115 research outputs found
Insights on the functions and ecophysiological relevance of the diverse carbonic anhydrases in microalgae
Carbonic anhydrases (CAs) exist in all kingdoms of life. They are metalloenzymes, often containing zinc, that catalyze the interconversion of bicarbonate and carbon dioxide—a ubiquitous reaction involved in a variety of cellular processes. So far, eight classes of apparently evolutionary unrelated CAs that are present in a large diversity of living organisms have been described. In this review, we focus on the diversity of CAs and their roles in photosynthetic microalgae. We describe their essential role in carbon dioxide-concentrating mechanisms and photosynthesis, their regulation, as well as their less studied roles in non-photosynthetic processes. We also discuss the presence in some microalgae, especially diatoms, of cambialistic CAs (i.e., CAs that can replace Zn by Co, Cd, or Fe) and, more recently, a CA that uses Mn as a metal cofactor, with potential ecological relevance in aquatic environments where trace metal concentrations are low. There has been a recent explosion of knowledge about this well-known enzyme with exciting future opportunities to answer outstanding questions using a range of different approaches
Regulation of carbon metabolism by environmental conditions: a perspective from diatoms and other chromalveolates
Diatoms belong to a major, diverse and species-rich eukaryotic clade, the Heterokonta, within the polyphyletic chromalveolates. They evolved as a result of secondary endosymbiosis with one or more Plantae ancestors, but their precise evolutionary history is enigmatic. Nevertheless, this has conferred them with unique structural and biochemical properties that have allowed them to flourish in a wide range of different environments and cope with highly variable conditions. We review the effect of pH, light and dark, and CO2 concentration on the regulation of carbon uptake and assimilation. We discuss the regulation of the Calvin-Benson-Bassham cycle, glycolysis, lipid synthesis, and carbohydrate synthesis at the level of gene transcripts (transcriptomics), proteins (proteomics) and enzyme activity. In contrast to Viridiplantae where redox regulation of metabolic enzymes is important, it appears to be less common in diatoms, based on the current evidence, but regulation at the transcriptional level seems to be widespread. The role of post-translational modifications such as phosphorylation, glutathionylation, etc., and of protein-protein interactions, has been overlooked and should be investigated further. Diatoms and other chromalveolates are understudied compared to the Viridiplantae, especially given their ecological importance, but we believe that the ever-growing number of sequenced genomes combined with proteomics, metabolomics, enzyme measurements, and the application of novel techniques will provide a better understanding of how this important group of algae maintain their productivity under changing conditions
Ecological imperatives for aquatic CO2-concentrating mechanisms
In aquatic environments, the concentration of inorganic carbon is spatially and temporally variable and CO2 can be substantially over-saturated or depleted. Depletion of CO2 plus low rates of diffusion cause inorganic carbon to be more limiting in aquatic than terrestrial environments and the frequency of species with a CCM, and their contribution to productivity is correspondingly greater. Aquatic photoautotrophs may have biochemical or biophysical CCMs and exploit CO2 from the sediment or the atmosphere. Though partly constrained by phylogeny, CCM activity is related to environmental conditions. CCMs are absent or down-regulated when their increased energy costs, lower CO2 affinity or altered mineral requirements outweigh their benefits. Aquatic CCMs are most widespread in environments with low CO2, high HCO3-, high pH and high light. Freshwater species are generally less effective at inorganic carbon removal than marine species but have a greater range of ability to remove carbon, matching the environmental variability in carbon availability. The diversity of CCMs in seagrasses and marine phytoplankton and detailed mechanistic studies on larger aquatic photoautotrophs are understudied. Strengthening the links between ecology and CCMs will increase our understanding of the mechanisms underlying ecological success and will place mechanistic studies in a clearer ecological context
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
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
A new widespread subclass of carbonic anhydrase in marine phytoplankton
Most aquatic photoautotrophs depend on CO2-concentrating mechanisms (CCMs) to maintain productivity at ambient concentrations of CO2, and carbonic anhydrase (CA) plays a key role in these processes. Here we present different lines of evidence showing that the protein LCIP63, identified in the marine diatom Thalassiosira pseudonana, is a CA. However, sequence analysis showed that it has a low identity with any known CA and therefore belongs to a new subclass that we designate as iota-CA. Moreover, LCIP63 unusually prefers Mn2+ to Zn2+ as a cofactor, which is potentially of ecological relevance since Mn2+ is more abundant than Zn2+ in the ocean. LCIP63 is located in the chloroplast and only expressed at low concentrations of CO2. When overexpressed using biolistic transformation, the rate of photosynthesis at limiting concentrations of dissolved inorganic carbon increased, confirming its role in the CCM. LCIP63 homologs are present in the five other sequenced diatoms and in other algae, bacteria, and archaea. Thus LCIP63 is phylogenetically widespread but overlooked. Analysis of the Tara Oceans database confirmed this and showed that LCIP63 is widely distributed in marine environments and is therefore likely to play an important role in global biogeochemical carbon cycling
