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

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.     

Inhibition of apoptosis in human laryngeal cancer cells by E6 and E7 oncoproteins of human papillomavirus 16

The carcinogenesis of human papillomaviruses type 16 (HPV-16) is mainly due to its two oncoproteins, E6 and E7. Their carcinogenic features in term of their relationship with Bcl-2 family are still unclear. We thus aimed to analyze the expression of Bcl-2 family members, Bcl-2, Bax, and Bak in laryngeal cancer cells transfected with the E6 or E7 and to determine the sensitivity of these cells to apoptotic stimuli. We employed two human laryngeal cancer cell lines, UMSCC12 and UMSCC11A in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1. We found that E6 and E7 inhibited apoptosis induced by TNF-alpha/CHX in both UMSCC11A and UMSCC12 cells, enhanced the stability of Bcl-2 protein and increased the degradation of Bak protein. Furthermore, it was found that HPV-16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer. The alteration of Bak by E6 and E7 was not through the influence on the Bak promoter, as the luciferase assay showed that neither E6 nor E7 changed the Bak promoter activity. We conclude that the evasion of apoptosis mediated by HPV-16 E6 and E7 is associated with increased Bcl-2 and decreased Bak in laryngeal carcinogenesis and that the decreased level of Bak by E6 and E7 is not caused by the regulation of the Bak promoter but by reducing its protein stability.     

The quantum efficiency of photosystem II and its relation to non-photochemical quenching of chlorophyll fluorescence; the effect of measuring-and growth temperature

The relation between the quantum yield of oxygen evolution of open photosystem II reactions centers (_p), calculated according to Weis and Berry (1987), and non-photochemical quenching of chlorophyll fluorescence of plants grown at 19°C and 7°C was measured at 19°C and 7°C. The relation was linear when measured at 19°C, but when measured at 7°C a deviation from linearity was observed at high values of non-photochemical quenching. In plants grown at 7°C this deviation occurred at higher values of non-photochemical quenching than in plants grown at 19°C. The deviations at high light intensity and low temperature are ascribed to an increase in an inhibition-related, non-photochemical quenching component (q_I).The relation between the quantum yield of excitation capture of open photosystem II reaction centers (_exe), calculated according to Genty et al. (1989), and non-photochemical quenching of chlorophyll fluorescence was found to be non-linear and was neither influenced by growth temperature nor by measuring temperature.At high PFD the efficiency of overall steady state electron transport measured by oxygen-evolution, correlated well with the product of q _N and the efficiency of excitation capture (_exe) but it deviated at low PFD. The deviations at low light intensity are attributed to the different populations of chloroplasts measured by gas exchange and chlorophyll fluorescence and to the light gradient within the leaf.Abbreviations F₀ basic fluorescence - F₀ basic fluorescence, thylakoid in energized state - F_m maximal fluorescence - F_m maximum fluorescence in energized state - F_s steady state fluorescence - F_v maximal variable fluorescence - PFD photon flux density - PS II_rc Photosystem II reaction center - q_F0 quenching of basic fluorescence - q_E energy related quenching - q_N non-photochemical quenching:-q_f-total quenching - q_I inhibition-related quenching - q_p photochemical quenching - q_r quenching due to state transition - R_d dark respiration - _p PS II efficiency of excitation capture of open PS II_rc - _pe extrapolated minimal value of _p - _p0 extrapolated maximal value of _p - _si quantum efficiency of linear electron transport, calculated from gas exchange measurements based on incident light - _sf quantum efficiency of linear electron transport, calculated from fluorescence measurements, based on incident measuring light     

Lipid composition in leaves of cucumber genotypes as affected by different temperature regimes and grafting

The lipid composition of leaves has been investigated in different genotypes of cucumber ( Cucumis sativus L.), which differ in temperature requirement for cultivation. In addition the effects of hardening by low but non-chilling temperature, soil heating and grafting (on the chilling-resistant C. ficifolia L.) on lipid composition have been studied. Content and composition of phospholipids and sterols were determined as well as phospholipid/sterol ratio, and fatty acid composition of total lipids and the different phospholipids.
The effects of genetic differentiation and of the various culture treatments on lipid composition of the leaves were very different. Genetic differentiation was evident as higher levels of Iinolenic acid in several phospholipids in the more cold-tolerant cultivars. Hardening the plants by low temperature resulted in a higher phospholipid level (especially phosphatidyl choline), more unsaturated phospholipid, and lowering of the sterol/phiospholipid ratio, all properties which may contribute to a higher membrane fluidity and lower growth temperature limit. Soil healing reduced the phospholipid level of the leaves slightly, and a higher content of 3- trans -hexadece-noic acid in phosphatidyl glycerol was observed. Grafting cucumber on the cold-resistant rootstock of C. ficifolia also raised the level of trans -hexadecenoic acid in phosphatidyl glycerol. The role of this fatty acid in the functioning of the chloroplast is discussed.     

