The eVect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll-protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chillingsensitive (CS) tomato. Dark-chilling did not inXuence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a Xuorescence intensity and an increase of the ratio of Xuorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves. Chilling of pea leaves induced an increase of the relative contribution of LHCII and PSII Xuorescence. A substantial decrease of the LHCII/PSII Xuorescence accompanied by an increase of that from LHCI/PSI was observed in thylakoids from chilled tomato leaves; both were attenuated by photoactivation. Chlorophyll Xuorescence of bright grana discs in chloroplasts from dark-chilled leaves, detected by confocal laser scanning microscopy, was more condensed in pea but signiWcantly dispersed in tomato, compared with control samples. The chloroplast images from transmission-electron microscopy revealed that dark-chilling induced an increase of the degree of grana stacking only in pea chloroplasts. Analyses of O-J-D-I-P Xuorescence induction curves in leaves of CS tomato before and after recovery from chilling indicate changes in electron transport rates at acceptor-and donor side of PS II and an increase in antenna size. In CT pea leaves these eVects were absent, except for a small but irreversible eVect on PSII activity and antenna size. Thus, the diVerences in chloroplast structure between CS and CT plants, induced by dark-chilling are a consequence of diVerent thylakoid supercomplexes rearrangements.Keywords Chloroplast and thylakoid membrane structure · Chilling-sensitive · Chilling tolerant · Chlorophyll-protein complexes · Cation-induced thylakoid stacking · O-J-D-I-P chlorophyll Xuorescence · Confocal laser scanning microscopy · Dark-chilling stress · Pea · Tomato Dedicated to Prof. Zbigniew Kaniuga on the 25th anniversary of his initiation of studies on chilling-induced stress in plants.

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.

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