Regulation of glyceraldehyde-3-phosphate dehydrogenase in the eustigmatophyte Pseudocharaciopsis ovalis is intermediate between a chlorophyte and a diatom

The regulation of NADPH-dependent GAPDH was analysed in the chromalveolate (eustigmatophyte) Pseudocharaciopsis ovalis and compared with the well-studied chlorophyte Chlamydomonas reinhardtii and with another chromalveolate(diatom), Asterionella formosa. Optimal pH for GAPDH activity in P. ovalis and C. reinhardtii ranged between 8 and 9, but in A. formosa ranged between 6.2 and 8.1. Assuming dark pH values of about 7 in the plastids of all three species, GAPDH would be down-regulated in the dark in C. reinhardtii and P. ovalis, but fully active in A. formosa. The time required for halfmaximal GAPDH activity on transfer to reducing conditions, was significantly different in each species: 1.4, 4.0 and 5.9 min in A. formosa, P. ovalis and C. reinhardtii respectively. Under oxidized conditions in P. ovalis and A. formosa, NADPH caused a large inhibition in GAPDH activity even at very low concentrations (10 to 20 mM) unlike in C. reinhardtii. This inhibition was relieved by addition of a reducing agent suggesting that NADPH can control GAPDH activity under dark-light transitions. A small increase of GAPDH activity with NADP at concentrations higher than 0.5mM was observed with P. ovalis and C. reinhardtii, while a greater than 1.5-fold stimulation was observed in A. formosa. Regulation of GAPDH in P. ovalis was intermediate between the diatom and the chlorophyte and the possible evolutionary reasons for this are discussed

Production of neutral lipids in Asterionella formosa and regulation of metabolism

La diatomée d'eau douce A. formosa peut produire des lipides neutres en plus ou moins grandes quantités en fonction des conditions de culture. Ainsi, nous avons montré par exemple qu'une carence en silice stimule la production de triacylglycérols (TAGs) mais génère une diminution de la biomasse. En revanche, nous avons montré que l'addition de bicarbonate et de phytohormones augmente à la fois la biomasse et la production de TAGs. L'ajout de phytohormones dans les milieux de culture de cette diatomée résulte en une augmentation de l'activité d'enzymes dans les extraits et notamment celles du cycle de Benson-Calvin. Parmi ces enzymes, la GAPDH est une enzyme dont l'activité augmente significativement. Nous avons montré que chez A. formosa, cette enzyme forme un complexe ternaire avec la CP12 et la Férrédoxine NADP Réductase (FNR) et non pas avec la CP12 et la phosphoribulokinase comme chez la plupart des organismes photosynthétiques. La régulation de cette enzyme en est de fait modifiée. La phytohormone, 24-épibrassinolide conduit à une augmentation d'activité de la GAPDH qui résulte de la dissociation du complexe GAPDH-CP12 et la GAPDH n'est plus redox régulée. La GAPDH chez les diatomées est donc régulée par des interactions protéineprotéine.A. formosa, a freshwater diatom, can produce different amounts of neutral lipids such as triacylglycerols (TAGs) under different growth conditions. We showed that as it is well-known for diatoms, starvation for silica increased the production of TAGs but decreased biomass. However, the addition of bicarbonate or phytohormones into the growth medium increased both biomass and TAGs. Addition of phytohormones increased the activities of enzymes in particular those of the Benson-Calvin cycle. Among the target enzymes of the Benson-Calvin cycle, GAPDH was strongly affected. We purified this enzyme and demonstrated that, in the diatom A. formosa, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the Calvin cycle, forms a complex with the small chloroplast protein CP12 and Ferredoxin NADP Reductase (FNR), which is involved in the photochemical phase of photosynthesis. In cells treated with the phytohormone, 24-epibrassinolide, GAPDH was "free", not redox-regulated and not associated anymore with CP12. Therefore GAPDH from this diatom is regulated by protein-protein interaction but the GAPDH/CP12/FNR complex replaces the one formed between GAPDH, CP12 and phosphoribulokinase found in most photoautotrophs

Glyceraldehyde-3-phosphate dehydrogenase is regulated by ferredoxin-NADP reductase in the diatom Asterionella formosa