Temperature-induced alterations of in vivo chlorophyll a fluorescence induction in cucumber as affected by DCMU

Induction of chlorophyll a fluorescence and photosynthesis as affected by temperature were measured in cucumber leaf discs. Abrupt changes of the maximal variable fluorescence, Fv(p), and photosynthesis were observed around 9° and 21°C when the temperature was decreased from 30° to 0°C. The temperature-dependent maximal fluorescence of DCMU-treated leaf discs showed a single change around 21°C. Temperature-induced chlorophyll a fluorescence alterations are discussed in relation to electron transport activity of the two photosystems and photosynthetic activity of the cucumber leaf discs.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Fm maximal fluorescence - Fv(p) maximal variable fluorescence - qE energy-dependent fluorescence quenching - qQ Qa-dependent fluorescence quenching     

Photoinhibition of photosystem II in vivo is preceded by down-regulation through light-induced acidification of the lumen: Consequences for the mechanism of photoinhibition in vivo

The mechanism of photoinhibition of photosystem II (PSII) was studied in intact leaf discs of Spinacia oleracea L. and detached leaves of Vigna unguiculata L. The leaf material was exposed to different photon flux densities (PFDs) for 100 min, while non-photochemical (q_N) and photochemical quenching (q_p) of chlorophyll fluorescence were monitored. The ‘energy’ and redox state of PSII were manipulated quite independently of the PFD by application of different temperatures (5–20° C), [CO₂] and [O₂] at different PFDs. A linear or curvilinear relationship between q_p and photoinhibition of PSII was observed. When [CO₂] and [O₂] were both low (30 μl · l^?1 and 2%, respectively), PSII was less susceptible at a given q_p than at ambient or higher [CO₂] and photoinhibition became only substantial when q_p decreased below 0.3. When high levels of energy-dependent quenching (q_E) (between 0.6 and 0.8) were reached, a further increase of the PFD or a further decrease of the metabolic demand for ATP and NADPH led to a shift from q_E to photoinhibitory quenching (q_I). This shift indicated that photoinhibition was preceded by down-regulation through light-induced acidification of the lumen. We propose that photoinhibition took place in the centers down-regulated by q_E. The shift from q_E to q_I occurred concomitant with q_P decreasing to zero. The results clearly show that photoinhibition does not primarily depend on the photon density in the antenna, but that photoinhibition depends on the energy state of the membrane in combination with the redox balance of PSII. The results are discussed with regard to the mechanism of photoinhibition of PSII, considering, in particular, effects of light-induced acidification on the donor side of PSII. Interestingly, cold-acclimation of spinach leaves did not significantly affect the relationship between q_P, q_E and photoinhibition of PSII at low temperature.     

Chilling-induced photoinhibition in nine isolates of Valonia utricularis (Chlorophyta) from different climate regions

Chilling induced inhibition of photosynthesis was studied in nine isolates of the marine tropical to warm-temperate green macrophyte Valonia utricularis (Roth) C. Agardh. According to their temperature requirements for growth and survival, the isolates belong to a cold-tolerant Atlantic/Mediterranean group and a cold-sensitive Indo-west Pacific group. After 5 hours exposure to 5 degrees C under moderate light, all isolates experienced similar substantial photoinhibition, which approached steady state levels after a decline in Fv/Fm to about 40% of the initial values. After return to optimal temperature and dim light conditions, Fv/Fm values increased with biphasic kinetics. A fast phase with half-life times of less than 30 minutes (dynamic photoinhibition) was followed by a slow phase lasting a few hours, indicating repair of photodamaged PSII reaction centres (chronic photoinhibition). In the Atlantic/Mediterranean isolates the fast phase accounted for more than 80 % of the recovery response, showing that these isolates were able to cope with the applied low temperature stress by down-regulating their PSII reaction centres. In contrast, the two isolates from the Seychelles were predominantly photodamaged. In a second experiment, three isolates (Corsica, Seychelles, Japan) were exposed to a similar relative amount of cold stress (0, 10, 15 degrees C, respectively). The Japanese isolate and the isolate from the Seychelles showed significantly less inhibition compared to 5 degrees C exposure, but no significant difference was found in the Corsican isolate. However, the degree of low temperature stress had no significant influence on the relative contributions of dynamic and chronic photoinhibition. Only two of the seven investigated isolates had a lower final inhibition level when grown at sub-optimal temperatures than at optimal temperatures. However, all sub-optimally grown Atlantic/Mediterranean isolates exhibited faster recovery kinetics from chilling-induced photoinhibition than optimally grown plants. This is related to a faster recovery from chronic photoinhibition than to a higher relative contribution of dynamic photoinhibition. A specific role of the photoprotective pigments of the xanthophyll cycle, leading to an acclimation response in the Atlantic/Mediterranean isolates may be involved. We conclude that ecotypic differentiation in V. utricularis is mirrored in different degrees of susceptibility to low temperature stress.     

