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Summary: Marine planktonic diatoms are among the most important contributors to phytoplankton blooms and marine net primary production. Their ecological success has been attributed to their ability to rapidly respond to changing environmental conditions.Here, we report common molecular mechanisms used by the model marine diatom Thalassiosira pseudonana to respond to 10 diverse environmental stressors using RNA‐Seq analysis.We identify a specific subset of 1076 genes that are differentially expressed in response to stressors that induce an imbalance between energy or resource supply and metabolic capacity, which we termed the diatom environmental stress response (d‐ESR). The d‐ESR is primarily composed of genes that maintain proteome homeostasis and primary metabolism. Photosynthesis is strongly regulated in response to environmental stressors but chloroplast‐encoded genes were predominantly upregulated while the nuclear‐encoded genes were mostly downregulated in response to low light and high temperature.In aggregate, these results provide insight into the molecular mechanisms used by diatoms to respond to a range of environmental perturbations and the unique role of the chloroplast in managing environmental stress in diatoms. This study facilitates our understanding of the molecular mechanisms underpinning the ecological success of diatoms in the ocean. [ABSTRACT FROM AUTHOR]

Summary: CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life‐cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism.Homologous recombination was induced in T. pseudonana using sequence‐specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT).Up to c. 85% of NAT‐resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell‐size regulation in centric diatoms.Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms. [ABSTRACT FROM AUTHOR]

Summary: Housekeeping genes (HKGs) are constitutively expressed with low variation across tissues/conditions. They are thought to be highly conserved and fundamental to cellular maintenance, with distinctive genomic features.Here, we identify 1505 HKGs in the unicellular marine diatom Thalassiosira pseudonana based on an RNA‐seq analysis of 232 samples taken under 12 experimental conditions over 0–72 h. We identify promising internal reference genes (IRGs) for T. pseudonana from the most stably expressed HKGs.A comparative analysis indicates < 18% of HKGs in T. pseudonana have orthologs in other eukaryotes, including other diatom species. Contrary to work on human tissues, T. pseudonana HKGs are longer than non‐HKGs, due to elongated introns. More ancient HKGs tend to be shorter than more recent HKGs, and expression levels of HKGs decrease more rapidly with gene length relative to non‐HKGs.Our results indicate that HKGs are highly variable across the tree of life and thus unlikely to be universally fundamental for cellular maintenance. We hypothesize that the distinct genomic features of HKGs of T. pseudonana may be a consequence of selection pressures associated with high expression and low variance across conditions. [ABSTRACT FROM AUTHOR]

Summary: Chitin is generally considered to be present in centric diatoms but not in pennate species. Many aspects of chitin biosynthetic pathways have not been explored in diatoms.We retrieved chitin metabolic genes from pennate (Phaeodactylum tricornutum) and centric (Thalassiosira pseudonana) diatom genomes. Chitin deacetylase (CDA) genes from each genome (PtCDA and TpCDA) were overexpressed in P. tricornutum. We performed comparative analysis of their sequence structure, phylogeny, transcriptional profiles, localization and enzymatic activities.The chitin relevant proteins show complex subcellular compartmentation. PtCDA was likely acquired by horizontal gene transfer from prokaryotes, whereas TpCDA has closer relationships with sequences in Opisthokonta. Using transgenic P. tricornutum lines expressing CDA‐green fluorescent protein (GFP) fusion proteins, PtCDA predominantly localizes to Golgi apparatus whereas TpCDA localizes to endoplasmic reticulum/chloroplast endoplasmic reticulum membrane. CDA‐GFP overexpression upregulated the transcription of chitin synthases and potentially enhanced the ability of chitin synthesis. Although both CDAs are active on GlcNAc5, TpCDA is more active on the highly acetylated chitin polymer DA60.We have addressed the ambiguous characters of CDAs from P. tricornutum and T. pseudonana. Differences in localization, evolution, expression and activities provide explanations underlying the greater potential of centric diatoms for chitin biosynthesis. This study paves the way for in vitro applications of novel CDAs. [ABSTRACT FROM AUTHOR]

Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity., To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterized alongside lipid accumulation., Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes., Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal-derived biofuels. [ABSTRACT FROM AUTHOR]

Diatoms are widespread in aquatic ecosystems where they may be limited by the supply of inorganic carbon. Their carbon dioxide-concentrating mechanisms ( CCMs) 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, were 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 phosphoenolpyruvate ( PEP) carboxylase ( PEPC) and the PEP-regenerating enzyme, pyruvate phosphate dikinase ( PPDK), were lower in cells grown at low than at high CO2. The ratios of PEPC and PPDK to ribulose bisphosphate carboxylase were substantially lower than 1, 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. [ABSTRACT FROM AUTHOR]

The mechanisms of carbon concentration in marine diatoms are controversial. At low CO2, decreases in O2 evolution after inhibition of phosphoenolpyruvate carboxylases ( PEPCs), and increases in PEPC transcript abundances, have been interpreted as evidence for a C4 mechanism in Thalassiosira pseudonana, but the ascertainment of which proteins are responsible for the subsequent decarboxylation and PEP regeneration steps has been elusive., We evaluated the responses of T. pseudonana to steady-state differences in CO2 availability, as well as to transient shifts to low CO2, by integrated measurements of photosynthetic parameters, transcript abundances and quantitative proteomics., On shifts to low CO2, two PEPC transcript abundances increased and then declined on timescales consistent with recoveries of Fv/ Fm, non-photochemical quenching ( NPQ) and maximum chlorophyll a-specific carbon fixation ( Pmax), but transcripts for archetypical decarboxylation enzymes phosphoenolpyruvate carboxykinase ( PEPCK) and malic enzyme ( ME) did not change. Of 3688 protein abundances measured, 39 were up-regulated under low CO2, including both PEPCs and pyruvate carboxylase ( PYC), whereas ME abundance did not change and PEPCK abundance declined., We propose a closed-loop biochemical model, whereby T. pseudonana produces and subsequently decarboxylates a C4 acid via PEPC2 and PYC, respectively, regenerates phosphoenolpyruvate ( PEP) from pyruvate in a pyruvate phosphate dikinase-independent (but glycine decarboxylase ( GDC)-dependent) manner, and recuperates photorespiratory CO2 as oxaloacetate ( OAA). [ABSTRACT FROM AUTHOR]

A correction to the article "The Nature of the CO2-Concentrating Mechanisms in a Marine Diatom, Thalassiosira pseudonana," which appeared in issue 209, is presented.

Diatoms are widespread in aquatic ecosystems where they may be limited by the supply of inorganic carbon. Their carbon dioxide-concentrating mechanisms (CCMs) 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, were 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 phosphoenolpyruvate (PEP) carboxylase (PEPC) and the PEP-regenerating enzyme, pyruvate phosphate dikinase (PPDK), were lower in cells grown at low than at high CO2 . The ratios of PEPC and PPDK to ribulose bisphosphate carboxylase were substantially lower than 1, 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., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)