Spectral Radiation Dependent Photoprotective Mechanism in the Diatom Pseudo-nitzschia multistriata

Phytoplankton, such as diatoms, experience great variations of photon flux density (PFD) and light spectrum along the marine water column. Diatoms have developed some rapidly-regulated photoprotective mechanisms, such as the xanthophyll cycle activation (XC) and the non-photochemical chlorophyll fluorescence quenching (NPQ), to protect themselves from photooxidative damages caused by excess PFD. In this study, we investigate the role of blue fluence rate in combination with red radiation in shaping photoacclimative and protective responses in the coastal diatom Pseudo-nitzschia multistriata. This diatom was acclimated to four spectral light conditions (blue, red, blue-red, blue-red-green), each of them provided with low and high PFD. Our results reveal that the increase in the XC pool size and the amplitude of NPQ is determined by the blue fluence rate experienced by cells, while cells require sensing red radiation to allow the development of these processes. Variations in the light spectrum and in the blue versus red radiation modulate either the photoprotective capacity, such as the activation of the diadinoxanthin-diatoxanthin xanthophyll cycle, the diadinoxanthin de-epoxidation rate and the capacity of non-photochemical quenching, or the pigment composition of this diatom. We propose that spectral composition of light has a key role on the ability of diatoms to finely balance light harvesting and photoprotective capacity.     

Growth form defines physiological photoprotective capacity in intertidal benthic diatoms

In intertidal marine sediments, characterized by rapidly fluctuating and often extreme light conditions, primary production is frequently dominated by diatoms. We performed a comparative analysis of photophysiological traits in 15 marine benthic diatom species belonging to the four major morphological growth forms (epipelon (EPL), motile epipsammon (EPM-M) and non-motile epipsammon (EPM-NM) and tychoplankton (TYCHO)) found in these sediments. Our analyses revealed a clear relationship between growth form and photoprotective capacity, and identified fast regulatory physiological photoprotective traits (that is, non-photochemical quenching (NPQ) and the xanthophyll cycle (XC)) as key traits defining the functional light response of these diatoms. EPM-NM and motile EPL showed the highest and lowest NPQ, respectively, with EPM-M showing intermediate values. Like EPL, TYCHO had low NPQ, irrespective of whether they were grown in benthic or planktonic conditions, reflecting an adaptation to a low light environment. Our results thus provide the first experimental evidence for the existence of a trade-off between behavioural (motility) and physiological photoprotective mechanisms (NPQ and the XC) in the four major intertidal benthic diatoms growth forms using unialgal cultures. Remarkably, although motility is restricted to the raphid pennate diatom clade, raphid pennate species, which have adopted a non-motile epipsammic or a tychoplanktonic life style, display the physiological photoprotective response typical of these growth forms. This observation underscores the importance of growth form and not phylogenetic relatedness as the prime determinant shaping the physiological photoprotective capacity of benthic diatoms.     

Changes in the condition of photosynthetic apparatus of a diatom alga <Emphasis Type="Italic">Thallassiosira weisflogii</Emphasis> during photoadaptation and photodamage

Two populations of a diatom alga Thallassiosira weisflogii were grown at photon flux densities (PFD) of 0.8 and 8 μmol/(m² s). For both diatom populations, the recovery of chlorophyll fluorescence parameters (F ₀, F _m, F _v/F _m, and NPQ) was monitored after nondestructive irradiation by visible light at PFD of 40 μmol/(m² s) and after high-intensity irradiation by visible light (1000–4000 μmol/(m² s)). The exposure of diatoms to PFD of 40 μmol/(m² s)—higher than PFD used for algal growth but still nondamaging to photosynthetic apparatus—induced nonphotochemical quenching (NPQ), which was stronger in algae grown at higher PFD (8 μmol/(m² s)) than in algae grown at low light. After irradiation with high-intensity light, the recovery of chlorophyll fluorescence parameters was more pronounced in algae grown at elevated PFD level. During short-term irradiation of diatoms with high-intensity visible light (1000 μmol/(m² s)), a stronger NPQ was observed in the culture adapted to high irradiance. After the treatment of algae with dithiothreitol (an inhibitor of carotenoid deepoxidase in the diadinoxanthin cycle) or NH₄Cl (an agent abolishing the proton gradient at thylakoid membranes), a short exposure of algae to PFD of 40 μmol/(m² s) induced hardly any nonphotochemical quenching. The results indicate the dominant contribution of xanthophyll cycle carotenoids to energy-dependent quenching.     

