Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom

The diadinoxanthin cycle (DD-cycle) in chromophyte algae involves the interconversion of two carotenoids, diadinoxanthin (DD) and diatoxanthin (DT). We investigated the kinetics of light-induced DD-cycling in the marine diatom Phaeodactylum tricornutum and its role in dissipating excess excitation energy in PS II. Within 15 min following an increase in irradiance, DT increased and was accompanied by a stoichiometric decrease in DD. This reaction was completely blocked by dithiothreitol (DTT). A second, time-dependent, increase in DT was detected 20 min after the light shift without a concomitant decrease in DD. DT accumulation from both processes was correlated with increases in non-photochemical quenching of chlorophyll fluorescence. Stern-Volmer analyses suggests that changes in non-photochemical quenching resulted from changes in thermal dissipation in the PS II antenna and in the reaction center. The increase in non-photochemical quenching was correlated with a small decrease in the effective absorption cross section of PS II. Model calculations suggest however that the changes in cross section are not sufficiently large to significantly reduce multiple excitation of the reaction center within the turnover time of steady-state photosynthetic electron transport at light saturation. In DTT poisoned cells, the change in non-photochemical quenching appears to result from energy dissipation in the reaction center and was associated with decreased photochemical efficiency. D1 protein degradation was slightly higher in samples poisoned with DTT than in control samples. These results suggest that while DD-cycling may dynamically alter the photosynthesis-irradiance response curve, it offers limited protection against photodamage of PS II reaction centers at irradiance levels sufficient to saturate steady-state photosynthesis.Abbreviations CAP chloramphenicol - D1 PS II reaction center protein - DD diadinoxanthin - DD cycle-diadinoxanthin cycle - DT diatoxanthin - DTT dithiothreitol - FCP fucoxanthin chlorophyll a-c protein - F_m maximum fluorescence yield in the dark-adapted state - F_o minimum fluorescence yield in the dark-adapted state - F_m and F_o maximum and minimum fluorescence yields respectively in some light adapted state - F_v maximum variable fluorescence yield in the dark-adapted state - I_k Irradiance at the intercept of the initial slope of the photosynthesis-irradiance curve and the maximum photosynthetic rate - k_D first order rate constant for nonradiative de-excitation of excitions in the PS II antenna - k_d first order rate constant for non-radiative de-excitation of excitons in the PS II reaction center - k_F first order rate constant for fluorescence - k_T first order rate constant for exciton transfer to the reaction center - k_t first order rate constant for exciton transfer from the reaction center to the antenna - Rubisco ribulose bisphosphate carboxylase - SV_m Stern-Volmer quenching coefficient of the maximum fluorescence yield - SV_o Stern-Volmer quenching coefficient of the miniximum fluorescence yield - _{PS II} apparent absorption cross-section of PS II - _arr average interval between exciton arrival to the PS II reaction center (ms) - _rem average interval between electron turnover during photosynthesis in the PS II reaction center (ms) - _d the probability that an exciton is non-radiatively dissipated in the reaction center - _T the probability that an exciton in the antenna is transferred to the reaction center - _t the probability that an exciton is transferred back from the reaction center to the antenna     

Rapid colour changes in Euglena sanguinea (Euglenophyceae) caused by internal lipid globule migration

The accumulation of red pigments, frequently carotenoids, under chronic stress is a response observed in diverse kinds of eukaryotic photoautotrophs. It is thought that red pigments protect the chlorophyll located underneath by a light-shielding mechanism. However, the synthesis or degradation of carotenoids is a slow process and this response is usually only observed when the stress is maintained over long periods of time. In contrast, rapid colour changes have been reported in the euglenophyte Euglena sanguinea. Here we study the ecophysiological process behind this phenomenon through chlorophyll fluorescence, and pigment, colour and ultrastructural analyses. Reddening in E. sanguinea was due to the presence of a large amount of free and esterified astaxanthin (representing 80% of the carotenoid pool). The process was highly dynamic, shifting from green to red in 8 min (and vice-versa in 20 min). This change was not due to de novo carotenogenesis, but to the relocation of cytoplasmic lipid globules where astaxanthin accumulates. Thus, red globules were observed to migrate from the centre of the cell to peripheral locations when exposed to high light. Globule migration seems to be so efficient that other classical photoprotective mechanisms are not operative in this species. Despite the presence and operation of the diadino-diatoxanthin cycle, non-photochemical quenching was almost undetectable. Since E. sanguinea forms extensive floating colonies, reddening can be observed at a much greater scale than at the cellular level and the mechanism described here is one of the fastest and most dramatic colour changes attributable to photosynthetic organisms at cell and landscape level. In conclusion E. sanguinea shows an extremely dynamic and efficient photoprotective mechanism, based more on organelle migration than on carotenoid biosynthesis, which prevents excess light absorption by chlorophylls reducing the need for other protective processes related to energy dissipation.     

Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana

Thylakoids of the diatom Cyclotella meneghiniana were separated by discontinuous gradient centrifugation into photosystem (PS) I, PSII, and fucoxanthin-chlorophyll protein (FCP) fractions. FCPs are homologue to light harvesting complexes of higher plants with similar function in e.g. brown algae and diatoms. Still, it is unclear if FCP complexes are specifically associated with either PSI or PSII, or if FCP complexes function as one antenna for both photosystems. However, a trimeric FCP complex, FCPa, and a higher FCP oligomer, FCPb, have been described for C. meneghiniana, already. In this study, biochemical and spectroscopical evidences are provided that reveal a different subset of associated Fcp polypeptides within the isolated photosystem complexes. Whereas the PSII associated Fcp antenna resembles FCPa since it contains Fcp2 and Fcp6, at least three different Fcp polypeptides are associated with PSI. By re-solubilisation and a further purification step Fcp polypeptides were partially removed from PSI and both fractions were analysed again by biochemical and spectroscopical means, as well as by HPLC. Thereby a protein related to Fcp4 and a so far undescribed 17 kDa Fcp were found to be strongly coupled to PSI, whereas presumably Fcp5, a subunit of the FCPb complex, is only loosely bound to the PSI core. Thus, an association of FCPb and PSI is assumed.     

