Expression of the Vitreoscilla hemoglobin gene in Nannochloropsis oceanica regulates intracellular oxygen balance under high-light.

Nannochloropsis oceanica is widely used as a model photosynthetic chassis to produce fatty acids and carotenoid pigments. However, intense light typically causes excessive generation of reactive oxygen species (ROS) and photorespiration in microalgal cells, which results in decreased cell growth rate and unsaturated fatty acid content. In this study, the Vitreoscilla hemoglobin gene (vgb) was introduced into N. oceanica cells and expressed by using the light-harvesting complex promoter and its signal peptide. Compared with wild type (WT), the growth rate of transformants increased by 7.4%-18.5%, and the eicosapentaenoic acid content in an optimal transformant increased by 21.0%. Correspondingly, the intracellular ROS levels decreased by 56.9%-70.0%, and the catalase content in transformants was about 1.8 times that of WT. The photorespiration level of transformants was reduced by the measurement and calculation of the dissolved oxygen concentration under the condition of light-dark transition. The expression level of the key genes related to the photorespiration pathway in transformants was more than 80% lower than that in WT. These results indicated that Vitreoscilla hemoglobin could improve microalgal growth by reducing ROS damage and modulating photorespiration under stress conditions.

Role of an ancient light-harvesting protein of PSI in light absorption and photoprotection.

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

Photosynthesis Regulation in Response to Fluctuating Light in the Secondary Endosymbiont Alga Nannochloropsis gaditana.

In nature, photosynthetic organisms are exposed to highly dynamic environmental conditions where the excitation energy and electron flow in the photosynthetic apparatus need to be continuously modulated. Fluctuations in incident light are particularly challenging because they drive oversaturation of photosynthesis with consequent oxidative stress and photoinhibition. Plants and algae have evolved several mechanisms to modulate their photosynthetic machinery to cope with light dynamics, such as thermal dissipation of excited chlorophyll states (non-photochemical quenching, NPQ) and regulation of electron transport. The regulatory mechanisms involved in the response to light dynamics have adapted during evolution, and exploring biodiversity is a valuable strategy for expanding our understanding of their biological roles. In this work, we investigated the response to fluctuating light in Nannochloropsis gaditana, a eukaryotic microalga of the phylum Heterokonta originating from a secondary endosymbiotic event. Nannochloropsis gaditana is negatively affected by light fluctuations, leading to large reductions in growth and photosynthetic electron transport. Exposure to light fluctuations specifically damages photosystem I, likely because of the ineffective regulation of electron transport in this species. The role of NPQ, also assessed using a mutant strain specifically depleted of this response, was instead found to be minor, especially in responding to the fastest light fluctuations.

Red-shifted light-harvesting system of freshwater eukaryotic alga Trachydiscus minutus (Eustigmatophyta, Stramenopila).

Survival of phototrophic organisms depends on their ability to collect and convert enough light energy to support their metabolism. Phototrophs can extend their absorption cross section by using diverse pigments and by tuning the properties of these pigments via pigment-pigment and pigment-protein interaction. It is well known that some cyanobacteria can grow in heavily shaded habitats by utilizing far-red light harvested with far-red-absorbing chlorophylls d and f. We describe a red-shifted light-harvesting system based on chlorophyll a from a freshwater eustigmatophyte alga Trachydiscus minutus (Eustigmatophyceae, Goniochloridales). A comprehensive characterization of the photosynthetic apparatus of T. minutus is presented. We show that thylakoid membranes of T. minutus contain light-harvesting complexes of several sizes differing in the relative amount of far-red chlorophyll a forms absorbing around 700 nm. The pigment arrangement of the major red-shifted light-harvesting complex is similar to that of the red-shifted antenna of a marine alveolate alga Chromera velia. Evolutionary aspects of the algal far-red light-harvesting complexes are discussed. The presence of these antennas in eustigmatophyte algae opens up new ways to modify organisms of this promising group for effective use of far-red light in mass cultures.

Excitation energy transfer in the far-red absorbing violaxanthin/vaucheriaxanthin chlorophyll a complex from the eustigmatophyte alga FP5.