PHOTOPHYSIOLOGICAL PROPERTIES OF THE MARINE PICOEUKARYOTE PICOCHLORUM RCC 237 (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

The photophysiological properties of strain RCC 237 belonging to the marine picoplanktonic genus Picochlorum, first described by Henley et al., were investigated under different photon flux densities (PFD), ranging from 40 to 400 μmol photons· m^?2·s^?1, mainly focusing on the development of the xanthophyll cycle and its relationship with the nonphotochemical quenching of fluorescence (NPQ). The functioning of the xanthophyll cycle and its photoprotective role was investigated by applying a progressive increase of PFD and using dithiotreitol and norflurazon to block specific enzymatic reactions in order to study in depth the relationship between xanthophyll cycle and NPQ. These two processes were significantly related only during the gradually increasing light periods and not during stable light periods, where NPQ and zeaxanthin were decoupled. This result reveals that NPQ is a photoprotective process developed by algae only when cells are experiencing increasing PFD or in response to stressful light variations, for instance after a sudden light shift. Results showed that the photobiological properties of Picochlorum strain RCC 237 seem to be well related to the surface water characteristics, as it is able to maintain its photosynthetic characteristics under different PFDs and to quickly activate the xanthophyll cycle under high light.     

Behavioural versus physiological photoprotection in epipelic and epipsammic benthic diatoms

Benthic diatoms are dominant primary producers in intertidal marine sediments, which are characterized by widely fluctuating and often extreme light conditions. To cope with sudden increases in light intensity, benthic diatoms display both behavioural and physiological photoprotection mechanisms. Behavioural photoprotection is restricted to raphid pennate diatoms, which possess a raphe system that enables motility and hence positioning in sediment light gradients (e.g. via vertical migration into the sediment). The main physiological photoprotection mechanism is to dissipate excess light energy as heat, measured as Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence. A trade-off between vertical migration and physiological photoprotection (NPQ) in benthic diatoms has been hypothesized before, but this has never been formally tested. We exposed five epipelic diatom species (which move in between sediment particles) and four epipsammic diatom species (which live in close association with individual sand grains) to high light conditions, and characterized both NPQ and the relative magnitude of the migratory response to high light. Our results reveal the absence of a significant downward migratory response in an araphid diatom, but also in several raphid epipsammic diatoms, while all epipelic species showed a significant migratory response upon high light exposure. In all epipsammic species the upregulation of NPQ was rapid and pronounced; NPQ relaxation in low light conditions, however, occurred faster in the araphid diatom, compared with the raphid epipsammic species. In contrast, all epipelic species lacked a strong and flexible NPQ response and showed higher susceptibility to photodamage when not able to migrate. While overall our results support the vertical migration-NPQ trade-off, the lack of strong relationships between the capacity for vertical migration and NPQ within the epipsammic and epipelic groups suggests that other factors as well, such as cell size, substrate type and photoacclimation, may influence photoprotective strategies.     

THE IMPACT OF NONPHOTOCHEMICAL QUENCHING OF FLUORESCENCE ON THE PHOTON BALANCE IN DIATOMS UNDER DYNAMIC LIGHT CONDITIONS1

The nonphotochemical quenching (NPQ) of fluorescence is an important photoprotective mechanism in particular under dynamic light conditions. Its photoprotective potential was suggested to be a functional trait of algal diversity. In the present study, the influence of the photoprotective capacity on the growth balance was investigated in two diatoms, which possess different NPQ characteristics. It was hypothesized that under fluctuating light conditions Cyclotella meneghiniana Kütz. would benefit from its large and flexible NPQ potential, whereas the comparably small NPQ capacity in Skeletonema costatum (Grev.) Cleve should exert an unfavorable impact on growth. The results of the study clearly falsify this hypothesis. Although C. meneghiniana possesses a fast NPQ component, this diatom was not able to recover its full NPQ capacity under fluctuating light. On the other hand, the induction of NPQ at relatively low irradiance in S. costatum resulted in rather small differences in the fraction of energy dissipation by the NPQ mechanism in the comparison of both diatoms. Larger differences were found in the metabolic characteristics. Both diatoms differed in their biomass composition, with a higher content of lipids in C. meneghiniana but higher amounts of carbohydrates in S. costatum. Finally, the lower degree of reduction in the biomass compensated for the higher respiration rates in S. costatum and resulted in a higher quantum efficiency of biomass production. An indirect correlation between the photoprotective and the metabolic capacity is discussed.     

