Two forms of sustained xanthophyll cycle-dependent energy dissipation in overwintering Euonymus kiautschovicus

Seasonal differences in PSII efficiency (F_v/F_m), the conversion state of the xanthophyll cycle (Z + A)/ (V + A + Z), and leaf adenylate status were investigated in Euonymus kiautschovicus. On very cold days in winter, F_v/F_m assessed directly in the field remained low and Z + A high throughout day and night in both sun and shade leaves. Pre-dawn transfer of leaves from subfreezing temperatures in the field to room temperature revealed that recovery (increases in F_v/F_m and conversion of Z + A to violaxanthin) consisted of one, rapid phase in shade leaves, whereas in sun leaves a rapid phase was followed by a slow phase requiring days. The pre-dawn ATP/ADP ratio, as well as that determined at midday, was similar when comparing overwintering leaves with those sampled in the summer, although pre-dawn levels of ATP + ADP were elevated in all leaves during winter relative to summer. After a natural transition to warmer days during the winter, pre-dawn F_v/F_m and Z + A in shade leaves had returned to values typical for summer, whereas in sun leaves F_v/F_m and Z + A levels remained intermediate between the cold day in winter and the summer day. Thus two distinct forms of sustained (Z + A)-dependent energy dissipation were identified based upon their differing characteristics. The form that was sustained on cold days but relaxed rapidly upon warming occurred in all leaves and may result from maintenance of a low lumenal pH responsible for the nocturnal engagement of (Z + A)-dependent thermal dissipation exclusively on very cold days in the winter. The form that was sustained even upon warming and correlated with slow Z + A to violaxanthin conversion occurred only in sun leaves and may represent a sustained engagement of (Z + A)-dependent energy dissipation associated with an altered PSII protein composition. In the latter, warm-sustained form, uncoupler or cycloheximide infiltration had no effect on the slow phase of recovery, but lincomycin infiltration inhibited the slow increase in F_v/F_m and the conversion of Z + A to violaxanthin.     

Mechanisms shaping the synergism of zeaxanthin and PsbS in photoprotective energy dissipation in the photosynthetic apparatus of plants

Safe operation of photosynthesis is vital to plants and is ensured by the activity of processes protecting chloroplasts against photo-damage. The harmless dissipation of excess excitation energy is considered to be the primary photoprotective mechanism and is most effective in the combined presence of PsbS protein and zeaxanthin, a xanthophyll accumulated in strong light as a result of the xanthophyll cycle. Here we address the problem of specific molecular mechanisms underlying the synergistic effect of zeaxanthin and PsbS. The experiments were conducted with Arabidopsis thaliana, using wild-type plants, mutants lacking PsbS (npq4), and mutants affected in the xanthophyll cycle (npq1), with the application of molecular spectroscopy and imaging techniques. The results lead to the conclusion that PsbS interferes with the formation of densely packed aggregates of thylakoid membrane proteins, thus allowing easy exchange and incorporation of xanthophyll cycle pigments into such structures. It was found that xanthophylls trapped within supramolecular structures, most likely in the interfacial protein region, determine their photophysical properties. The structures formed in the presence of violaxanthin are characterized by minimized dissipation of excitation energy. In contrast, the structures formed in the presence of zeaxanthin show enhanced excitation quenching, thus protecting the system against photo-damage.     

Duration of irradiation rather than quantity and frequency of high irradiance inhibits photosynthetic processes in the lichen <Emphasis Type="Italic">Lasallia pustulata</Emphasis>

Lichen thalli were exposed to 4 regimes differing in irradiance and duration of irradiation. Photosynthetic efficiency of thalli was monitored by chlorophyll fluorescence parameters and xanthophyll cycle analysis. Maximal quantum yield of photosystem 2 (F_V/F_M) decreased gradually with time in long-term treatment. The effect of additional short-term high irradiance (HI) treatment applied each 24 h was not significant. Nevertheless, short-term HI applied repeatedly on thalli kept in the dark led to a significant decrease of F_V/F_M. Non-photochemical quenching recorded during the long-term treatment corresponded to the content of zeaxanthin (Z). In short-term treatment, however, proportion of Z (and antheraxanthin) to total amount of xanthophyll cycle pigments recovered to the initial values every 24 h after each repeated short-term HI event in thalli kept in dark. Thus duration of irradiation rather than irradiance and frequency of HI events is important for a decrease in primary photosynthetic processes in wet thalli of Lasallia pustulata. Rapidly responding photoprotective mechanisms, such as conversion of xanthophyll cycle pigments, are involved mainly in short-term irradiation events, even at HI.     

Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a_D1 or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.     