This work highlights spectroscopic investigations on a new representative of photosynthetic antenna complexes in the LHC family, a putative violaxanthin/vaucheriaxanthin chlorophyll a (VCP) antenna complex from a freshwater Eustigmatophyte alga FP5. A representative VCP-like complex, named as VCP-B3 was studied with both static and time-resolved spectroscopies with the aim of obtaining a deeper understanding of excitation energy migration within the pigment array of the complex. Compared to other VCP representatives, the absorption spectrum of the VCP-B3 is strongly altered in the range of the chlorophyll a Qy band, and is substantially red-shifted with the longest wavelength absorption band at 707 nm at 77 K. VCP-B3 shows a moderate xanthophyll-to-chlorophyll a efficiency of excitation energy transfer in the 50-60% range, 20-30% lower from comparable VCP complexes from other organisms. Transient absorption studies accompanied by detailed data fitting and simulations support the idea that the xanthophylls that occupy the central part of the complex, complementary to luteins in the LHCII, are violaxanthins. Target analysis suggests that the primary route of xanthophyll-to-chlorophyll a energy transfer occurs via the xanthophyll S1 state.

Optimum Production Conditions, Purification, Identification, and Antioxidant Activity of Violaxanthin from Microalga Eustigmatos cf. polyphem (Eustigmatophyceae).

Violaxanthin is a major xanthophyll pigment in the microalga Eustigmatos cf. polyphem, but the amount produced after propagation can vary depending upon culture conditions. In this study, the effects of cultivation time, nitrogen concentration, light intensity, and culture mode on violaxanthin production were investigated. The results showed that this microalga vigorously grew and maintained a high level of violaxanthin in the fed-batch culture, and the highest violaxanthin productivity of 1.10 ± 0.03 mg L-1 d-1 was obtained under low light illumination with 18 mM of initial nitrogen supply for ten days. Additionally, violaxanthin was purified from E. cf. polyphem by silica gel chromatography and preparative high-performance liquid chromatography (PHPLC), and identified with high-resolution mass spectrometry (HRMS). The antioxidant activity of the purified violaxanthin was evaluated by three tests in vitro: reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2-azobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical assays. The strongest inhibition of purified violaxanthin occurred during the scavenging of ABTS⁺ radicals, with EC50 of 15.25 µg mL-1. In conclusion, this is the first report to investigate the effects of different culture conditions on violaxanthin accumulation in E. cf. polyphem and provide a novel source for the production of violaxanthin that can be used for food and pharmaceutical applications.

Simultaneous quantum yield measurements of carbon uptake and oxygen evolution in microalgal cultures.

The photosynthetic quantum yield (Φ), defined as carbon fixed or oxygen evolved per unit of light absorbed, is a fundamental but rarely determined biophysical parameter. A method to estimate Φ for both net carbon uptake and net oxygen evolution simultaneously can provide important insights into energy and mass fluxes. Here we present details for a novel system that allows quantification of carbon fluxes using pH oscillation and simultaneous oxygen fluxes by integration with a membrane inlet mass spectrometer. The pHOS system was validated using Phaeodactylum tricornutum cultured with continuous illumination of 110 µmole quanta m-2 s-1 at 25°C. Furthermore, simultaneous measurements of carbon and oxygen flux using the pHOS-MIMS and photon flux based on spectral absorption were carried out to explore the kinetics of Φ in P. tricornutum during its acclimation from low to high light (110 to 750 µmole quanta m-2 s-1). Comparing results at 0 and 24 hours, we observed strong decreases in cellular chlorophyll a (0.58 to 0.21 pg cell-1), Fv/Fm (0.71 to 0.59) and maximum ΦCO2 (0.019 to 0.004) and ΦO2 (0.028 to 0.007), confirming the transition toward high light acclimation. The Φ time-series indicated a non-synchronized acclimation response between carbon uptake and oxygen evolution, which has been previously inferred based on transcriptomic changes for a similar experimental design with the same diatom that lacked physiological data. The integrated pHOS-MIMS system can provide simultaneous carbon and oxygen measurements accurately, and at the time-resolution required to resolve high-resolution carbon and oxygen physiological dynamics.

Dimeric Structure of the Blue Light Sensor Protein Photozipper in the Active State.

The light oxygen voltage-sensing (LOV) domain plays a crucial role in blue light (BL) sensing in plants and microorganisms. LOV domains are usually associated with the effector domains and regulate the activities of effector domains in a BL-dependent manner. Photozipper (PZ) is monomeric in the dark state. BL induces reversible dimerization of PZ and subsequently increases its affinity for the target DNA sequence. In this study, we report the analyses of PZ by pulsed electron-electron double resonance (PELDOR). The neutral flavin radical was formed by BL illumination in the presence of dithiothreitol in the LOV-C254S (without the bZIP domain) and PZ-C254S mutants, where the cysteine residue responsible for adduct formation was replaced with serine. The magnetic dipole interactions of 3 MHz between the neutral radicals were detected in both LOV-C254S and PZ-C254S, indicating that these mutants are dimeric in the radical state. The PELDOR simulation showed that the distance between the radical pair is close to that estimated from the dimeric crystal structure in the "light state" [Heintz, U., and Schlichting, I. (2016) eLife 5, e11860], suggesting that in the radical state, LOV domains in PZ-C254S form a dimer similar to that of LOV-C254S, which lacks the bZIP domain.

