In vivo estimation of pigment composition and optical absorption cross-sectionby spectroradiometry in four aquatic photosynthetic micro-organisms

International audienceThe objective of the present study was to estimate in vivo pigment composition and to retrieve absorption cross-section values, a∗, of photosynthetic micro-organisms using a non-invasive technique of reflectance spectrometry. To test the methodology, organisms from different taxonomical groups and different pigment composition were used (Spirulina platensis a Cyanophyta, Porphyridium cruentum a Rhodophyta, Dunaliella tertiolecta a Chlorophyta and Entomoneis paludosa a Bacillariophyta) and photoacclimated to two different irradiance levels: 25 μmol photon m−2 s−1 (Low Light, LL) and 500 μmol photon m−2 s−1 (High Light, HL). Second derivative spectra from reflectance were used to identify pigment in vivo absorption bands that were linked to specific pigments detected by high performance liquid chromatography. Whereas some absorption bands such as those induced by Chlorophyll (Chl) a (416, 440, 625 and around 675 nm) were ubiquous, others were taxonomically specific (e.g. 636 nm for Chl c in E. paludosa) and/or photo-physiological dependent (e.g. 489 nm for zeaxanthin in the HL-acclimated S. platensis). The optical absorption cross-section, a∗, was retrieved from reflectance data using a radiative transfer model previously developed for microphytobenthos. Despite the cellular Chl a decrease observed from LL to HL (up to 88% for S. platensis), the a∗ increased, except for P. cruentum. This was attributed to a ‘package effect’ and to a greater absorption by photoprotective carotenoids that did not contribute to the energy transfer to the core Chl a

The Role of Sustained Photoprotective Non-photochemical Quenching in Low Temperature and High Light Acclimation in the Bloom-Forming Arctic Diatom Thalassiosira gravida

Thalassiosira gravida is a major Arctic diatom responsible for the under-ice spring bloom. We investigated T. gravida physiological plasticity growing it at two temperatures (0 and 5°C) and under different light intensities typically found in its natural environment. T. gravida showed remarkable thermal- and photo-acclimatory plasticity including: low light saturation parameter for growth (KE) and photosynthesis (EK), low μmax but relatively high Chl a/C, low C/N, and decreasing light-saturated carbon fixation rate (PmC) with increasing growth irradiance. T. gravida also showed remarkable photoprotective features, namely a strong sustained non-photochemical quenching (NPQs, hour kinetics relaxation) supported by a high amount of xanthophyll cycle pigments. T. gravida growth remained possible under a wide range of irradiances but photosynthetic plasticity was higher at moderately low light (up to ~50 μmol photons m−2 s−1), nevertheless corresponding to the mean in situ conditions under which it predominates, i.e., underneath the spring thin-ice punctuated with melting ponds. The potential role of NPQs in the photophysiological plasticity of T. gravida is discussed

Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms

Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum

Diatoms are phytoplanktonic organisms that grow successfully in the ocean where light conditions are highly variable. Studies of the molecular mechanisms of light acclimation in the marine diatom Phaeodactylum tricornutum show that carotenoid de-epoxidation enzymes and LHCX1, a member of the light-harvesting protein family, both contribute to dissipate excess light energy through non-photochemical quenching (NPQ). In this study, we investigate the role of the other members of the LHCX family in diatom stress responses. Our analysis of available genomic data shows that the presence of multiple LHCX genes is a conserved feature of diatom species living in different ecological niches. Moreover, an analysis of the levels of four P. tricornutum LHCX transcripts in relation to protein expression and photosynthetic activity indicates that LHCXs are differentially regulated under different light intensities and nutrient starvation, mostly modulating NPQ capacity. We conclude that multiple abiotic stress signals converge to regulate the LHCX content of cells, providing a way to fine-tune light harvesting and photoprotection. Moreover, our data indicate that the expansion of the LHCX gene family reflects functional diversification of its members which could benefit cells responding to highly variable ocean environments

Green Edge ice camp campaigns : understanding the processes controlling the under-ice Arctic phytoplankton spring bloom