Gloeobacter violaceus— investigation of an unusual photosynthetic apparatus. Absence of the long wavelength emission of photosystem I in 77 K fluorescence spectra

The deeply purple cyanobacterium Gloeobacter violaceus is subject of this investigation. It does not contain thylakoids, and the photosynthetic apparatus is located in the only membrane of the cell, the plasma membrane. Upon excitation with blue light, the 77 K fluorescence emission spectra of neither intact cells (excited with 427 nm) nor of the isolated plasma membrane (excited with 430 nm), show the expected long wavelength photosystem I emission characteristic for low energy chlorophylls. Maximal fluorescence emission was observed at 688 nm. independent on the excitation wavelength, 427 (430) nm blue light, exciting mainly chlorophyll, or 550 nm green light, exciting mainly phycoerythin. The ratio of P700 to chlorophyll was 175. O₂-evolution was 160 μmol mg_-1 chlorophyll h_-1 in saturating white light; the compensation point was reached at 6 μmol m₂ s_-1 in cultures grown at 25 μmol m₂ s_-1. Dark O₂ uptake was 50 μmol mg_-1 chlorophyll h_-1. During adaptation to increasing white light intensities Gloeobacter reduces the amount of phycocyanin and chlorophyll per cell and strongly increases the concentration of carotenoids relative to chlorophyll. The carotenoid concentration per cell increases with increasing light intensity. Apparently, part of the carotenoids is not located in the plasma membrane.     

Correlation between Photoinhibition Sensitivity and the Rates and Relative Extents of Xanthophyll Cycle De-epoxidation in Chlorina Mutants of Barley (Hordeum vulgare L.)

We compared photoinhibition sensitivity to high irradiance (HI) in wild-type barley (wt) and both its chlorina f ₁₀₄-nuclear gene mutant, that restricts chlorophyll (Chl) a and Chl b synthesis, and its f ₂-nuclear gene mutant, that inhibits all Chl b synthesis. Both F_v/F_m and _PS2 decreased more significantly in f ₂ than f ₁₀₄ and wt with duration of HI exposure. Chl degraded more rapidly in the f ₂ than in either f ₁₀₄ or wt. Most sensitivity to photoinhibition was exhibited for f ₂, whereas there was little difference in response to HI between the f ₁₀₄ and wt. The highest de-epoxidation (DES) value at every time point of exposure to HI was measured for f ₂, whereas the wt had the lowest value among the three strains. There were two lifetime components resolved for the conversion of violaxanthin (V) to zeaxanthin plus antheraxanthin (Z + A). The most rapid lifetime was around 6 min and the slower lifetime was >140 min, in both the mutants and wt. However, the wt and f ₁₀₄ both displayed larger amplitudes of both de-epoxidation lifetimes than f ₂. The difference between the final de-epoxidation state (DES = [Z + A]/[V + A + Z]) in the light compared to the dark expressed as DES for wt, f ₁₀₄, and f ₂ was 0.630, 0.623, and 0.420, respectively. The slow lifetime component and overall larger DES in the wt and f ₁₀₄ correlated with more photoprotection, as indicated by relatively higher F_v/F_m and _PS2, compared to the f ₂. Hence the photoprotection against photoinhibition has no relationship with the absolute DES value, but there is a strong relationship with de-epoxidation rate and relative extent or DES.     

A new fluorescence band F689 in photosystem II revealed by picosecond analysis at 4-77 K: Function of two terminal energy sinks F689 and F695 in PS II

We performed picosecond time-resolved fluorescence spectroscopy in spinach photosystem II (PS II) particles at 4, 40, and 77 K and identified a new fluorescence band, F689. F689 was identified in addition to the well-known F685 and F695 bands in both analyses of decay-associated spectra and global Gaussian deconvolution of time-resolved spectra. Its fast decay suggests the energy transfer directly from F689 to the reaction center chlorophyll P680. The contribution of F689, which increases only at low temperature, explains the unusually broad and variable bandwidth of F695 at low temperature. Global analysis revealed the three types of excitation energy transfer/dissipation processes: (1) energy transfer from the peripheral antenna to the three core antenna bands F685, F689, and F695 with time constants of 29 and 171 ps at 77 and 4 K, respectively; (2) between the three core bands (0.18 and 0.82 ns); and (3) the decays of F689 (0.69 and 3.02 ns) and F695 (2.18 and 4.37 ns). The retardations of these energy transfer rates and the slow F689 decay rate produced the strong blue shift of the PS II fluorescence upon the cooling below 77 K.     

Disentangling two non-photochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77 K

Diatoms, which are primary producers in the oceans, can rapidly switch on/off efficient photoprotection to respond to fast light-intensity changes in moving waters. The corresponding thermal dissipation of excess-absorbed-light energy can be observed as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Fluorescence-induction measurements on Cyclotella meneghiniana diatoms show two NPQ processes: qE₁ relaxes rapidly in the dark while qE₂ remains present upon switching to darkness and is related to the presence of the xanthophyll-cycle pigment diatoxanthin (Dtx). We performed picosecond fluorescence measurements on cells locked in different (quenching) states, revealing the following sequence of events during full development of NPQ. At first, trimers of light-harvesting complexes (fucoxanthin–chlorophyll a/c proteins), or FCPa, become quenched, while being part of photosystem II (PSII), due to the induced pH gradient across the thylakoid membrane. This is followed by (partial) detachment of FCPa from PSII after which quenching persists. The pH gradient also causes the formation of Dtx which leads to further quenching of isolated PSII cores and some aggregated FCPa. In subsequent darkness, the pH gradient disappears but Dtx remains present and quenching partly pertains. Only in the presence of some light the system completely recovers to the unquenched state.