Light History Influences the Response of Fluvial Biofilms to Zn Exposure

Fluvial biofilms are subject to multistress situations in natural ecosystems, such as the co‐occurrence of light intensity changes and metal toxicity. However, studies simultaneously addressing both factors are rare. This study evaluated in microcosm conditions the relationship between short‐term light intensity changes and Zn toxicity on fluvial biofilms with long‐term photoacclimation to different light conditions. Biofilms that had long‐term photoacclimation to 25 μmol photons · m^?2 · s^?1 (low light [LL] biofilms), 100 μmol photons · m^?2 · s^?1 (medium light [ML] biofilms), and 500 μmol photons · m^?2 · s^?1 (high light [HL] biofilms) were characterized by different structural (Chlorophyll‐a [Chl‐a], total biomass‐AFDW, EPS, algal groups, and diatom taxonomy) and physiological attributes (ETR‐I curves and photosynthetic pigments). HL biofilms showed higher light saturation intensity and a higher production of xanthophylls than LL biofilms. In contrast, LL biofilms had many structural differences; a higher proportion of diatoms and lower AFDW and EPS contents than ML and HL biofilms. A clear effect of light intensity changes on Zn toxicity was also demonstrated. Zn toxicity was enhanced when a sudden increase in light intensity also occurred, mainly with LL biofilms, causing higher inhibition of both the Φ′_PSII and the Φ_PSII. A decoupling of NPQ from de‐epoxidation reaction (DR) processes was also observed, indicating substantial damage to photoprotective mechanisms functioning in biofilms (i.e., xanthophyll cycle of diatoms) due to Zn toxicity. This study highlights the need to take into account environmental stress (e.g., light intensity changes) to better assess the environmental risks of chemicals (e.g., metals).     

In diatoms, the transthylakoid proton gradient regulates the photoprotective non-photochemical fluorescence quenching beyond its control on the xanthophyll cycle

In diatoms, the non-photochemical fluorescence quenching (NPQ) regulates photosynthesis during light fluctuations. NPQ is associated with an enzymatic xanthophyll cycle (XC) which is controlled by the light-driven transthylakoid proton gradient (delta pH). In this report, special illumination conditions and chemicals were used to perturb the kinetics of the delta pH build-up, of the XC and of NPQ. We found that the delta pH-related acidification of the lumen is also needed for NPQ to develop by switching the xanthophylls to an 'activated' state, probably via the protonation of light-harvesting antenna proteins. It confirms the NPQ model previously proposed for diatoms.     

Microscale imaging sheds light on species-specific strategies for photo-regulation and photo-acclimation of microphytobenthic diatoms

Intertidal microphytobenthic (MPB) biofilms are key sites for coastal primary production, predominantly by pennate diatoms exhibiting photo-regulation via non-photochemical quenching (NPQ) and vertical migration. Movement is the main photo-regulation mechanism of motile (epipelic) diatoms and because they can move from light, they show low-light acclimation features such as low NPQ levels, as compared to non-motile (epipsammic) forms. However, most comparisons of MPB species-specific photo-regulation have used low light acclimated monocultures, not mimicking environmental conditions. Here we used variable chlorophyll fluorescence imaging, fluorescent labelling in sediment cores and scanning electron microscopy to compare the movement and NPQ responses to light of four epipelic diatom species from a natural MPB biofilm. The diatoms exhibited different species-specific photo-regulation features and a large NPQ range, exceeding that reported for epipsammic diatoms. This could allow epipelic species to coexist in compacted light niches of MPB communities. We show that diatom cell orientation within MPB can be modulated by light, where diatoms oriented themselves more perpendicular to the sediment surface under high light vs. more parallel under low light, demonstrating behavioural, photo-regulatory response by varying their light absorption cross-section. This highlights the importance of considering species-specific responses and understanding cell orientation and photo-behaviour in MPB research.     

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.     

Changes in the photosynthetic reaction centre II in the diatom Phaeodactylum tricornutum result in non-photochemical fluorescence quenching

Diatoms are an important group of primary producers in the aquatic environment. They are able to acclimate to fast changes in the light intensity by various mechanisms including a rise in non-photochemical fluorescence quenching (NPQ). The latter has been attributed to the xanthophyll cycle (XC) following activation of diadinoxanthin de-epoxidase by the acidification of the thylakoid lumen. To examine whether fluorescence quenching in the diatom Phaeodactylum tricornutum depends on the ΔpH generated by the photosynthetic electron transport, we arrested the latter by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). This treatment hardly affected the NPQ or XC, even when methylviologen was present. Dissipation of the ΔpH by 2,4-dinitrophenol inhibited the XC but did not alter NPQ. Similar results, i.e. inhibition of the XC but normal fluorescence quenching, were observed when the experiments were performed at 3°C. Measurements of thermoluminescence showed that excess light treatment caused a marked decline in the signals obtained as a result of recombination of Q_B^- with the S₃ state of the Mn cluster; this was also observed in cells treated with DCMU (recombination of Q_A^- with S₂). Light treatment also diminished the Q_A^- re-oxidation signals. The data suggest that changes in PSII core centre itself due to exposure to excess light conditions play an important part in the acclimation of P. tricornutum to the changing light conditions.     