The Green Edge initiative was developed to investigate the processes controlling the primary productivity and fate of organic matter produced during the Arctic phytoplankton spring bloom (PSB) and to determine its role in the ecosystem. Two field campaigns were conducted in 2015 and 2016 at an ice camp located on landfast sea ice southeast of Qikiqtarjuaq Island in Baffin Bay (67.4797∘ N, 63.7895∘ W). During both expeditions, a large suite of physical, chemical and biological variables was measured beneath a consolidated sea-ice cover from the surface to the bottom (at 360 m depth) to better understand the factors driving the PSB. Key variables, such as conservative temperature, absolute salinity, radiance, irradiance, nutrient concentrations, chlorophyll a concentration, bacteria, phytoplankton and zooplankton abundance and taxonomy, and carbon stocks and fluxes were routinely measured at the ice camp. Meteorological and snow-relevant variables were also monitored. Here, we present the results of a joint effort to tidy and standardize the collected datasets, which will facilitate their reuse in other Arctic studies

Écophysiologie fonctionnelle des diatomées planctoniques et benthiques : du photosystème à l’écosystème

The biological properties of microalgal communities, and in particular their productivity, largely explain the structuring and the functioning of most, if not all, marine ecosystems and their food-webs. The assessment of the microalgal productivity is rendered difficult by the stochastiscity of environmental driving forces, and especially of light, which directly impact the photosynthetic process. Hence, in order to better understand the mechanistic of the relationship between microalgae and their environment, it is necessary to decipher the regulation of the cellular bioenergetics at the molecular level, and to take into account its functional diversity as well as its impact on the ecophysiology and ultimately on the ecology of communities in situ. This level of knowledge has become essential for, for instance, the reliable quantification and the realistic modeling of marine primary production and of its spatial and temporal dynamics, an information which helps to rationally manage the exploitation of marine biological resources with a strong social and economic impact. This is the context in which I performed my researches during the last 17 years, and because they are one of the most important group of microalgae, and by far the most productive one, I focused my researches on diatoms

Écophysiologie fonctionnelle des diatomées planctoniques et benthiques : du photosystème à l’écosystème

The biological properties of microalgal communities, and in particular their productivity, largely explain the structuring and the functioning of most, if not all, marine ecosystems and their food-webs. The assessment of the microalgal productivity is rendered difficult by the stochastiscity of environmental driving forces, and especially of light, which directly impact the photosynthetic process. Hence, in order to better understand the mechanistic of the relationship between microalgae and their environment, it is necessary to decipher the regulation of the cellular bioenergetics at the molecular level, and to take into account its functional diversity as well as its impact on the ecophysiology and ultimately on the ecology of communities in situ. This level of knowledge has become essential for, for instance, the reliable quantification and the realistic modeling of marine primary production and of its spatial and temporal dynamics, an information which helps to rationally manage the exploitation of marine biological resources with a strong social and economic impact. This is the context in which I performed my researches during the last 17 years, and because they are one of the most important group of microalgae, and by far the most productive one, I focused my researches on diatoms

Stratégies d’adaptation des diatomées phytoplanctoniques aux variations de l’intensité lumineuse rencontrées dans leur environnement naturel

