Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll–protein complexes in bean thylakoid membranes

Abstract

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 degrees C and subsequent 3-h photoactivation under white light (200 mumol photons m(-2) s(-1)) at 22 degrees C. Although, no change in chlorophyll (Chl) content and Chl a/b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI-PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry (F(v)/F(m)) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.

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

“…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.…”

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

“…Steady-state Xuorescence spectra of isolated thylakoids at 25°C (298 K) and 120 K were determined as described previously (Garstka et al 2005). In order to determine the eVects of chilling and subsequent photoactivation on the relative contribution of speciWc CP complexes to the overall Xuorescence pattern, Xuorescence spectra of thylakoids at 120 K were normalized to the same area (100) under the spectrum (Andreeva et al 2003).…”

“…Thylakoid membranes and intact chloroplasts were prepared as described previously (Garstka et al 2005). Thylakoids were always freshly prepared before each experiments, kept on ice in the dark and used within 2 h for both electrophoresis and Xuorescence spectroscopy.…”

“…However, the mechanism involved in dark-chilling-induced chloroplast injury has still not been elucidated (Kratsch and Wise 2000). Garstka et al (2005) have reported that in CS bean dark-chilling-induced disturbance of chloroplast structure might be a consequence of rearrangements of CP supercomplexes-LHCI-PSI and LHCII trimers. These eVects were reversed to a large extent after post-chilling photoactivation, with the exception of LHCII, which remained partly in the aggregated form (Garstka et al 2005).…”