Photoprotection strategies of the alga Nannochloropsis gaditana.

Nannochloropsis spp. are algae with high potential for biotechnological applications due to their capacity to accumulate lipids. However, little is known about their photosynthetic apparatus and acclimation/photoprotective strategies. In this work, we studied the mechanisms of non-photochemical quenching (NPQ), the fast response to high light stress, in Nannochloropsis gaditana by "locking" the cells in six different states during quenching activation and relaxation. Combining biochemical analysis with time-resolved fluorescence spectroscopy, we correlated each NPQ state with the presence of two well-known NPQ components: de-epoxidized xanthophylls and stress-related antenna proteins (LHCXs). We demonstrated that after exposure to strong light, the rapid quenching that takes place in the antennas of both photosystems was associated with the presence of LHCXs. At later stages, quenching occurs mainly in the antennas of PSII and correlates with the amount of de-epoxidised xanthophylls. We also observed changes in the distribution of excitation energy between photosystems, which suggests redistribution of excitation between photosystems as part of the photo-protective strategy. A multistep model for NPQ induction and relaxation in N. gaditana is discussed.

The plastoquinone pool of Nannochloropsis oceanica is not completely reduced during bright light pulses.

The lipid-producing model alga Nannochloropsis oceanica has a distinct photosynthetic machinery. This organism possesses chlorophyll a as its only chlorophyll species, and has a high ratio of PSI to PSII. This high ratio of PSI to PSII may affect the redox state of the plastoquinone pool during exposure to light, and consequently may play a role in activating photoprotection mechanisms. We utilized pulse-amplitude modulated fluorometry to investigate the redox state of the plastoquinone pool during and after bright light pulses. Our data indicate that even very intense (5910 µmol photons s-1m-2 of blue light having a wavelength of 440 nm) light pulses of 0.8 second duration are not sufficient to completely reduce the plastoquinone pool in Nannochloropsis. In order to achieve extensive reduction of the plastoquinone pool by bright light pulses, anaerobic conditions or an inhibitor of the photosynthetic electron transport chain has to be utilized. The implication of this finding for the application of the widely used saturating pulse method in algae is discussed.

Photoacclimation of photosynthesis in the Eustigmatophycean Nannochloropsis gaditana.

Nannochloropsis is an eukaryotic alga of the phylum Heterokonta, originating from a secondary endosymbiotic event. In this work, we investigated how the photosynthetic apparatus responds to growth in different light regimes in Nannochloropsis gaditana. We found that intense illumination induces the decrease of both photosystem I and II contents and their respective antenna sizes. Cells grown in high light showed a larger capacity for electron transport, with enhanced cyclic electron transport around photosystem I, contributing to photoprotection from excess illumination. Even when exposed to excess light intensities for several days, N. gaditana cells did not activate constitutive responses such as nonphotochemical quenching and the xanthophyll cycle. These photoprotection mechanisms in N. gaditana thus play a role in acclimation to fast changes in illumination within a time range of minutes, while regulation of the electron flow capacity represents a long-term response to prolonged exposure to excess light.

Light Remodels Lipid Biosynthesis in Nannochloropsis gaditana by Modulating Carbon Partitioning between Organelles.

The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid, and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomo-Ser and triacylglycerols, together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the down-regulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion, and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in N. gaditana and lay the foundations for a hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the subcellular level.

Blue-light-regulated transcription factor, Aureochrome, in photosynthetic stramenopiles.

During the course of evolution through various endosymbiotic processes, diverse photosynthetic eukaryotes acquired blue light (BL) responses that do not use photosynthetic pathways. Photosynthetic stramenopiles, which have red algae-derived chloroplasts through secondary symbiosis, are principal primary producers in aquatic environments, and play important roles in ecosystems and aquaculture. Through secondary symbiosis, these taxa acquired BL responses, such as phototropism, chloroplast photo-relocation movement, and photomorphogenesis similar to those which green plants acquired through primary symbiosis. Photosynthetic stramenopile BL receptors were undefined until the discovery in 2007, of a new type of BL receptor, the aureochrome (AUREO), from the photosynthetic stramenopile alga, Vaucheria. AUREO has a bZIP domain and a LOV domain, and thus BL-responsive transcription factor. AUREO orthologs are only conserved in photosynthetic stramenopiles, such as brown algae, diatoms, and red tide algae. Here, a brief review is presented of the role of AUREOs as photoreceptors for these diverse BL responses and their biochemical properties in photosynthetic stramenopiles.

