Light-Modulated Responses of Growth and Photosynthetic Performance to Ocean Acidification in the Model Diatom Phaeodactylum tricornutum

Ocean acidification (OA) due to atmospheric CO₂ rise is expected to influence marine primary productivity. In order to investigate the interactive effects of OA and light changes on diatoms, we grew Phaeodactylum tricornutum, under ambient (390 ppmv; LC) and elevated CO₂ (1000 ppmv; HC) conditions for 80 generations, and measured its physiological performance under different light levels (60 µmol m⁻² s⁻¹, LL; 200 µmol m⁻² s⁻¹, ML; 460 µmol m⁻² s⁻¹, HL) for another 25 generations. The specific growth rate of the HC-grown cells was higher (about 12–18%) than that of the LC-grown ones, with the highest under the ML level. With increasing light levels, the effective photochemical yield of PSII (F_v′/F_m′) decreased, but was enhanced by the elevated CO₂, especially under the HL level. The cells acclimated to the HC condition showed a higher recovery rate of their photochemical yield of PSII compared to the LC-grown cells. For the HC-grown cells, dissolved inorganic carbon or CO₂ levels for half saturation of photosynthesis (K_1/2 DIC or K_1/2 CO₂) increased by 11, 55 and 32%, under the LL, ML and HL levels, reflecting a light dependent down-regulation of carbon concentrating mechanisms (CCMs). The linkage between higher level of the CCMs down-regulation and higher growth rate at ML under OA supports the theory that the saved energy from CCMs down-regulation adds on to enhance the growth of the diatom.     

Interactive Effects of Ocean Acidification and Nitrogen-Limitation on the Diatom Phaeodactylum tricornutum

Climate change is expected to bring about alterations in the marine physical and chemical environment that will induce changes in the concentration of dissolved CO₂ and in nutrient availability. These in turn are expected to affect the physiological performance of phytoplankton. In order to learn how phytoplankton respond to the predicted scenario of increased CO₂ and decreased nitrogen in the surface mixed layer, we investigated the diatom Phaeodactylum tricornutum as a model organism. The cells were cultured in both low CO₂ (390 μatm) and high CO₂ (1000 μatm) conditions at limiting (10 μmol L⁻¹) or enriched (110 μmol L⁻¹) nitrate concentrations. Our study shows that nitrogen limitation resulted in significant decreases in cell size, pigmentation, growth rate and effective quantum yield of Phaeodactylum tricornutum, but these parameters were not affected by enhanced dissolved CO₂ and lowered pH. However, increased CO₂ concentration induced higher rETR_max and higher dark respiration rates and decreased the CO₂ or dissolved inorganic carbon (DIC) affinity for electron transfer (shown by higher values for K_{1/2 DIC} or K_{1/2 CO2}). Furthermore, the elemental stoichiometry (carbon to nitrogen ratio) was raised under high CO₂ conditions in both nitrogen limited and nitrogen replete conditions, with the ratio in the high CO₂ and low nitrate grown cells being higher by 45% compared to that in the low CO₂ and nitrate replete grown ones. Our results suggest that while nitrogen limitation had a greater effect than ocean acidification, the combined effects of both factors could act synergistically to affect marine diatoms and related biogeochemical cycles in future oceans.     

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.     

Towards the Industrial Production of Omega-3 Long Chain Polyunsaturated Fatty Acids from a Genetically Modified Diatom Phaeodactylum tricornutum