Diatoms are a widespread and ecologically important group of heterokont algae that contribute about 20% to global productivity. Previous work has shown that regulation of key Calvin cycle enzymes in diatoms differs from that of the Plantae, and that in crude extracts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be inhibited by NADPH under oxidizing conditions. Here, chromatography, mass spectrometry and sequence analysis showed that in the freshwater diatom, Asterionella formosa, GAPDH can interact with ferredoxin-NADP reductase (FNR) from the primary phase of photosynthesis, and the small chloroplast protein, CP12. In contrast, the ternary complex between GAPDH, phosphoribulokinase (PRK) and CP12, that is widespread in Plantae and cyanobacteria, was absent. Surface plasmon resonance measurements confirmed that GAPDH and FNR are able to interact. Activity measurements under oxidizing conditions, showed that NADPH can inhibit GAPDH-CP12 in the presence of FNR from A. formosa or Spinacia oleracea, explaining the earlier observed inhibition within crude extracts. Diatom plastids have distinctive attributes including the lack of the oxidative pentose phosphate pathway and so cannot produce NADPH in the dark. The observed down-regulation of GAPDH may allow NADPH to be re-routed towards other reductive processes contributing to their ecological success

Effect of environmental conditions on various enzyme activities and triacylglycerol contents in cultures of the freshwater diatom, Asterionella formosa (Bacillariophyceae)

International audienceA detailed analysis of triacylglycerols (TAGs) contents, fatty acid patterns and key enzyme activities in the freshwater diatom Asterionella formosa was performed under various conditions, including nitrate, iron and silicon limitation (stress conditions), or bicarbonate and phytohormones supplementation (stimulation conditions). Of all the conditions tested, the addition of bicarbonate produced the greatest increase (5-fold) in TAGs contents compared to the control while the biomass increased. The addition of phytohormones also allowed a significant increase in TAGs of about 3-fold while the biomass increased. Silicon, unlike iron and nitrate limitation, also triggered a significant increase in TAGs contents of 3.5-fold but negatively affected the biomass. Analysis of fatty acid profiles showed that the mono-unsaturated C16:1 fatty acid was the most abundant in A. formosa, followed by C16:0, C14:0 and eicosapentaenoic acid (EPA; C20:5 n-3). EPA levels were found to increase under nitrate and iron limitation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), phosphofructokinase (PFK), glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) activities differed with growth conditions. Most enzymes were up-regulated in stimulated cells while in the case of stressed cells, the pattern of activities was more variable. Detailed analysis of all enzyme activities showed that the most important enzyme among those tested was GAPDH which could be a good candidate for genetic engineering of high lipid-producing algae. This study provides a better understanding of key enzymes and biochemical pathways involved in lipid accumulation processes in diatoms

Flavodiiron Proteins Promote Fast and Transient O 2 Photoreduction in Chlamydomonas

International audienceDuring oxygenic photosynthesis, the reducing power generated by light energy conversion is mainly used to reduce carbon dioxide. In bacteria and archae, flavodiiron (Flv) proteins catalyze O 2 or NO reduction, thus protecting cells against oxidative or nitrosative stress. These proteins are found in cyanobacteria, mosses, and microalgae, but have been lost in angiosperms. Here, we used chlorophyll fluorescence and oxygen exchange measurement using [ 18 O]-labeled O 2 and a membrane inlet mass spectrometer to characterize Chlamydomonas reinhardtii flvB insertion mutants devoid of both FlvB and FlvA proteins. We show that Flv proteins are involved in a photo-dependent electron flow to oxygen, which drives most of the photosynthetic electron flow during the induction of photosynthesis. As a consequence, the chlorophyll fluorescence patterns are strongly affected in flvB mutants during a light transient, showing a lower PSII operating yield and a slower nonphotochemical quenching induction. Photoautotrophic growth of flvB mutants was indistinguishable from the wild type under constant light, but severely impaired under fluctuating light due to PSI photo damage. Remarkably, net photosynthesis of flv mutants was higher than in the wild type during the initial hour of a fluctuating light regime, but this advantage vanished under long-term exposure, and turned into PSI photo damage, thus explaining the marked growth retardation observed in these conditions. We conclude that the C. reinhardtii Flv participates in a Mehler-like reduction of O 2 , which drives a large part of the photosynthetic electron flow during a light transient and is thus critical for growth under fluctuating light regimes

DYRKP kinase regulates cell wall degradation in Chlamydomonas by inducing matrix metalloproteinase expression