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
External α-carbonic anhydrase and solute carrier 4 are required for bicarbonate uptake in a freshwater angiosperm
The freshwater monocot Ottelia alismoides is the only known species to operate three CO2-concentrating mechanisms (CCMs): constitutive bicarbonate (HCO3–) use, C4 photosynthesis, and facultative Crassulacean acid metabolism, but the mechanism of HCO3– use is unknown. We found that the inhibitor of an anion exchange protein, 4,4'-diisothio-cyanatostilbene-2,2'-disulfonate (DIDS), prevented HCO3– use but also had a small effect on CO2 uptake. An inhibitor of external carbonic anhydrase (CA), acetazolamide (AZ), reduced the affinity for CO2 uptake but also prevented HCO3– use via an effect on the anion exchange protein. Analysis of mRNA transcripts identified a homologue of solute carrier 4 (SLC4) responsible for HCO3– transport, likely to be the target of DIDS, and a periplasmic α-carbonic anhydrase 1 (α-CA1). A model to quantify the contribution of the three different pathways involved in inorganic carbon uptake showed that passive CO2 diffusion dominates inorganic carbon uptake at high CO2 concentrations. However, as CO2 concentrations fall, two other pathways become predominant: conversion of HCO3– to CO2 at the plasmalemma by α-CA1 and transport of HCO3– across the plasmalemma by SLC4. These mechanisms allow access to a much larger proportion of the inorganic carbon pool and continued photosynthesis during periods of strong carbon depletion in productive ecosystems
In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts
Galactolipids are the main lipids from plant photosynthetic membranes and they can be digested by pancreatic lipase related protein 2 (PLRP2), an enzyme found in the pancreatic secretion in many animal species. Here, we used transmission Fourier-transform infrared spectroscopy (FTIR) to monitor continuously the hydrolysis of galactolipids by PLRP2, in situ and in real time. The method was first developed with a model substrate, a synthetic monogalactosyl diacylglycerol with 8-carbon acyl chains (C8-MGDG), in the form of mixed micelles with a bile salt, sodium taurodeoxycholate (NaTDC). The concentrations of the residual substrate and reaction products (monogalactosylmonoglyceride, MGMG; monogalactosylglycerol, MGG; octanoic acid) were estimated from the carbonyl and carboxylate vibration bands after calibration with reference standards. The results were confirmed by thin layer chromatography analysis (TLC) and specific staining of galactosylated compounds with thymol and sulfuric acid. The method was then applied to the lipolysis of more complex substrates, a natural extract of MGDG with long acyl chains, micellized with NaTDC, and intact chloroplasts isolated from spinach leaves. After a calibration performed with α-linolenic acid, the main fatty acid (FA) found in plant galactolipids, FTIR allowed quantitative measurement of chloroplast lipolysis by PLRP2. A full release of FA from membrane galactolipids was observed, that was not dependent on the presence of bile salts. Nevertheless, the evolution of amide vibration band in FTIR spectra suggested the interaction of membrane proteins with NaTDC and lipolysis products
Diatom teratologies as biomarkers of contamination: Are all deformities ecologically meaningful?
Contaminant-related stress on aquatic biota is difficult to assess when lethal impacts are not observed. Diatoms, by displaying deformities (teratologies) in their valves, have the potential to reflect sub-lethal responses to environmental stressors such as metals and organic compounds. For this reason, there is great interest in using diatom morphological aberrations in biomonitoring. However, the detection and mostly the quantification of teratologies is still a challenge; not all studies have succeeded in showing a relationship between the proportion of abnormal valves and contamination level along a gradient of exposure. This limitation in part reflects the loss of ecological information from diatom teratologies during analyses when all deformities are considered. The type of deformity, the severity of aberration, species proneness to deformity formation, and propagation of deformities throughout the population are key components and constraints in quantifying teratologies. Before a metric based on diatom deformities can be used as an indicator of contamination, it is important to better understand the “ecological signal” provided by this biomarker. Using the overall abundance of teratologies has proved to be an excellent tool for identifying contaminated and non-contaminated environments (presence/absence), but refining this biomonitoring approach may bring additional insights allowing for a better assessment of contamination level along a gradient. The dilemma: are all teratologies significant, equal and/or meaningful in assessing changing levels of contamination? This viewpoint article examines numerous interrogatives relative to the use of diatom teratologies in water quality monitoring, provides selected examples of differential responses to contamination, and proposes solutions that may refine our understanding and quantification of the stress. This paper highlights the logistical problems associated with accurately evaluating and interpreting teratologies and stimulates more discussion and research on the subject to enhance the sensitivity of this metric in bioassessments
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