The marine diatom Phaeodactylum tricornutum can accumulate up to 30% of the omega-3 long chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA) and, as such, is considered a good source for the industrial production of EPA. However, P. tricornutum does not naturally accumulate significant levels of the more valuable omega-3 LC-PUFA docosahexaenoic acid (DHA). Previously, we have engineered P. tricornutum to accumulate elevated levels of DHA and docosapentaenoic acid (DPA) by overexpressing heterologous genes encoding enzyme activities of the LC-PUFA biosynthetic pathway. Here, the transgenic strain Pt_Elo5 has been investigated for the scalable production of EPA and DHA. Studies have been performed at the laboratory scale on the cultures growing in up to 1 L flasks a 3.5 L bubble column, a 550 L closed photobioreactor and a 1250 L raceway pond with artificial illumination. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt_Elo5 and wild type P. tricornutum grown in 3.5 L bubble columns. The highest content of DHA (7.5% of total fatty acids, TFA) in transgenic strain was achieved in cultures grown in seawater salts, Instant Ocean (IO), supplemented with F/2 nutrients (F2N) under continuous light. After identifying the optimal conditions for omega-3 LC-PUFA accumulation in the small-scale experiments we compared EPA and DHA levels of the transgenic strain grown in a larger fence-style tubular photobioreactor and a raceway pond. We observed a significant production of DHA over EPA, generating an EPA/DPA/DHA profile of 8.7%/4.5%/12.3% of TFA in cells grown in a photobioreactor, equivalent to 6.4 μg/mg dry weight DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond at ambient temperature but supplemented with artificial illumination (110 μmol photons m^-2s^-1 ) on a 16:8h light:dark cycle, in natural seawater and F/2 nutrients was 24.8% EPA and 10.3% DHA. Transgenic strain grown in RP produced the highest levels of EPA (12.8%) incorporated in neutral lipids. However, the highest partitioning of DHA in neutral lipids was observed in cultures grown in PBR (7.1%). Our results clearly demonstrate the potential for the development of the transgenic Pt_Elo5 as a platform for the commercial production of EPA and DHA.     

Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum

Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.     

Regulation of Transpiration in Avena. Responses to Red and Blue Light Steps

The transpiration responses of primary Arena leaves to red and blue light steps were investigated. The response to a red light step was a so-called slow response (with a rise time of about 8 min). The response to a blue light step consisted of both a slow, and a rapid response (with a rise time of about 2 min). CO₂-free air outside the leaf eliminated only the slow response, i.e. in CO₂-free ait the plant responded to blue light steps but not to red ones. A short exposure of red light prior to a blue light step enhanced the rapid response. The same enhancement of the rapid response could be achieved by means of a temporary pretreatment with CO₂-free air. The magnitude of the rapid response was dependent on the blue light irradiance and no threshold effects could be detected. — The experimental results are discussed by means of a model, based on stomatal regulation by, ion transport between the subsidiary cells and the guard cells. It is suggested that the slow transpiration response is due to CO₂-regulation of the stomatal aperture and that the rapid response reflect a CO₂-independent blue light sensitive process, which acts directly on the ion transport through the subsidiary and guard cell membranes.     

Potassium and Photosynthesis in the Marine Diatom Phaeodactylum tricornutum as Related to Washes with Sodium Chloride

Three washes of Phaeodactylum tricornutum with potassium free sodium chloride solution reduced the potassium content of the cells to approximately 7% of the control value at pH 7.0. There was a concomitant reduction of the light induced evolution of oxygen to a value of approximately 20% of the control value. This reduction was less at pH 8.0. Addition of potassium to the washed cells gave rise, after 15 min, to a partial regain of the photosynthetic activity and of cellular potassium. Activities of the two photosystems as assayed here were not dependent on the maintainance of a high potassium content in the cells. The level of ribulose-1,5-diphosphate carboxylase activity was the same whether the cells had been washed with potassium free or potassium containing saline. Ruptured cells rapidly lost their ability to catalyse the photoreduction of dichlorophenolindophenol by water.     

The Effects of Excess Irradiance on Photosynthesis in the Marine Diatom Phaeodactylum tricornutum