Diatoms play an important role in the carbon cycle. They contribute up to 40 % of theoceanic primary production. They are dominant in turbulent waters where the vertical mixingexposes them to large and rapid changes in light intensity. The collection of solar energy used forphotosynthesis is achieved, in diatoms, by light-harvesting complexes (LHCF) which differ in manyrespects from those of higher plants. For the latter, the photoregulatory mechanisms are rather wellknown. Such is not the case in diatoms. My thesis project has been to study the adaptation strategiesof planktonic diatoms to changing light. On the short term photoprotection takes place, while forlonger terms, photoacclimation is occurring.In a first part of my work, we obtained with special culture conditions, cells specificallyenriched in diadinoxanthin (DD). This pigment is involved in a crucial photoprotective mechanismwhich consists in a switch of the LCHF function from efficient transfer of the absorbed energy tothe reaction centers, to dissipation of excess energy. This modification is brought by the nonphotochemicalquenching of the chlorophyll fluorescence (NPQ) and regulated by the deepoxidationof the DD into diatoxanthin (DT) (the so-called xanthophyll cycle). It induces adecrease in the amount of energy transferred to the photosystem II (PS II) reaction centers. Thecomparison between the NPQ and the xanthophyll cycle developed in the cells with different DDpool sizes led to the conclusion that NPQ is an efficient photoprotective mechanism which isproportional to the DT pool size. This unique material also provided additional informations on themolecular mechanisms involved in the NPQ regulation. The LHCF of the two types of cells havebeen purified. Their polypeptide composition and pigment content have been analyzed with aspecial concern about the localization of the DD and DT molecules.In a second part of my work, other photoprotective mechanisms have been studied with aspecific interest for the alternative routes of electron transfer which store or utilize the reducingpower generated in excess during high light illumination. We were able to demonstrate theoccurrence of a cyclic electron flow around PS II and also the storage of a reduced compoundduring the high light illumination, used later by chlororespiration at lower intensity or in the dark.These two mechanisms can contribute to photoprotection.We also studied photoacclimation of planktonic diatoms and more precisely the capacity ofenergy dissipation in cells acclimated to high light. In these cells, the correlation between NPQ andDT is modified. Our results suggest different possible localizations for DT in the thylakoidmembrane and an indirect role for DT in NPQ.Lastly, we verified that the photoprotective dissipation of energy studied in Phaeodactylumtricornutum also occurred in several other planktonic diatoms of ecological interest (such asThalassiosira weisflogii). These results validate the choice of P. tricornutum as a model organism.Another aspect of my work concerned the use of fluorescence as a non invasive method toevaluate the phytoplanktonic content of aquatic ecosystems. A trichromatic fluorometer wasconceived and built in the lab. It was used to follow the dynamics of freshwater planktonicpopulations for an early detection of blooms with a special focus on cyanobacteria. The set-up canbe used to discriminate between four phytoplanktonic groups (green algae, diatoms and two typesof cyanobacteria). It is also able to evaluate the physiological state of a whole population. This workwas done in collaboration with the French company Suez-Ondeo Services.Les diatomées planctoniques ont un rôle prépondérant dans le cycle du carbone puisqu’ellessont responsables de 40 % de la production primaire océanique. Elles dominent dans les eauxturbulentes où le mélange vertical les expose à des changements d’intensité lumineuse rapides etimportants. L’absorption de l’énergie lumineuse utilisée pour la photosynthèse est assurée par descomplexes pigments-protéines chlorophylliens (LHCF) qui sont relativement différents de ceux desplantes vertes. Chez ces dernières, les mécanismes de photorégulation sont assez bien connus,contrairement aux diatomées. Mon travail de thèse a porté sur les stratégies d’adaptation desdiatomées planctoniques aux variations lumineuses à court terme (la photoprotection) et à longterme (la photoacclimatation).Dans un premier temps, nous avons obtenu, grâce à des conditions de cultures particulières,des cellules spécifiquement enrichies en diadinoxanthine (DD). Ce pigment est impliqué dans unmécanisme photoprotecteur essentiel qui se manifeste par le passage rapide des LHCF de leurfonction “normale” de transfert efficace de l’énergie absorbée aux centres réactionnels à unefonction de dissipation de l’énergie lumineuse. Ce changement fonctionnel est illustré par unquenching non-photochimique de la fluorescence chlorophyllienne (NPQ) régulé par la déépoxydationréversible de la DD en diatoxanthine (DT) (cycle des xanthophylles). Il entraîne ladiminution de la proportion d’énergie transmise au centres réactionnels photosystème II (PS II). Lacomparaison de la dissipation d’énergie et du cycle des xanthophylles dans les deux types de culturea permis de démontrer de manière indubitable l’efficacité du NPQ dans la photoprotection. Cematériel unique nous a également apporté de précieuses informations sur les mécanismesmoléculaires impliqués dans la régulation du NPQ. Les complexes pigments-protéines ont étépurifiés chez les deux types de cultures. Leur contenu polypeptidique et pigmentaire a étécaractérisé avec une attention toute particulière pour la localisation de la DD.Dans un second temps, les processus photoprotecteurs autres ont été examinés et un accentparticulier a été donné aux voies alternatives à la voie photosynthétique qui permettent d’utiliseret/ou de stocker l’énergie en excès. Nous avons mis en évidence l’existence d’un transport cycliquedes électrons au sein du PS II ainsi que le stockage d’un composé réduit à la lumière utilisé ensuitecomme carburant à l’obscurité par la chlororespiration. Ces deux mécanismes sont bien connus pourcontribuer à la photoprotection de la cellule.Nous avons également étudié la photoacclimatation des diatomées planctoniques en axantnotre approche sur les capacités de photoprotection des diatomées acclimatées à forte intensitélumineuse. Chez ces algues, la relation entre le cycle de la DD et le développement du NPQ estmodifiée. Ceci suggère plusieurs localisations possibles de la DT au sein de l’appareilphotosynthétique et un rôle indirect de la DT dans le NPQ.Enfin, nous avons montré que les mécanismes de photoprotection étudiés en détail chezPhaeodactylum tricornutum poussée dans différentes conditions de lumière existent également àdes degrés divers chez plusieurs autres diatomées planctoniques d’importance écologique (tel queThalassiosira weisflogii). Cette comparaison a permis de valider notre choix de P. tricornutumcomme organisme modèle.Une autre partie de ce travail a consisté à la mise en application pratique de l’observablequ’est l’émission de fluorescence chlorophyllienne qui a été utilisée dans l’étude de laphotorégulation chez les diatomées. Ceci a abouti à l’élaboration d’un fluorimètre pour le suivi de ladynamique des peuplements phytoplanctoniques d’eau douce en vue de la détection précoce desproliférations algales avec une attention pour les cyanobactéries. L’appareil est capable de mesurerla biomasse, de distinguer entre quatre groupes phytoplanctoniques (algues vertes, diatomées, deuxtypes de cyanobactéries) et d’évaluer la physiologie du peuplement. Ce travail a été réalisé encollaboration avec la société Suez-Ondeo Services

