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CN-PAGE as a tool for separating pigment–protein complexes and studying their thermal stability in spruce and barley thylakoid membranes
Kurasová, Irena ; Svrčinová, K. ; Karlický, Václav ; Špunda, Vladimír
The central aim of our study was to develop a method for solubilization and native electrophoretic (colourless native polyacrylamide gel electrophoresis; CN-PAGE) separation of pigment–protein complexes (PPCs) embedded in thylakoid membranes (tBMs) isolated from spruce. Subsequently, we focused on studying the effect of temperature on the composition and PPC stability of two different species: barley and spruce. We found that the mild detergent n-dodecyl β-D-maltoside (β-DM) is suitable for PPC solubilization of spruce tBMs, but longer solubilization and a higher ratio of detergent to total chlorophyll are needed for spruce than are needed for barley. We also unified CN-PAGE protocols to optimize the separation of spruce and barley PPCs that resulted in the separation of photosystem I (PSI) and photosystem II (PSII) supercomplexes (SCs), PSI and PSII core dimers, PSII core monomers, trimeric and monomeric light-harvesting complexes of PSII, and bands with free pigments. Studying the effect of elevated temperature on PPCs using CN-PAGE revealed different thermal stability of PPCs in spruce and barley tBMs. Pronounced PPCs changes were observed at temperatures at or above 40°C. We observed partial disappearance of PSII SCs bands at 44°C in barley and at 52°C in spruce. In addition, spruce PSI SCs exhibited slightly higher thermal stability than did barley PSI SCs. The increased thermal stability of spruce tBMS in comparison to that of barley tBM was also confirmed by the circular dichroism spectra of isolated tBMs at different temperatures (Karlický et al. 2015).
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The thermostability of photosystem II photochemistry is related to maintenance of thylakoid membranes organization
Karlický, Václav ; Kurasová, I. ; Špunda, Vladimír
For higher plant photosynthetic reactions, responses to the temperature changes are important, particularly if we consider global warming and the increasing frequency of extreme temperature fluctuations. High temperature stress decreases photosynthetic assimilation through the inactivation of photosystem II (PSII), the most heat-sensitive component of the oxygen-evolving complex. We have recently found higher thermostability of spruce PSII photochemistry compared to such control plants as Arabidopsis species and barley. In this work, we have therefore attempted to describe the causes of this effect on the level of the organization of pigment–protein complexes (PPCs) in spruce thylakoid membranes using circular dichroism (CD) spectroscopy. We have confirmed higher maximum efficiency of PSII photochemistry (FV/FM) for spruce needles in comparison to barley leaves. Temperature-dependent CD spectra have also demonstrated higher (by about 6°C) PSII thermostability of chiral macro-organization of PPCs in spruce thylakoid membranes compared to those in barley. However, thermal disruption of PPCs did not reveal significant differences. Our results demonstrate that the stability of PSII macro-organization in different plant species correlates with the thermostability of PSII photochemistry in intact needles/leaves and so effective PSII photochemistry is related to the maintenance of PSII macro-organization under high temperature stress.
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Response of epidermal blue-green fluorescence emission from Barley leaves to uv radiation stress
Karlický, Václav ; Nezval, J. ; Štroch, Michal ; Špunda, Vladimír
Blue-green fl uorescence (BGF) is emitted by plant tissue when excited by UV radiation. Th e emission spectrum of BGF varies with plant species and is aff ected by the physiological status of the plant. BGF has been attributed mainly to cell wall-bound ferulic acid, although a contribution from fl avonoids and other simple phenols has also been suggested. Th e BGF signal is of interest due to its potential application in the remote sensing of plant stress: such techniques use ratios of blue and green to chlorophyll red and far-red fl uorescence emission intensities to estimate plant stress. In this work we investigated the response of BGF to UV-radiation stress and characterized changes in the shape of BGF emission spectra. We found these changes to be more signifi cant during the acclimation to UV-B radiation in comparison with UV-A or high irradiance of PAR. Furthermore, the spectral changes in BGF emission were due to a decline of fl uorescence at a wavelength of 435 nm compared with bands around 455 nm and 520 nm. In view of this, we have tried to distinguish individual fl uorescent components using more detailed fl uorescence analysis and to explain the causes of these spectral changes.
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