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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

Representation of amplicon melting temperature (Tm) values, in°C, plotted against Cp values (in cycles) obtained for field samples at the three sampled depths: Surface (orange), DCM (green) and DCM+40 (blue).For comparison, the graphs include the corresponding values from supplementary S3 Table. HL amplicons: Prochlorococcus strains MED4 (M4), MIT9515 (M5) and EQPAC1-C (EQ). LL amplicons: Prochlorococcus strains MIT9313 (M3), NATL2A (NA), and the Synechococcus strain WH7803 (WH).
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pone.0133207.g003: Representation of amplicon melting temperature (Tm) values, in°C, plotted against Cp values (in cycles) obtained for field samples at the three sampled depths: Surface (orange), DCM (green) and DCM+40 (blue).For comparison, the graphs include the corresponding values from supplementary S3 Table. HL amplicons: Prochlorococcus strains MED4 (M4), MIT9515 (M5) and EQPAC1-C (EQ). LL amplicons: Prochlorococcus strains MIT9313 (M3), NATL2A (NA), and the Synechococcus strain WH7803 (WH).

Mentions: A combined analysis of Tm values of the different amplicons and their corresponding Cp values was used to estimate the sequence heterogeneity (and hence, genetic variability) within each sample (Fig 3). rnpB and psbA HL amplicons showed an extreme homogeneity of Tm values for all 182 samples, irrespective of the depth or of their relative abundance (Fig 3). In fact, all samples showed essentially the same Tm value, 81 and 82°C, respectively, within a margin of few tenths of°C (Table 5). The situation was similar for HL rbcL amplicons, although some samples, particularly at DCM+40, showed clearly differentiated Tm values (Fig 3, central left panel). In this case, 13% of DCM+40 samples showed enough HL rbcL amplicon heterogeneity to be resolved in two Tm peaks, a phenomenon that was only episodic in samples from 3 m or DCM and unobserved with the other two HL amplicons at any depth (Table 5). These atypical amplicons corresponded to samples with very low abundance in HL sequences—i.e., high Cp values (Fig 3). However, we consider that this Tm variability is still consistent with the natural variation of Prochlorococcus strains, as HL rbcL amplicons with a low Tm value were also observed for the cultured EQPAC1-C strain (marked as "EQ" in Fig 3, S3 Table in supplementary material).


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)

Representation of amplicon melting temperature (Tm) values, in°C, plotted against Cp values (in cycles) obtained for field samples at the three sampled depths: Surface (orange), DCM (green) and DCM+40 (blue).For comparison, the graphs include the corresponding values from supplementary S3 Table. HL amplicons: Prochlorococcus strains MED4 (M4), MIT9515 (M5) and EQPAC1-C (EQ). LL amplicons: Prochlorococcus strains MIT9313 (M3), NATL2A (NA), and the Synechococcus strain WH7803 (WH).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133207.g003: Representation of amplicon melting temperature (Tm) values, in°C, plotted against Cp values (in cycles) obtained for field samples at the three sampled depths: Surface (orange), DCM (green) and DCM+40 (blue).For comparison, the graphs include the corresponding values from supplementary S3 Table. HL amplicons: Prochlorococcus strains MED4 (M4), MIT9515 (M5) and EQPAC1-C (EQ). LL amplicons: Prochlorococcus strains MIT9313 (M3), NATL2A (NA), and the Synechococcus strain WH7803 (WH).
Mentions: A combined analysis of Tm values of the different amplicons and their corresponding Cp values was used to estimate the sequence heterogeneity (and hence, genetic variability) within each sample (Fig 3). rnpB and psbA HL amplicons showed an extreme homogeneity of Tm values for all 182 samples, irrespective of the depth or of their relative abundance (Fig 3). In fact, all samples showed essentially the same Tm value, 81 and 82°C, respectively, within a margin of few tenths of°C (Table 5). The situation was similar for HL rbcL amplicons, although some samples, particularly at DCM+40, showed clearly differentiated Tm values (Fig 3, central left panel). In this case, 13% of DCM+40 samples showed enough HL rbcL amplicon heterogeneity to be resolved in two Tm peaks, a phenomenon that was only episodic in samples from 3 m or DCM and unobserved with the other two HL amplicons at any depth (Table 5). These atypical amplicons corresponded to samples with very low abundance in HL sequences—i.e., high Cp values (Fig 3). However, we consider that this Tm variability is still consistent with the natural variation of Prochlorococcus strains, as HL rbcL amplicons with a low Tm value were also observed for the cultured EQPAC1-C strain (marked as "EQ" in Fig 3, S3 Table in supplementary material).

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