Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum

Siaut, Magali; Heijde, Marc; Mangogna, Manuela; Montsant, Anton; Coesel, Sacha; Allen, Andrew E.; Manfredonia, Alessandro; Falciatore, Angela; Bowler, Chris

doi:10.1016/j.gene.2007.05.022

Cited by 290 publications

(283 citation statements)

References 45 publications

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“…The Pt-DPH coding sequence was obtained after PCR amplification from cDNA using the oligos Phy_DraI-Fw and Phy_XhoI_Rv, inserted into the pENTR1A vector (Invitrogen) in DraI/XhoI, and recombined with the pDEST-C-HA vector (Siaut et al, 2007). The Pt-DPH-HA fragment was amplified from the pDEST-Pt-DPH-HA plasmid using CodPromPhy_Fw and PhyHA_AT_Rv oligos and then fused to the Pt-DPH 59 upstream region amplified from the genomic DNA with the PhyProm_Fw2 and the PromCodPhy_Rv primer pairs, and the Pt-DPH terminator with the oligos Pt-PhyAt_Fw and Pt-PhyAt_Rv0, using the PhyProm_Fw3 and Pt-PhyAt_Rv1 primers (primer sequences are listed in Supplemental Table 1).…”

Section: Construction Of Pt-dph Reporter Linesmentioning

confidence: 99%

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Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

et al. 2016

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The absorption of visible light in aquatic environments has led to the common assumption that aquatic organisms sense and adapt to penetrative blue/green light wavelengths but show little or no response to the more attenuated red/far-red wavelengths. Here, we show that two marine diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana, possess a bona fide red/far-red light sensing phytochrome (DPH) that uses biliverdin as a chromophore and displays accentuated red-shifted absorbance peaks compared with other characterized plant and algal phytochromes. Exposure to both red and far-red light causes changes in gene expression in P. tricornutum, and the responses to far-red light disappear in DPH knockout cells, demonstrating that P. tricornutum DPH mediates far-red light signaling. The identification of DPH genes in diverse diatom species widely distributed along the water column further emphasizes the ecological significance of far-red light sensing, raising questions about the sources of far-red light. Our analyses indicate that, although far-red wavelengths from sunlight are only detectable at the ocean surface, chlorophyll fluorescence and Raman scattering can generate red/far-red photons in deeper layers. This study opens up novel perspectives on phytochrome-mediated far-red light signaling in the ocean and on the light sensing and adaptive capabilities of marine phototrophs.

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Section: Construction Of Pt-dph Reporter Linesmentioning

confidence: 99%

“…Primer efficiencies were determined as described (Pfaffl, 2001). RPS and/or TBP were used as reference genes and normalization performed as described (Siaut et al, 2007).…”

Section: Rna Extraction and Gene Expression Analysismentioning

confidence: 99%

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

et al. 2016

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“…The joint action of glutamine synthetase (GS) and glutamate synthase (GOGAT) is thought to be the main route of ammonium assimilation into amino acids and other nitrogenous compounds (Dortch et al, 1979;Clayton and Ahmed, 1986;Zehr and Falkowski, 1988). Diatoms possess a plastid-localized GSII that is of red algal origin (Robertson et al, 1999;Robertson and Tartar, 2006;Siaut et al, 2007) and thought to be responsible for the assimilation of ammonium produced by nitrate reduction. Transcript levels of glnII (encoding GSII) are higher in diatom cells assimilating nitrate than in those assimilating ammonium directly (Takabayashi et al, 2005).…”

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confidence: 99%

“…They include a cytosolic NAD(P)H-dependent nitrite reductase [NAD(P)H-NiR] that is homologous to nirB of bacteria and fungi, along with a mitochondrial GSIII (Robertson and Alberte, 1996;Armbrust et al, 2004;Allen et al, 2006;Siaut et al, 2007). Neither of these enzymes has been found in green algae or plants.…”

