Coordinated phosphate uptake by extracellular alkaline phosphatase and solute carrier transporters in marine diatoms.

Marine diatoms express genes encoding potential phosphate transporter and alkaline phosphatase (APase) under phosphate-limited (-P) condition. This indicates that diatoms use high-affinity phosphate uptake system with organic phosphate hydration. The function of molecules playing roles for Pi uptake was determined in this study. Pi uptake and APase activity of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, were monitored during acclimation to -P condition. The transcript levels of Pi transporter were analyzed, and Pi transporters were localized with GFP tagging in diatom cells. KO mutants of plasma membrane solute carrier proteins (SLC34s) or APase were established, and their phenotype was evaluated. Some Na+ /Pi transporter candidates, SLC34s in P. tricornutum and T. pseudonana, increased transcript under -P condition. Whole-cell Pi transport was specifically stimulated by sodium ion but independent of potassium, lithium, or proton. Genome-editing KO of PtSLC34-5 and APase (Pt49678) in P. tricornutum was highly inhibitory for Pi uptake, and KO of TpSLC34-2 was also highly inhibitory for Pi uptake in T. pseudonana. SLC34s and APase were co-expressed under -P conditions in marine diatoms. SLC34s play a major role in the initial acclimation stage of diatom cells to -P condition and APase plays an increasing role in the prolonged Pi-starved condition.

Localization and characterization θ carbonic anhydrases in Thalassiosira pseudonana.

Carbonic anhydrase (CA) is a crucial component for the operation of CO2-concentrating mechanisms (CCMs) in the majority of aquatic photoautotrophs that maintain the global primary production. In the genome of the centric marine diatom, Thalassiosira pseudonana, there are four putative gene sequences that encode θ-type CA, which was a type of CA recently identified in marine diatoms and green algae. In the present study, specific subcellular locations of four θCAs, TpθCA1, TpθCA2, TpθCA3, and TpθCA4 were determined by expressing GFP-fused proteins of these TpθCAs in T. pseudonana. As a result, C-terminal GFP fusion proteins of TpθCA1, TpθCA2, and TpθCA3 were all localized in the chloroplast; TpθCA2 was at the central chloroplast area, and the other two TpθCAs were throughout the chloroplast. Immunogold-labeling transmission electron microscopy was further performed for the transformants expressing TpθCA1:GFP and TpθCA2:GFP with anti-GFP-monoclonal antibody. TpθCA1:GFP was localized in the free stroma area, including the peripheral pyrenoid area. TpθCA2:GFP was clearly located as a lined distribution at the central part of the pyrenoid structure, which was most likely the pyrenoid-penetrating thylakoid. Considering the presence of the sequence encoding the N-terminal thylakoid-targeting domain in the TpθCA2 gene, this localization was likely the lumen of the pyrenoid-penetrating thylakoid. On the other hand, TpθCA4:GFP was localized in the cytoplasm. Transcript analysis of these TpθCAs revealed that TpθCA2 and TpθCA3 were upregulated in atmospheric CO2 (0.04% CO2, LC) levels, while TpθCA1 and TpθCA4 were highly induced under 1% CO2 (HC) condition. The genome-editing knockout (KO) of TpθCA1, by CRISPR/Cas9 nickase, gave a silent phenotype in T. pseudonana under LC-HC conditions, which was in sharp agreement with the case of the previously reported TpθCA3 KO. In sharp contrast, TpθCA2 KO is so far unsuccessful, suggesting a housekeeping role of TpθCA2. The silent phenotype of KO strains of stromal CAs suggests that TpαCA1, TpθCA1, and TpθCA3 may have functional redundancy, but different transcript regulations in response to CO2 of these stromal CAs suggest in part their independent roles.

Multiple plasma membrane SLC4s contribute to external HCO3- acquisition during CO2 starvation in the marine diatom Phaeodactylum tricornutum.

The availability of CO2 is one of the restrictions on aquatic photosynthesis. Solute carrier (SLC) 4-2, a plasma membrane HCO3- transporter has previously been identified in the marine diatom Phaeodactylum tricornutum. In this study, we discovered two paralogs, PtSLC4-1 and PtSLC4-4, that are both localized at the plasma membrane. Their overexpression stimulated HCO3- uptake, and this was inhibited by the anion channel blocker 4,4´-diisothiocyanostilbene-2,2´-disulfonic (DIDS). Similarly to SLC4-2, PtSLC4-1 specifically required Na+ of ~100 mM for its maximum HCO3- transport activity. Unlike PtSLC4-1 and PtSLC4-2, the HCO3- transport of PtSLC4-4 depended equally on Na+, K+, or Li+, suggesting its broad selectivity for cations. Transcript analyses indicated that PtSLC4-1 was the most abundant HCO3- transporter under CO2 concentrations below atmospheric levels, while PtSLC4-4 showed little transcript induction under atmospheric CO2 but transient induction to comparable levels to PtSLC4-1 during the initial acclimation stage from high CO2 (1%) to very low CO2 (<0.002%). Our results strongly suggest a major HCO3- transport role of PtSLC4-1 with a relatively minor role of PtSLC4-2, and that PtSLC4-4 operates under severe CO2 limitation unselectively to cations when the other SLC4s do not function to support HCO3- uptake.

Metabolic Innovations Underpinning the Origin and Diversification of the Diatom Chloroplast.

: Of all the eukaryotic algal groups, diatoms make the most substantial contributions to photosynthesis in the contemporary ocean. Understanding the biological innovations that have occurred in the diatom chloroplast may provide us with explanations to the ecological success of this lineage and clues as to how best to exploit the biology of these organisms for biotechnology. In this paper, we use multi-species transcriptome datasets to compare chloroplast metabolism pathways in diatoms to other algal lineages. We identify possible diatom-specific innovations in chloroplast metabolism, including the completion of tocopherol synthesis via a chloroplast-targeted tocopherol cyclase, a complete chloroplast ornithine cycle, and chloroplast-targeted proteins involved in iron acquisition and CO2 concentration not shared between diatoms and their closest relatives in the stramenopiles. We additionally present a detailed investigation of the chloroplast metabolism of the oil-producing diatom Fistuliferasolaris, which is of industrial interest for biofuel production. These include modified amino acid and pyruvate hub metabolism that might enhance acetyl-coA production for chloroplast lipid biosynthesis and the presence of a chloroplast-localised squalene synthesis pathway unknown in other diatoms. Our data provides valuable insights into the biological adaptations underpinning an ecologically critical lineage, and how chloroplast metabolism can change even at a species level in extant algae.