A new type of flexible CP12 protein in the marine diatom <i>Thalassiosira pseudonana</i>

International audienceBackground: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. Methods: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionizationmass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. Results: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. Conclusions: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle

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

Responses of the marine diatom Thalassiosira pseudonana to changes in CO2 concentration: a proteomic approach

The concentration of CO2 in many aquatic systems is variable, often lower than the KM of the primary carboxylating enzyme Rubisco, and in order to photosynthesize efficiently, many algae operate a facultative CO2 concentrating mechanism (CCM). Here we measured the responses of a marine diatom, Thalassiosira pseudonana, to high and low concentrations of CO2 at the level of transcripts, proteins and enzyme activity. Low CO2 caused many metabolic pathways to be remodeled. Carbon acquisition enzymes, primarily carbonic anhydrase, stress, degradation and signaling proteins were more abundant while proteins associated with nitrogen metabolism, energy production and chaperones were less abundant. A protein with similarities to the Ca2+/ calmodulin dependent protein kinase II_association domain, having a chloroplast targeting sequence, was only present at low CO2. This protein might be a specific response to CO2 limitation since a previous study showed that other stresses caused its reduction. The protein sequence was found in other marine diatoms and may play an important role in their response to low CO2 concentration

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

Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state

International audienceThe redox switch protein CP12 is a key player of the regulation of the Benson–Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold

Inorganic carbon uptake in a freshwater diatom, Asterionella formosa (Bacillariophyceae): from ecology to genomics

Inorganic carbon availability can limit primary productivity and control species composition of freshwater phytoplankton. This is despite the presence of CO2-concentrating mechanisms (CCMs) in some species that maximize inorganic carbon uptake. We investigated the effects of inorganic carbon on the seasonal distribution, growth rates and photosynthesis of a freshwater diatom, Asterionella formosa, and the nature of its CCM using genomics. In a productive lake, the frequency of A. formosa declined with CO2 concentration below air-equilibrium. In contrast, CO2 concentrations at 2.5-times air-equilibrium did not increase growth rate, cell C-quota or the ability to remove inorganic carbon. A pH-drift experiment strongly suggested that HCO3− as well as CO2 could be used. Calculations combining hourly inorganic carbon concentrations in a lake with known CO2 and HCO3− uptake kinetics suggested that rates of photosynthesis of A. formosa would be approximately carbon saturated and largely dependent on CO2 uptake when CO2 was at or above air-equilibrium. However, during summer carbon depletion, HCO3− would be the major form of carbon taken up and carbon saturation will fall to around 30%. Genes encoding proteins involved in CCMs were identified in the nuclear genome of A. formosa. We found carbonic anhydrases from subclasses α, β, γ and θ, as well as solute carriers from families 4 and 26 involved in HCO3− transport, but no periplasmic carbonic anhydrase. A model of the components of the CCM and their location in A. formosa showed that they are more similar to Phaeodactylum tricornutum than to Thalassiosira pseudonana, two marine diatoms

RSH enzyme diversity for (p)ppGpp metabolism in Phaeodactylum tricornutum and other diatoms

International audienceThe nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae

Golden magic: RSH enzymes for (p)ppGpp metabolism in the diatom PhaeodactylumPhaeodactylum tricornutumtricornutum

The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the golden-coloured marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red alga