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.     

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (∼ 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A₁ instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.     

Seasonal variation in pigmentation of the dinoflagellate Peridinium gatunense (Dinophyceae) in Lake Kinneret, Israel

1. Pigment composition was measured in natural phytoplankton samples from Lake Kinneret, Israel. From March through June 1998, the dinoflagellate Peridinium gatunense Nygaard mostly contributed more than 95% of the algal biomass. Peak densities were found in April, close to the water surface, with >10⁹ cells m^?3, chlorophyll (Chl) a concentration of 380 mg m^?3 and areal Chl‐a density of >1300 mg m^?2. 2. Cellular concentrations of Chl‐a changed between 201 and 282 pg cell^?1, but did not show a defined temporal fluctuation. 3. The mass ratio of Chl‐c to Chl‐a changed from March to June between 0.16 and 0.22, and the peridinin to Chl‐a ratio changed from 0.25 to 0.41. Neither ratio showed a clear pattern of seasonal change. Conversely, there was a progressive increase in diadinoxanthin and β‐carotene ratios to Chl‐a through the season, parallel to the increase in photon flux impinging upon the lake surface. The diadinoxanthin to Chl‐a ratio changed from 0.11 to 0.28 and the β‐carotene to Chl‐a ratio varied from 0.03 to 0.08 from March through June. 4. Diatoxanthin was not detected in natural samples. However, it was present in experiments with P. gatunense cultures, when concentration of diatoxanthin increased rapidly, concurrent with a decrease in diadinoxanthin and β‐carotene concentrations, while Chl‐c and peridinin ratios to Chl‐a were almost stable with photon flux increase. 5. The seasonal variation in cellular pigmentation of P. gatunense in Lake Kinneret suggests that accumulation of photoprotective pigments is essential for optimisation of photosynthetic activity of this large dinoflagellate.     

GENERAL FEATURES OF PHOTOPROTECTION BY ENERGY DISSIPATION IN PLANKTONIC DIATOMS (BACILLARIOPHYCEAE)1

Planktonic diatoms (Bacillariophyceae) have to cope with large fluctuations of light intensity and periodic exposure to high light. After a shift to high light, photoprotective dissipation of excess energy characterized by the nonphotochemical quenching of fluorescence (NPQ) and the concomitant deepoxidation of diadinoxanthin to diatoxanthin (DT) were measured in four different planktonic marine diatoms (Bacillariophyceae): Skeletonema costatum (Greville) Cleve, Cylindrotheca fusiformis Reimann et Lewin, Thalassiosira weissflogii (Grunow) Fryxell et Hasle, and Ditylum brightwellii (West) Grunow in comparison to the model organism Phaeodactylum tricornutum Böhlin. Upon a sudden increase of light intensity, deepoxidation was rapid and de novo synthesis of DT also occurred. In all species, NPQ was linearly related to the amount of DT formed during high light. In this report, we focused on the role of DT in the dissipation of energy that takes place in the light‐harvesting complex. In S. costatum for the same amount of DT, less NPQ was formed than in P. tricornutum and as a consequence the photoprotection of PSII was less efficient. The general features of photoprotection by harmless dissipation of excess energy in planktonic diatoms described here partly explain why diatoms are well adapted to light intensity fluctuations.     

PIGMENT TURNOVER IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII. II. THE 14CO2-LABELING KINETICS OF CAROTENOIDS1

The biosynthesis and turnover of the pigments fucoxanthin, diadinoxanthin (DD), and diatoxanthin (DT) were studied in exponentially growing cultures of the diatom Thalassiosira weissflogii (Grunow) Fryxell and Hasle to investigate the dependence of pigment turnover on algal growth rates and light intensity. ¹⁴C-bicarbonate was used as a tracer. The labeling kinetics of fucoxanthin and DT were described satisfactorily by a simple precursor-pigment model with two free parameters, the precursor and pigment turnover rate. At growth irradiances < 200 μE · m^?2· s^?1, labeling kinetics of DD indicated the presence of two kinetically distinct DD pools and at least one precursor pool. The average growth rate-normalized pigment turnover rate of fucoxanthin was 0. The growth rate-normalized turnover rate of DT, determined only at high light irradiances (> 200 μE·m^?2·s^?1), was 1.3. At high light irradiances, the growth rate-normalized turnover rate of DD was 1.8. At low light irradiances, the turnover rates of the two DD pools were 3.7 and 0, respectively. The corresponding pigment turnover times were on the order of days to weeks, depending on the growth rate of the cultures. A comparison of pigment pool sizes, pigment turnover rates, and precursor turnover rates suggests that fucoxanthin is synthesized from a pool of DD and that DD and DT are synthesized from a common precursor, possibly β-carotene. No evidence was seen for dynamic xanthophyll cycling. This suggests that the commonly known “xanthophyll cycle” is the simple unidirectional conversion of DD into DT, or of DT into DD, in response to rapid irradiance changes.