Abstract The cell wall of plants and algae is an important cell structure that protects cells from changes in the external physical and chemical environment. This extracellular matrix composed of polysaccharides and glycoproteins, is needed to be remodeled continuously throughout the life cycle. However, compared to matrix polysaccharides, little is known about the mechanisms regulating the formation and degradation of matrix glycoproteins. We report here that a plant kinase belonging to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family present in all eukaryotes regulates cell wall degradation in the model microalga Chlamydomonas reinhardtii by inducing the expression of matrix metalloproteinases (MMPs). In the absence of DYRKP, daughter cells fail to degrade the parental cell wall, and form multicellular structures. On the other hand, the complementation line of DYRKP was shown to degrade the parental cell wall normally. Transcriptomic and proteomic analyses indicate a marked down-regulation of MMP expression in the dyrkp mutants. Additionally, the expression of MMP was confirmed to be consistent with the expression pattern of DYRKP. Our findings show that DYRKP, by ensuring timely MMP expression, enables the successful execution of the cell cycle. Altogether, this study provides new insight into the life cycle regulation in plants and algae. IN A NUTSHELL Background Plants and algae have different types of polysaccharides in their cell walls, but they have glycoproteins in common. Glycoprotein synthesis and degradation must be tightly regulated to ensure normal growth and differentiation. However, little is known about the regulatory mechanism of glycoprotein degradation in both plants and algae. The cell cycle of Chlamydomonas reinhardtii begins anew with the hatching of daughter cells, and the role of matrix metalloproteinases (MMPs) is known to be important in this process. In our previous study, we observed that a knockout mutant of the plant kinase belonging to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRKP) formed a palmelloid structure and failed to hatch. Questions What is the role of DYRKP in microalgae? Specifically, why does the dyrkp mutant form a palmelloid structure? Palmelloid is usually observed in dividing cells or after exposure to stresses. We therefore hypothesized that the palmelloid phenotype observed in dyrkp mutant could either be due to a defect in cell hatching or due to an increased stress state in the mutant population. Findings We answered these questions by comparative studies in different culture conditions and by examining additional dyrkp knockout mutants generated by CRISPR-Cas9 in various background strains with more or less intact cell walls. Palmelloid formation in the dyrkp mutant was observed under optimal growth (mixo- or auto-trophic condition) and very low light conditions. Interestingly, unlike the parent strain, in which only cell wall fragments are observed in old cultures, the parental cell wall of the dyrkp mutant remained almost intact even after the release of daughter cells. Also, the cell division rate of the cell wall-less dyrkp mutants was similar to their background strain. These results suggest that dyrkp mutants have a problem in degrading the parental cell walls. Indeed, proteomic and transcriptomic analyses revealed reduced levels of protease families in the dyrkp mutant, and in particular with a significantly lower amount of several key members of the MMP family. Through the analysis of complementation lines, we confirmed that the DYRKP was required for strong and rapid expression of MMPs. Next steps We are pursuing research to understand what the phosphorylation clients of DYRKP are and how they regulate the expression of the MMPs identified in this study. One sentence summary The DYRKP kinase induces the expression of matrix metalloproteinases involved in the degradation of the parental cell wall, allowing prompt hatching of daughter cells after cell division

Synthesis and Evaluation of the Antibacterial Activities of 13-Substituted Berberine Derivatives

International audienceThe biological activities of berberine, a natural plant molecule, are known to be affected by structural modifications, mostly at position 9 and/or 13. A series of new 13-substituted berberine derivatives were synthesized and evaluated in term of antimicrobial activity using various microorganisms associated to human diseases. Contrarily to the original molecule berberine, several derivatives were found strongly active in microbial sensitivity tests against Mycobacterium, Candida albicans and Gram-positive bacteria, including naïve or resistant Bacillus cereus, Staphylococcus aureus and Streptococcus pyogenes with minimal inhibitory concentration (MIC) of 3.12 to 6.25 µM. Among the various Gram-negative strains tested, berberine's derivatives were only found active on Helicobacter pylori and Vibrio alginolyticus (MIC values of 1.5-3.12 µM). Cytotoxicity assays performed on human cells showed that the antimicrobial berberine derivatives caused low toxicity resulting in good therapeutic index values. In addition, a mechanistic approach demonstrated that, contrarily to already known berberine derivatives causing either membrane permeabilization, DNA fragmentation or interacting with FtsZ protein, active derivatives described in this study act through inhibition of the synthesis of peptidoglycan or RNA. Overall, this study shows that these new berberine derivatives can be considered as potent and safe anti-bacterial agents active on human pathogenic microorganisms, including ones resistant to conventional antibiotics