Stratégies d’adaptation des diatomées phytoplanctoniques aux variations de l’intensité lumineuse rencontrées dans leur environnement naturel

Diatoms play an important role in the carbon cycle. They contribute up to 40 % of theoceanic primary production. They are dominant in turbulent waters where the vertical mixingexposes them to large and rapid changes in light intensity. The collection of solar energy used forphotosynthesis is achieved, in diatoms, by light-harvesting complexes (LHCF) which differ in manyrespects from those of higher plants. For the latter, the photoregulatory mechanisms are rather wellknown. Such is not the case in diatoms. My thesis project has been to study the adaptation strategiesof planktonic diatoms to changing light. On the short term photoprotection takes place, while forlonger terms, photoacclimation is occurring.In a first part of my work, we obtained with special culture conditions, cells specificallyenriched in diadinoxanthin (DD). This pigment is involved in a crucial photoprotective mechanismwhich consists in a switch of the LCHF function from efficient transfer of the absorbed energy tothe reaction centers, to dissipation of excess energy. This modification is brought by the nonphotochemicalquenching of the chlorophyll fluorescence (NPQ) and regulated by the deepoxidationof the DD into diatoxanthin (DT) (the so-called xanthophyll cycle). It induces adecrease in the amount of energy transferred to the photosystem II (PS II) reaction centers. Thecomparison between the NPQ and the xanthophyll cycle developed in the cells with different DDpool sizes led to the conclusion that NPQ is an efficient photoprotective mechanism which isproportional to the DT pool size. This unique material also provided additional informations on themolecular mechanisms involved in the NPQ regulation. The LHCF of the two types of cells havebeen purified. Their polypeptide composition and pigment content have been analyzed with aspecial concern about the localization of the DD and DT molecules.In a second part of my work, other photoprotective mechanisms have been studied with aspecific interest for the alternative routes of electron transfer which store or utilize the reducingpower generated in excess during high light illumination. We were able to demonstrate theoccurrence of a cyclic electron flow around PS II and also the storage of a reduced compoundduring the high light illumination, used later by chlororespiration at lower intensity or in the dark.These two mechanisms can contribute to photoprotection.We also studied photoacclimation of planktonic diatoms and more precisely the capacity ofenergy dissipation in cells acclimated to high light. In these cells, the correlation between NPQ andDT is modified. Our results suggest different possible localizations for DT in the thylakoidmembrane and an indirect role for DT in NPQ.Lastly, we verified that the photoprotective dissipation of energy studied in Phaeodactylumtricornutum also occurred in several other planktonic diatoms of ecological interest (such asThalassiosira weisflogii). These results validate the choice of P. tricornutum as a model organism.Another aspect of my work concerned the use of fluorescence as a non invasive method toevaluate the phytoplanktonic content of aquatic ecosystems. A trichromatic fluorometer wasconceived and built in the lab. It was used to follow the dynamics of freshwater planktonicpopulations for an early detection of blooms with a special focus on cyanobacteria. The set-up canbe used to discriminate between four phytoplanktonic groups (green algae, diatoms and two typesof cyanobacteria). It is also able to evaluate the physiological state of a whole population. This workwas done in collaboration with the French company Suez-Ondeo Services.Les diatomées planctoniques ont un rôle prépondérant dans le cycle du carbone puisqu’ellessont responsables de 40 % de la production primaire océanique. Elles dominent dans les eauxturbulentes où le