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Clade-Specific Quantitative Analysis of Photosynthetic Gene Expression in Prochlorococcus.

Fernández-Pinos MC, Casado M, Caballero G, Zinser ER, Dachs J, Piña B - PLoS ONE (2015)

Bottom Line: After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths.Sequence and quantitative analyses of the corresponding amplicons showed the presence of high-light (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences.Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Chemistry, IDAEA-CSIC, Barcelona, Catalonia, Spain.

ABSTRACT
Newly designed primers targeting rbcL (CO2 fixation), psbA (photosystem II) and rnpB (reference) genes were used in qRT-PCR assays to assess the photosynthetic capability of natural communities of Prochlorococcus, the most abundant photosynthetic organism on Earth and a major contributor to primary production in oligotrophic oceans. After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths. Sequence and quantitative analyses of the corresponding amplicons showed the presence of high-light (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences. Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons. Laboratory exposure of Prochlorococcus MED4 (HL) and MIT9313 (LL) strains to organic pollutants (PAHs and organochlorine compounds) showed a decrease of rbcL transcript abundances, and of the rbcL to psbA ratios for both strains. We propose this technique as a convenient assay to evaluate effects of environmental stressors, including pollution, on the oceanic Prochlorococcus photosynthetic function.

No MeSH data available.


Related in: MedlinePlus

Oceanic stations from Malaspina 2010 circumnavigation selected for RNA analysis.Stations labeled in red correspond to those from which amplicon sequences were obtained.
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pone.0133207.g001: Oceanic stations from Malaspina 2010 circumnavigation selected for RNA analysis.Stations labeled in red correspond to those from which amplicon sequences were obtained.

Mentions: A total of 182 samples of oceanic water were collected at 62 stations in the Atlantic, Indian and Pacific Oceans during the Malaspina 2010 circumnavigation, from 14 December 2010 to 14 July 2011 aboard the R/V BioHesperides (Fig 1 and S2 Table). We sampled three depths at each station: 3 m depth, (Niskin bottle), deep chlorophyll maximum (DCM) depth, and DCM+40 m depth (both in Niskin bottles attached to a rosette—CTD system). Samples were collected between 8 h and 12 h am local time (10 h 45 min ± 46 min, S2 Table). This time period coincides with the peak of carbon fixation by Prochlorococcus, which occurs between dawn and midday [43, 72], as well as with the maximal expression of rbcL and psbA genes [44]. One liter of seawater from each sample was transferred from the Niskin bottle to a glass bottle, previously rinsed with milli-Q water and a small volume of the sampled seawater, prefiltered onto a 20-μm-pore-size net, and finally filtered onto 47-mm-diameter, 0.2-μm-pore-size PTFE filter at 80 mbar vacuum pressure. Filters were split into two halves. One half was preserved in lysis buffer (50 mM Tris-HCl, 40 mM EDTA, 0.75 M Sucrose) and stored at -20°C for genomic DNA extraction. The second half was preserved in RNAlater (Sigma-Aldrich, Saint Louis, MO) for RNA isolation analysis and preserved at -80°C. In all cases, we kept rigorously the time limit of 10 min between sample collection and the storage of the filter.


Clade-Specific Quantitative Analysis of Photosynthetic Gene Expression in Prochlorococcus.

Fernández-Pinos MC, Casado M, Caballero G, Zinser ER, Dachs J, Piña B - PLoS ONE (2015)

Oceanic stations from Malaspina 2010 circumnavigation selected for RNA analysis.Stations labeled in red correspond to those from which amplicon sequences were obtained.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526520&req=5

pone.0133207.g001: Oceanic stations from Malaspina 2010 circumnavigation selected for RNA analysis.Stations labeled in red correspond to those from which amplicon sequences were obtained.
Mentions: A total of 182 samples of oceanic water were collected at 62 stations in the Atlantic, Indian and Pacific Oceans during the Malaspina 2010 circumnavigation, from 14 December 2010 to 14 July 2011 aboard the R/V BioHesperides (Fig 1 and S2 Table). We sampled three depths at each station: 3 m depth, (Niskin bottle), deep chlorophyll maximum (DCM) depth, and DCM+40 m depth (both in Niskin bottles attached to a rosette—CTD system). Samples were collected between 8 h and 12 h am local time (10 h 45 min ± 46 min, S2 Table). This time period coincides with the peak of carbon fixation by Prochlorococcus, which occurs between dawn and midday [43, 72], as well as with the maximal expression of rbcL and psbA genes [44]. One liter of seawater from each sample was transferred from the Niskin bottle to a glass bottle, previously rinsed with milli-Q water and a small volume of the sampled seawater, prefiltered onto a 20-μm-pore-size net, and finally filtered onto 47-mm-diameter, 0.2-μm-pore-size PTFE filter at 80 mbar vacuum pressure. Filters were split into two halves. One half was preserved in lysis buffer (50 mM Tris-HCl, 40 mM EDTA, 0.75 M Sucrose) and stored at -20°C for genomic DNA extraction. The second half was preserved in RNAlater (Sigma-Aldrich, Saint Louis, MO) for RNA isolation analysis and preserved at -80°C. In all cases, we kept rigorously the time limit of 10 min between sample collection and the storage of the filter.

Bottom Line: After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths.Sequence and quantitative analyses of the corresponding amplicons showed the presence of high-light (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences.Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Chemistry, IDAEA-CSIC, Barcelona, Catalonia, Spain.

ABSTRACT
Newly designed primers targeting rbcL (CO2 fixation), psbA (photosystem II) and rnpB (reference) genes were used in qRT-PCR assays to assess the photosynthetic capability of natural communities of Prochlorococcus, the most abundant photosynthetic organism on Earth and a major contributor to primary production in oligotrophic oceans. After optimizing sample collection methodology, we analyzed a total of 62 stations from the Malaspina 2010 circumnavigation (including Atlantic, Pacific and Indian Oceans) at three different depths. Sequence and quantitative analyses of the corresponding amplicons showed the presence of high-light (HL) and low-light (LL) Prochlorococcus clades in essentially all 182 samples, with a largely uniform stratification of LL and HL sequences. Synechococcus cross-amplifications were detected by the taxon-specific melting temperatures of the amplicons. Laboratory exposure of Prochlorococcus MED4 (HL) and MIT9313 (LL) strains to organic pollutants (PAHs and organochlorine compounds) showed a decrease of rbcL transcript abundances, and of the rbcL to psbA ratios for both strains. We propose this technique as a convenient assay to evaluate effects of environmental stressors, including pollution, on the oceanic Prochlorococcus photosynthetic function.

No MeSH data available.


Related in: MedlinePlus