The response of Phaeodactylum tricornutum to excess light was remarkably similar to that observed in higher plants and green algae and was characterized by complex changes in minimal fluorescence yields of fully dark-adapted samples and declines in maximum variable fluorescence levels and oxygen evolution rates. In our study the parallel decreases in the effective rate constant for photosystem II (PSII) photochemistry, the variable fluorescence yield of a dark-adapted sample, and light-limited O2 evolution rates after short (0-10 min) exposures to photoinhibitory conditions could not be attributed to damage or down-regulation of PSII reaction centers. Instead, these changes were consistent with the presence of nonphotochemical quenching of PSII excitation energy in the antennae. This quenching was analogous to that component of nonphotochemical quenching studied in higher plants that is associated with photoinhibition of photosynthesis and/or processes protecting against photoinhibition in that it did not relax readily in the dark and persisted in the absence of a bulk transthylakoid proton gradient. The quenching was most likely associated with photoprotective processes in the PSII antenna that reduced the extent of photoinhibitory damage, particularly after longer exposures. Our results suggest that a large population of damaged, slowly recovering PSII centers did not form in Phaeodactylum even after 60 min of exposure to excess actinic light.     

Improved Blue,Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.     

Dynamics of Lipid Biosynthesis and Redistribution in the Marine Diatom Phaeodactylum tricornutum Under Nitrate Deprivation

One approach to achieve continuous overproduction of lipids in microalgal ??cell factories?? relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO₂ fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using ¹³C-bicarbonate, that cell growth in nitrate (NO ₃ ^? )-deprived cultures resulted predominantly in de novo lipid synthesis (60?% over 3?days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of ¹²C occurred in all previously existing TAGs in NO ₃ ^? -deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO ₃ ^? -deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO ₃ ^? replete cultures. Liquid chromatography-tandem mass spectrometry mass spectra and ¹³C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 ¹³C carbons, indicating substantial incorporation of ¹³C-bicarbonate into fatty acid chains under both nutrient states.     

Flowering of Cultivated Green and SAN 9789-Treated Chenopodium rubrum Plants Exposed to White,Blue, and Red Light

Green plants and plants devoid of photosynthetic pigments were compared with regard to their ability to flower under various growth conditions. Green plants of Chenopodium rubrum L. and plants treated with norflurazon SANDOZ-9789 (SAN) were grown on sucrose-containing media with or without hormones (GA₃, BA, IAA, ABA) under short-day photoperiodic or continuous illumination with white, blue, or red light. Green and SAN-treated albino plants produced flowers only under short-day conditions. The flowering of green plants was independent of the presence of sucrose and hormones in the medium as well as of the light quality. The albino plants produced flowers under white and blue light but did not flower in red light. The addition of GA₃ or BA to the medium induced flowering of albino plants exposed to red light. The functional interaction of photoreceptors in the flowering control is discussed.     

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.     

Circadian Rhythms in Stomatal Responsiveness to Red and Blue Light

Stomata of many plants have circadian rhythms in responsiveness to environmental cues as well as circadian rhythms in aperture. Stomatal responses to red light and blue light are mediated by photosynthetic photoreceptors; responses to blue light are additionally controlled by a specific blue-light photoreceptor. This paper describes circadian rhythmic aspects of stomatal responsiveness to red and blue light in Vicia faba. Plants were exposed to a repeated light:dark regime of 1.5:2.5 h for a total of 48 h, and because the plants could not entrain to this short light:dark cycle, circadian rhythms were able to "free run" as if in continuous light. The rhythm in the stomatal conductance established during the 1.5-h light periods was caused both by a rhythm in sensitivity to light and by a rhythm in the stomatal conductance established during the preceding 2.5-h dark periods. Both rhythms peaked during the middle of the subjective day. Although the stomatal response to blue light is greater than the response to red light at all times of day, there was no discernible difference in period, phase, or amplitude of the rhythm in sensitivity to the two light qualities. We observed no circadian rhythmicity in net carbon assimilation with the 1.5:2.5 h light regime for either red or blue light. In continuous white light, small rhythmic changes in photosynthetic assimilation were observed, but at relatively high light levels, and these appeared to be attributable largely to changes in internal CO2 availability governed by stomatal conductance.     

Effects of Metal Combinations on the Production of Phytochelatins and Glutathione by the Marine Diatom Phaeodactylum tricornutum

Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 μM Cu² and 0.45 μM Cd²⁺) to the culture medium induced the production of short-chained phytochelatins ((γ-Glu-Cys)_n-Gly where n = 2–5), whereas a single addition of Zn (2.2 μM Zn²⁺) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.