The Effect of Diel Temperature and Light Cycles on the Growth of Nannochloropsis oculata in a Photobioreactor Matrix

A matrix of photobioreactors integrated with metabolic sensors was used to examine the combined impact of light and temperature variations on the growth and physiology of the biofuel candidate microalgal species Nannochloropsis oculata. The experiments were performed with algal cultures maintained at a constant 20°C versus a 15°C to 25°C diel temperature cycle, where light intensity also followed a diel cycle with a maximum irradiance of 1920 µmol photons m⁻² s⁻¹. No differences in algal growth (Chlorophyll a) were found between the two environmental regimes; however, the metabolic processes responded differently throughout the day to the change in environmental conditions. The variable temperature treatment resulted in greater damage to photosystem II due to the combined effect of strong light and high temperature. Cellular functions responded differently to conditions before midday as opposed to the afternoon, leading to strong hysteresis in dissolved oxygen concentration, quantum yield of photosystem II and net photosynthesis. Overnight metabolism performed differently, probably as a result of the temperature impact on respiration. Our photobioreactor matrix has produced novel insights into the physiological response of Nannochloropsis oculata to simulated environmental conditions. This information can be used to predict the effectiveness of deploying Nannochloropsis oculata in similar field conditions for commercial biofuel production.     

Diversity in Xanthophyll Cycle Pigments Content and Related Nonphotochemical Quenching (NPQ) Among Microalgae: Implications for Growth Strategy and Ecology

Xanthophyll cycle-related nonphotochemical quenching (NPQ), which is present in most photoautotrophs, allows dissipation of excess light energy. Xanthophyll cycle-related NPQ depends principally on xanthophyll cycle pigments composition and their effective involvement in NPQ. Xanthophyll cycle-related NPQ is tightly controlled by environmental conditions in a species-/strain-specific manner. These features are especially relevant in microalgae living in a complex and highly variable environment. The goal of this study was to perform a comparative assessment of NPQ ecophysiologies across microalgal taxa in order to underline the specific involvement of NPQ in growth adaptations and strategies. We used both published results and data acquired in our laboratory to understand the relationships between growth conditions (irradiance, temperature, and nutrient availability), xanthophyll cycle composition, and xanthophyll cycle pigments quenching efficiency in microalgae from various taxa. We found that in diadinoxanthin-containing species, the xanthophyll cycle pigment pool is controlled by energy pressure in all species. At any given energy pressure, however, the diatoxanthin content is higher in diatoms than in other diadinoxanthin-containing species. XC pigments quenching efficiency is species-specific and decreases with acclimation to higher irradiances. We found a clear link between the natural light environment of species/ecotypes and quenching efficiency amplitude. The presence of diatoxanthin or zeaxanthin at steady state in all species examined at moderate and high irradiances suggests that cells maintain a light-harvesting capacity in excess to cope with potential decrease in light intensity.     

Ultrafast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms

The diatom algae, responsible for at least a quarter of the global photosynthetic carbon assimilation in the oceans, are capable of switching on rapid and efficient photoprotection, which helps them cope with the large fluctuations of light intensity in the moving waters. The enhanced dissipation of excess excitation energy becomes visible as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Intact cells of the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum, which show different NPQ induction kinetics under high light illumination, were investigated by picosecond time-resolved fluorescence under dark and NPQ-inducing high light conditions. The fluorescence kinetics revealed that there are two independent sites responsible for NPQ. The first quenching site is located in an FCP antenna system that is functionally detached from both photosystems, while the second quenching site is located in the PSII-attached antenna. Notwithstanding their different npq induction and reversal kinetics, both diatoms showed identical NPQ via both mechanisms in the steady-state. Their fluorescence decays in the dark-adapted states were different, however. A detailed quenching model is proposed for NPQ in diatoms.     

