of a variety of pigment-protein complexes. Here, we investigated the consequences of altered carotenoid composition for the functional organization of photosynthetic complexes in wild-type and various mutant strains of the cyanobacterium Synechocystis sp. PCC 6803. Although it is generally accepted that xanthophylls do not play a role in cyanobacterial photosynthesis in low-light conditions, we have found that the absence of xanthophylls leads to reduced oligomerization of photosystems I and II. This is remarkable because these complexes do not bind xanthophylls. Oligomerization is even more disturbed in crtH mutant cells, which show limited carotenoid synthesis; in these cells also the phycobilisomes are distorted despite the fact that these extramembranous light-harvesting complexes do not contain carotenoids. The number of phycocyanin rods connected to the phycobilisome core is strongly reduced leading to high amounts of unattached phycocyanin units. In the absence of carotenoids the overall organization of the thylakoid membranes is disturbed: Photosystem II is not formed, photosystem I hardly oligomerizes and the assembly of phycobilisomes remains incomplete. These data underline the importance of carotenoids in the structural and functional organization of the cyanobacterial photosynthetic machinery

Correction to: Parenchyma cell wall structure in twining stem of Dioscorea balcanica (vol 24, pg 4653, 2017)

In the original publication of the article, one of the project numbers was omitted in the Acknowledgments. The correct version is provided below.Original publication: [http://cer.ihtm.bg.ac.rs/handle/123456789/2090

Non-Photochemical Quenching and Energy Dissipation in Plants, Algae and Cyanobacteria

XXXVIII, 649 p. 170 illus., 88 illus. in color.on