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confidence: 99%

The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants

Hockin

Möck²,

Mulholland³

et al. 2011

Plant Physiology

325

304

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The availability of nitrogen varies greatly in the ocean and limits primary productivity over large areas. Diatoms, a group of phytoplankton that are responsible for about 20% of global carbon fixation, respond rapidly to influxes of nitrate and are highly successful in upwelling regions. Although recent diatom genome projects have highlighted clues to the success of this group, very little is known about their adaptive response to changing environmental conditions. Here, we compare the proteome of the marine diatom Thalassiosira pseudonana (CCMP 1335) at the onset of nitrogen starvation with that of nitrogen-replete cells using two-dimensional gel electrophoresis. In total, 3,310 protein spots were distinguishable, and we identified 42 proteins increasing and 23 decreasing in abundance (greater than 1.5-fold change; P , 0.005). Proteins involved in the metabolism of nitrogen, amino acids, proteins, and carbohydrates, photosynthesis, and chlorophyll biosynthesis were represented. Comparison of our proteomics data with the transcriptome response of this species under similar growth conditions showed good correlation and provided insight into different levels of response. The T. pseudonana response to nitrogen starvation was also compared with that of the higher plant Arabidopsis (Arabidopsis thaliana), the green alga Chlamydomonas reinhardtii, and the cyanobacterium Prochlorococcus marinus. We have found that the response of diatom carbon metabolism to nitrogen starvation is different from that of other photosynthetic eukaryotes and bears closer resemblance to the response of cyanobacteria.

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“…Values are means and error bars denote the range (n = 2). B, METE transcript abundance after the addition of different concentrations of vitamin B 12 for C. reinhardtii (as described above; solid line) and the marine diatom P. tricornutum (as normalized to Histone H4, 30S Ribosomal protein S1, and TATA box-binding protein as housekeeping genes [Siaut et al, 2007] …”

Section: Defining the Regulation Of Mete By B 12mentioning

confidence: 99%

Unraveling Vitamin B₁₂-Responsive Gene Regulation in Algae

Helliwell

Scaife²,

Sasso³

et al. 2014

Plant Physiol.

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Photosynthetic microalgae play a vital role in primary productivity and biogeochemical cycling in both marine and freshwater systems across the globe. However, the growth of these cosmopolitan organisms depends on the bioavailability of nutrients such as vitamins. Approximately one-half of all microalgal species requires vitamin B 12 as a growth supplement. The major determinant of algal B 12 requirements is defined by the isoform of methionine synthase possessed by an alga, such that the presence of the B 12 -independent methionine synthase (METE) enables growth without this vitamin. Moreover, the widespread but phylogenetically unrelated distribution of B 12 auxotrophy across the algal lineages suggests that the METE gene has been lost multiple times in evolution. Given that METE expression is repressed by the presence of B 12 , prolonged repression by a reliable source of the vitamin could lead to the accumulation of mutations and eventually gene loss. Here, we probe METE gene regulation by B 12 and methionine/folate cycle metabolites in both marine and freshwater microalgal species. In addition, we identify a B 12 -responsive element of Chlamydomonas reinhardtii METE using a reporter gene approach. We show that complete repression of the reporter occurs via a region spanning 2574 to 290 bp upstream of the METE start codon. A proteomics study reveals that two other genes (S-Adenosylhomocysteine hydrolase and Serine hydroxymethyltransferase2) involved in the methionine-folate cycle are also repressed by B 12 in C. reinhardtii. The strong repressible nature and high sensitivity of the B 12 -responsive element has promising biotechnological applications as a cost-effective regulatory gene expression tool.

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Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum

Cited by 290 publications

References 45 publications

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants

Unraveling Vitamin B₁₂-Responsive Gene Regulation in Algae

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Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum

Cited by 290 publications

References 45 publications

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

The Response of Diatom Central Carbon Metabolism to Nitrogen Starvation Is Different from That of Green Algae and Higher Plants

Unraveling Vitamin B12-Responsive Gene Regulation in Algae

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Unraveling Vitamin B₁₂-Responsive Gene Regulation in Algae