Effect of specific light supply rate on photosynthetic efficiency of Nannochloropsis salina in a continuous flat plate photobioreactor.

In this work, Nannochloropsis salina was cultivated in a continuous-flow flat-plate photobioreactor, working at different residence times and irradiations to study the effect of the specific light supply rate on biomass productivity and photosynthetic efficiency. Changes in residence times lead to different steady-state cell concentrations and specific growth rates. We observed that cultures at steady concentration were exposed to different values of light intensity per cell. This specific light supply rate was shown to affect the photosynthetic status of the cells, monitored by fluorescence measurements. High specific light supply rate can lead to saturation and photoinhibition phenomena if the biomass concentration is not optimized for the selected operating conditions. Energy balances were applied to quantify the biomass growth yield and maintenance requirements in N. salina cells.

Screening for Suitable Reference Genes for Quantitative Real-Time PCR in Heterosigma akashiwo (Raphidophyceae).

The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters. To understand the mechanisms of H. akashiwo bloom formation, gene expression analysis is often required. To accurately characterize the expression levels of a gene of interest, proper reference genes are essential. In this study, we assessed ten of the previously reported algal candidate genes (rpL17-2, rpL23, cox2, cal, tua, tub, ef1, 18S, gapdh, and mdh) for their suitability as reference genes in this species. We used qRT-PCR to quantify the expression levels of these genes in H. akashiwo grown under different temperatures, light intensities, nutrient concentrations, and time points over a diel cycle. The expression stability of these genes was evaluated using geNorm and NormFinder algorithms. Although none of these genes exhibited invariable expression levels, cal, tub, rpL17-2 and rpL23 expression levels were the most stable across the different conditions tested. For further validation, these selected genes were used to normalize the expression levels of ribulose-1, 5-bisphosphate carboxylase/oxygenase large unite (HrbcL) over a diel cycle. Results showed that the expression of HrbcL normalized against each of these reference genes was the highest at midday and lowest at midnight, similar to the diel patterns typically documented for this gene in algae. While the validated reference genes will be useful for future gene expression studies on H. akashiwo, we expect that the procedure used in this study may be helpful to future efforts to screen reference genes for other algae.

Metabolic switching: synergistic induction of carotenogenesis in the aerial microalga, Vischeria helvetica, under environmental stress conditions by inhibitors of fatty acid biosynthesis.

OBJECTIVES: To investigate the roles of fatty acid biosynthesis in carotenogenesis in the high-lipid accumulating aerial microalga Vischeria helvetica KGU-Y001, we cultured algal cells with fatty acid biosynthesis inhibitors. RESULTS: Under nitrogen-deficient, high-light (200 µmol photons m(-2) s(-1)) conditions, the alga accumulated 6.2 mg carotenoids g(-1) dry weight cells (DWC) after 1 week of culture. The total fatty acid content increased gradually, and reached 290 mg g(-1) DWC after 9 weeks. When algal cells were cultured with a fatty acid biosynthesis inhibitor (molinate) under nitrogen-deficient, high-light conditions for 1 week, carotenoid accumulation was synergistically increased to 2.4 times that in algal cells cultured without the inhibitor in nitrogen-deficient, low-light conditions (40 µmol photons m(-2) s(-1)). The synergistic induction of carotenogenesis was suppressed by an inhibitor of c-jun N-terminal kinase, a mitogen-activated protein kinase-like protein. CONCLUSION: In a commercial context, carotenoid production could be increased by using fatty acid biosynthesis inhibitors to redirect metabolic flux to carotenoid biosynthesis instead of fatty acid synthesis.

Rapid induction of omega-3 fatty acids (EPA) in Nannochloropsis sp. by UV-C radiation.