Rapid regulation of excitation energy in two pennate diatoms from contrasting light climates

Non-photochemical quenching (NPQ) is a fast acting photoprotective response to high light stress triggered by over excitation of photosystem II. The mechanism for NPQ in the globally important diatom algae has been principally attributed to a xanthophyll cycle, analogous to the well-described qE quenching of higher plants. This study compared the short-term NPQ responses in two pennate, benthic diatom species cultured under identical conditions but which originate from unique light climates. Variable chlorophyll fluorescence was used to monitor photochemical and non-photochemical excitation energy dissipation during high light transitions; whereas whole cell steady state 77 K absorption and emission were used to measure high light elicited changes in the excited state landscapes of the thylakoid. The marine shoreline species Nitzschia curvilineata was found to have an antenna system capable of entering a deeply quenched, yet reversible state in response to high light, with NPQ being highly sensitive to dithiothreitol (a known inhibitor of the xanthophyll cycle). Conversely, the salt flat species Navicula sp. 110-1 exhibited a less robust NPQ that remained largely locked-in after the light stress was removed; however, a lower amplitude, but now highly reversible NPQ persisted in cells treated with dithiothreitol. Furthermore, dithiothreitol inhibition of NPQ had no functional effect on the ability of Navicula cells to balance PSII excitation/de-excitation. These different approaches for non-photochemical excitation energy dissipation are discussed in the context of native light climate.     

PHOTOINHIBITION OF PSII IN EMILIANIA HUXLEYI (HAPTOPHYTA) UNDER HIGH LIGHT STRESS: THE ROLES OF PHOTOACCLIMATION,PHOTOPROTECTION, AND PHOTOREPAIR1

The response of the coccolithophorid Emiliania huxleyi (Lohmann) W. H. Hay et H. Mohler to acute exposure to high photon flux densities (PFD) was examined in terms of PSII photoinhibition, photoprotection, and photorepair. The time and light dependencies of these processes were characterized as a function of the photoacclimation state of the alga. Low‐light (LL) acclimated cells displayed a higher degree of photoinhibition, measured as decline in F_v/F_m, than high‐light (HL) acclimated cells. However, HL cultures were more susceptible to photodamage but also more capable of compensating for it by performing a faster repair cycle. The relation between gross photoinhibition (observed in the presence of an inhibitor of repair) and PFD to which the algae were exposed deviated from linearity at high PFD, which calls into question the universality of current concepts of photoinhibition in mechanistic models. The light dependence of the de‐epoxidation state (DPS) of the xanthophyll cycle (XC) pigments on the timescale of hours was the same in cells acclimated to LL and HL. However, HL cells were more efficient in realizing nonphotochemical quenching (NPQ) on short timescales, most likely due to a larger XC pool. LL cells displayed an increase in the PSII effective cross‐section (σ_PSII) as a result of photoinhibition, which was observed also in HL cells when net photoinhibition was induced by blocking the D1 repair cycle. The link between σ_PSII and photoinhibition suggests that the population of PSII reaction centers (RCIIs) of E. huxleyi shares a common antenna, according to a “lake” organization of the light‐harvesting complex.     

The super-excess energy dissipation in diatom algae: comparative analysis with higher plants

When grown at intermittent light regime, diatom alga Phaeodactylum tricornutum is able to form photoprotective non-photochemical chlorophyll fluorescence quenching (NPQ) three to five times larger than that observed in the higher plants. This quenching is sustained in the dark for 5 to 10 min, reverses completely within approximately 1 h and seems to be very tightly related to the presence of the zeaxanthin analogue, diatoxanthin. Addition of the uncoupler NH4Cl before illumination can completely abolish formation of NPQ, revealing the ΔpH-dependency of the xanthophyll cycle activity. Once established, NPQ can also be almost completely reversed by the uncoupler. However, the higher NPQ is formed the more time is required for its reversal. At the point when the fluorescence was approximately 90% recovered the level of illumination-induced diatoxanthin was found to be only partially reduced. This indicates that the proton gradient is a key triggering factor of NPQ. It was also noticed that NPQ in Phaeodactylum cells was absent even when majority of reaction centers were closed and the plastoquinone pool was significantly reduced. The absence of NPQ at these conditions could be due to very low levels of ΔpH. It is likely that in diatoms alternative sources of protons such as the PS I cyclic electron transfer and/or chlororespiration are important in generating the proton gradient sufficient to trigger NPQ. Absorption changes associated with the xanthophyll cycle activity were found to be larger than those for higher plants. The position of the positive maximum in the difference spectrum illuminated-minus-dark was 512–514 nm in comparison to the 505–508 nm for leaves. The 535 nm band associated with NPQ in plants is absent in Phaeodactylum. An uncoupler-sensitive absorption change at 522 nm was discovered. Kinetics of NPQ showed linear correlation with the 522 nm absorption change. This revised version was published online in June 2006 with corrections to the Cover Date.