mélange vertical les expose à des changements d’intensité lumineuse rapides etimportants. L’absorption de l’énergie lumineuse utilisée pour la photosynthèse est assurée par descomplexes pigments-protéines chlorophylliens (LHCF) qui sont relativement différents de ceux desplantes vertes. Chez ces dernières, les mécanismes de photorégulation sont assez bien connus,contrairement aux diatomées. Mon travail de thèse a porté sur les stratégies d’adaptation desdiatomées planctoniques aux variations lumineuses à court terme (la photoprotection) et à longterme (la photoacclimatation).Dans un premier temps, nous avons obtenu, grâce à des conditions de cultures particulières,des cellules spécifiquement enrichies en diadinoxanthine (DD). Ce pigment est impliqué dans unmécanisme photoprotecteur essentiel qui se manifeste par le passage rapide des LHCF de leurfonction “normale” de transfert efficace de l’énergie absorbée aux centres réactionnels à unefonction de dissipation de l’énergie lumineuse. Ce changement fonctionnel est illustré par unquenching non-photochimique de la fluorescence chlorophyllienne (NPQ) régulé par la déépoxydationréversible de la DD en diatoxanthine (DT) (cycle des xanthophylles). Il entraîne ladiminution de la proportion d’énergie transmise au centres réactionnels photosystème II (PS II). Lacomparaison de la dissipation d’énergie et du cycle des xanthophylles dans les deux types de culturea permis de démontrer de manière indubitable l’efficacité du NPQ dans la photoprotection. Cematériel unique nous a également apporté de précieuses informations sur les mécanismesmoléculaires impliqués dans la régulation du NPQ. Les complexes pigments-protéines ont étépurifiés chez les deux types de cultures. Leur contenu polypeptidique et pigmentaire a étécaractérisé avec une attention toute particulière pour la localisation de la DD.Dans un second temps, les processus photoprotecteurs autres ont été examinés et un accentparticulier a été donné aux voies alternatives à la voie photosynthétique qui permettent d’utiliseret/ou de stocker l’énergie en excès. Nous avons mis en évidence l’existence d’un transport cycliquedes électrons au sein du PS II ainsi que le stockage d’un composé réduit à la lumière utilisé ensuitecomme carburant à l’obscurité par la chlororespiration. Ces deux mécanismes sont bien connus pourcontribuer à la photoprotection de la cellule.Nous avons également étudié la photoacclimatation des diatomées planctoniques en axantnotre approche sur les capacités de photoprotection des diatomées acclimatées à forte intensitélumineuse. Chez ces algues, la relation entre le cycle de la DD et le développement du NPQ estmodifiée. Ceci suggère plusieurs localisations possibles de la DT au sein de l’appareilphotosynthétique et un rôle indirect de la DT dans le NPQ.Enfin, nous avons montré que les mécanismes de photoprotection étudiés en détail chezPhaeodactylum tricornutum poussée dans différentes conditions de lumière existent également àdes degrés divers chez plusieurs autres diatomées planctoniques d’importance écologique (tel queThalassiosira weisflogii). Cette comparaison a permis de valider notre choix de P. tricornutumcomme organisme modèle.Une autre partie de ce travail a consisté à la mise en application pratique de l’observablequ’est l’émission de fluorescence chlorophyllienne qui a été utilisée dans l’étude de laphotorégulation chez les diatomées. Ceci a abouti à l’élaboration d’un fluorimètre pour le suivi de ladynamique des peuplements phytoplanctoniques d’eau douce en vue de la détection précoce desproliférations algales avec une attention pour les cyanobactéries. L’appareil est capable de mesurerla biomasse, de distinguer entre quatre groupes phytoplanctoniques (algues vertes, diatomées, deuxtypes de cyanobactéries) et d’évaluer la physiologie du peuplement. Ce travail a été réalisé encollaboration avec la société Suez-Ondeo Services

What can Arctic diatoms do for you ?

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