Photooxidative inhibition of nitrate reductase during chilling

The effect of a temperature close to the freezing point (chilling) on the nitrate reductase system of leaf discs of Cucumis sativus L. cv. Kleine Groene Scherpe was determined in the absence and presence of light. The capacity of leaf discs in the light (250 μE m⁻²s⁻¹) at 20°C to increase in vivo and in vitro nitrate reductase activity, was unaffected by chilling pretreatment in the dark, but 4 h of chilling pretreatment in the light (250 μE m⁻²s⁻¹) decreased the capacity to less than 50% of the unchilled control. The chilling inhibition of the capacity to increase nitrate reductase activity was of a photooxidative nature since it only occurred in the presence of light and oxygen. Plants grown at a low light intensity (65 μE m⁻²s⁻¹) lost 95% of their capacity to increase nitrate reductase activity, while plants grown at 195 μE m⁻²s⁻¹ retained 80% of their nitrate reducing capacity after 6 h chilling pretreatment in the 250 μE m⁻²s⁻¹ light. Previously induced nitrate reductase activity was also affected by light during chilling. A lag phase of 7 h preceded a fast phase of decrease in activity. Both in vivo and in vitro activity decreased to 15% of the control value after 18 h of chilling in the light. It is concluded that the induction mechanism of nitrate reductase is primarily affected by photooxidation during chilling. The decrease in nitrate reductase activity is attributed to a decrease in the amount of activity enzyme.     

Inhibition of chilling-induced photooxidative damage to leaves of Cucumis sativus L. by treatment with amino alcohols

The effect of pretreatment of cucumber (Cucumis sativus L.) roots with choline chloride or ethanolamine on leaf phospholipid composition and light-induced leaf damage during chilling was studied. Photooxidative chlorophyll degradation was similarly inhibited by both amino alcohols. The decrease of the chlorophyll a/chlorophyll b ratio and the increase of polyunsaturated-fatty-acid degradation during chilling in the light were equally inhibited by pretreatment with choline chloride or ethanolamine. Treatment with choline chloride and ethanolamine caused, respectively, 43% and 26% increases in the total phospholipid contents of the leaves. After treatment with choline chloride, the phosphatidylcholine content was higher than the content of phosphatidylethanolamine; the reverse was true after treatment with ethanolamine. The chlorophyll concentration increased less than the phospholipid concentration, resulting in a decreased chlorophyll/phospholipid ratio of treated leaves. During chilling in the light, degradation of phosphatidylcholine, ethanolamine and phosphatidyl glycerol occurred. Phosphatidyl glycerol was less sensitive than phosphatidylcholine and ethanolamine. The degradation was equally inhibited by pretreatment with either amino alcohol. Possible connections between the phospholipid content of leaf membranes and the inhibition of chilling-induced photooxidative leaf damage are discussed.Abbreviations CC choline chloride - Chl chlorophyll - EA ethanolamine - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PG phosphatidyl glycerol     

Cell death induced by ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid in anaplastic thyroid carcinoma cells is via a mitochondrial-mediated pathway

The chemical compound ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F), isolated from the Chinese herbal medicine plant Pteris semipinnata L, has been known to exert antitumor activity. However, the molecular mechanism of the action is not understood. In this study we demonstrated that apoptotic cell death induced by 5F in FRO cells was concentration- and time-dependent. The rapid increase in intracellular reactive oxygen species (ROS) levels was involved in the mechanism of cell death. c-Jun N-terminal kinase (JNK) activation and G2 block were related to cell death induced by 5F. Extracellular signal-related kinase (ERK) and p38 were also activated, but as survival signals in response to 5F treatment to counteract the induction of cell death. In the process of the induction of apoptotic cell death, Bax translocated into mitochondria, a reduction in Δψ _m was observed and a release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria into the cytosol occurred, indicating that cell death induced by 5F was through a mitochondrial-mediated pathway.