Omega-3 fatty acids, such as eicosapentaenoic acid (EPA), provide substantial health benefits. As global fish stocks are declining and in some cases are contaminated with heavy metals, there is a need to find more sustainable land-based sources of these essential fatty acids. The oleaginous microalga Nannochloropsis sp. has been identified as a highly efficient producer of omega-3 fatty acids. In this study, we present a new process to rapidly induce biosynthesis of essential fatty acids, including EPA in Nannochloropsis sp. BR2. Short exposure to UV-C at a dose of 100 or 250 mJ/cm(2) led to a significant increase in total cellular lipid contents when compared to mock-treated controls. A low dosage of 100 mJ/cm(2) also led to a twofold increase in total EPA content within 24 h that constituted 30% of total fatty acids and up to 12% of total dry weight at higher dosages. UV-C radiation may find uses as an easily applicable external inducer for large-scale production of omega-3 production from microalgae.

Light-induced extracellular electron transport by the marine raphidophyte Chattonella marina.

There is increasing interest in extracellular electron transfer (EET) from organisms to receptors, particularly in anaerobic biofilms at mineral surfaces. Less attention has been given to EET by planktonic organisms in oxic environments where extracellular electron generation and transport might be expected to be of limited consequence. In this study, the EET activity of the photosynthetic marine raphidophyte, Chattonella marina, was examined using a mediatorless photosynthetic microbial fuel cell with results showing positive light response. Electron output by organisms present in cell suspension was substantially higher than those present in biofilms at the electrode surface. Indeed, current generation under light illumination of the C. marina suspension continued even when contact between the organisms and the electrodes was prevented by dialysis membrane, suggesting that soluble electron carriers secreted by C. marina were facilitating the EET process. Cyclic voltammetry measurements of the cell-free exudate showed redox peaks in the range of 0.1-0.5 V (vs Ag/AgCl), confirming that redox active species were present in the cell suspension. Facilitation of electron transfer from the planktonic organism to the anode by endogenous redox-active exudates appears to be critical to current generation. The ability of these exudates to remain in their reduced state in the presence of oxygen is possibly a function of the spin-restricted nature of oxygen-mediated exudate oxidation. Quantification of the EET processes operating in this planktonic system assists in understanding the means and extent to which C. marina induces redox transformations in the external medium with these transformations presumably of benefit to the survival of this organism, potentially including facilitation of iron uptake and induction of toxicity to other organisms.

Comparing the diel vertical migration of Karlodinium veneficum (dinophyceae) and Chattonella subsalsa (Raphidophyceae): PSII photochemistry, circadian control, and carbon assimilation.

Diel vertical migration (DVM) is thought to provide an adaptive advantage to some phytoplankton, and may help determine the ecological niche of certain harmful algae. Here we separately compared DVM patterns between two species of harmful algae isolated from the Delaware Inland Bays, Karlodinium veneficum and Chattonella subsalsa, in laboratory columns. We interpreted the DVM patterns of each species with Photosystem II (PSII) photochemistry, rates of carbon assimilation, and specific growth rates. Each species migrated differently, wherein K. veneficum migrated closer to the surface each day with high population synchrony, while C. subsalsa migrated near to the surface from the first day of measurements with low population synchrony. Both species appeared to downregulate PSII in high light at the surface, but by different mechanisms. C. subsalsa grew slower than K. veneficum in low light intensities (≈bottom of columns), and exhibited maximal rates of C-assimilation (Pmax) at surface light intensities, suggesting this species may prefer high light, potentially explaining this species' rapid surface migration. Contrastingly, K. veneficum showed declines in carbon assimilation at surface light intensities, and exhibited a smaller reduction in growth at low (bottom) light intensities (compared to C. subsalsa), suggesting that this species' step-wise migration was photoacclimative and determined daily migration depth. DVM was found to be under circadian control in C. subsalsa, but not in K. veneficum. However, there was little evidence for circadian regulation of PSII photochemistry in either species. Migration conformed to each species' physiology, and the results contribute to our understanding each alga's realized environmental niche.

The use of Design of Experiments and Response Surface Methodology to optimize biomass and lipid production by the oleaginous marine green alga, Nannochloropsis gaditana in response to light intensity, inoculum size and CO2.

Biodiesel produced from microalgal lipids is being considered as a potential source of renewable energy. However, a number of hurdles will have to be overcome if such a process is to become practical. One important factor is the volumetric production of biomass and lipid that can be achieved. The marine alga Nannochloropsis gaditana is under study since it is known to be highly oleaginous and has a number of other attractive properties. Factors that might be important in biomass and lipid production by this alga are light intensity, inoculum size and CO2. Here we have carried out for the first time a RSM-DOE study of the influence of these important culture variables and define conditions that maximize biomass production, lipid content (BODIPY® fluorescence) and total lipid production. Moreover, flow cytometry allowed the examination on a cellular level of changes that occur in cellular populations as they age and accumulate lipids.