The nature of the CO2-concentrating mechanisms in a marine diatom, Thalassiosira pseudonana

• Diatoms are widespread in aquatic ecosystems where they may be limited by the supply of inorganic carbon. Their carbon dioxide concentrating mechanisms (CCM) involving transporters and carbonic anhydrases (CAs) are well known, but the contribution of a biochemical CCM involving C4 metabolism is contentious. • The CCM(s) present in the marine centric diatom, Thalassiosira pseudonana, was studied in cells exposed to high or low concentrations of CO2, using a range of approaches. • At low CO2, cells possessed a CCM based on active uptake of CO2 (70% contribution) and bicarbonate, while at high CO2, cells were restricted to CO2. CA was highly and rapidly activated on transfer to low CO2 and played a key role because inhibition of external CA produced uptake kinetics similar to cells grown at high CO2. • The activities of PEP carboxylase (PEPCase) and the PEP regenerating enzyme, pyruvate phosphate dikinase (PPDK), were lower in cells grown at low than at high CO2. The ratios of PEPCase and PPDK to ribulose bisphosphate carboxylase were substantially lower than one even at low CO2. • Our data suggest that the kinetic properties of this species results from a biophysical CCM and not from C4 type metabolism

The role of C₄ metabolism in the marine diatom Phaeodactylum tricornutum

Diatoms are important players in the global carbon cycle. Their apparent photosynthetic affinity for ambient CO(2) is much higher than that of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), indicating that a CO(2)-concentrating mechanism (CCM) is functioning. However, the nature of the CCM, a biophysical or a biochemical C(4), remains elusive. Although (14)C labeling experiments and presence of complete sets of genes for C(4) metabolism in two diatoms supported the presence of C(4), other data and predicted localization of the decarboxylating enzymes, away from Rubisco, makes this unlikely. We used RNA-interference to silence the single gene encoding pyruvate-orthophosphate dikinase (PPDK) in Phaeodactylum tricornutum, essential for C(4) metabolism, and examined the photosynthetic characteristics. The mutants possess much lower ppdk transcript and PPDK activity but the photosynthetic K(1/2) (CO(2)) was hardly affected, thus clearly indicating that the C(4) route does not serve the purpose of raising the CO(2) concentration in close proximity of Rubisco in P. tricornutum. The photosynthetic V(max) was slightly reduced in the mutant, possibly reflecting a metabolic constraint that also resulted in a larger lipid accumulation. We propose that the C(4) metabolism does not function in net CO(2) fixation but helps the cells to dissipate excess light energy and in pH homeostasis

In silico predictions for fucoxanthin production by the diatom Phaeodactylum tricornutum

Diatoms and brown seaweeds are the main producers of fucoxanthin, an oxy-carotenoid with important biological functions related to its antioxidative properties. The diatom Phaeodactylum tricornutum appears in this scenario as a good source for fucoxanthin production. Its whole genome sequence was published in 2008, and some genome-scale metabolic models are currently available. This work intends to make use of the two most recent genome-scale metabolic models published to predict ways to increase fucoxanthin production, using constraint-based modeling and flux balance analysis. One of the models was completed with 31 downstream reactions of the methylerythritol 4-phosphate plastidic (MEP) pathway. Simulations and optimizations were performed regarding inorganic carbon and nitrogen sources in the two models and comparisons were made between them. Biomass growth was predicted to increase in all sources tested, i.e., CO2, HCO3?, NO3? and urea. However, the best results were obtained by combining CO2 plus HCO3? regarding inorganic carbon, and for urea as a nitrogen source, in both models tested. As a result of optimizations for fucoxanthin production, many of the knockout reactions brought on are involved in the metabolism of pyruvate, glutamine/glutamate and nitrogen assimilation.This work was supported by a grant from the National Council for Scientific and Technological Development (CNPq nº 490383/2013-0). The research fellowship from CNPq (grant nº 307099/2015-6) on behalf of M. Maraschin is acknowledged.info:eu-repo/semantics/publishedVersio

Biochemical and biophysical CO2 concentrating mechanisms in two species of freshwater macrophyte within the genus Ottelia (Hydrocharitaceae)

Two freshwater macrophytes, Ottelia alismoides and Ottelia acuminata, were grown at low (mean 5 µmol L-1) and high (mean 400 µmol L-1) CO2 concentrations under natural conditions. The ratio of PEPC to RubisCO was 1.8 in O. acuminata in both treatments. In O. alismoides, this ratio was 2.8 and 5.9 when grown at high and low CO2, respectively, as a result of a 2-fold increase of PEPC activity. The activity of PPDK was similar to and changed in-line with PEPC (1.9-fold change). The activity of the decarboxylating NADP-malic enzyme (ME) was very low in both species while NAD-ME activity was high and increased with PEPC activity in O. alismoides. These results suggest that O. alismoides might perform a type of C4 metabolism with NAD-ME decarboxylation, despite lacking Kranz anatomy. The C4-activity was still present at high CO2 suggesting that it could be constitutive. O. alismoides at low CO2 showed diel acidity variation of up to 34 μequiv g-1 FW indicating it may also operate a form of Crassulacean Acid Metabolism (CAM). pH-drift experiments showed that both species were able to use bicarbonate. In O. acuminata, the kinetics of carbon uptake were altered by CO2 growth conditions, unlike in O. alismoides. Thus the two species appear to regulate their carbon concentrating mechanisms differently in response to changing CO2. The Hydrocharitaceae have many species with evidence for C4, CAM, or a metabolism involving organic acids, and are worthy of further study

Functions of the Duplicated hik31 Operons in Central Metabolism and Responses to Light, Dark, and Carbon Sources in Synechocystis sp. Strain PCC 6803

There are two closely related hik31 operons involved in signal transduction on the chromosome and the pSYSX plasmid in the cyanobacterium Synechocystis sp. strain PCC 6803. We studied the growth, cell morphology, and gene expression in operon and hik mutants for both copies, under different growth conditions, to examine whether the duplicated copies have the same or different functions and gene targets and whether they are similarly regulated. Phenotype analysis suggested that both operons regulated common and separate targets in the light and the dark. The chromosomal operon was involved in the negative control of autotrophic events, whereas the plasmid operon was involved in the positive control of heterotrophic events. Both the plasmid and double operon mutant cells were larger and had division defects. The growth data also showed a regulatory role for the chromosomal hik gene under high-CO2 conditions and the plasmid operon under low-O2 conditions. Metal stress experiments indicated a role for the chromosomal hik gene and operon in mediating Zn and Cd tolerance, the plasmid operon in Co tolerance, and the chromosomal operon and plasmid hik gene in Ni tolerance. We conclude that both operons are differentially and temporally regulated. We suggest that the chromosomal operon is the primarily expressed copy and the plasmid operon acts as a backup to maintain appropriate gene dosages. Both operons share an integrated regulatory relationship and are induced in high light, in glucose, and in active cell growth. Additionally, the plasmid operon is induced in the dark with or without glucose