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. 2017 Jan 27;12(1):e0168991.
doi: 10.1371/journal.pone.0168991. eCollection 2017.

Photoinactivation of Photosystem II in Prochlorococcus and Synechococcus

Cole D Murphy  1 , Mitchell S Roodvoets  1 , Emily J Austen  2 , Allison Dolan  2 , Audrey Barnett  3 , Douglas A Campbell  2
Affiliations

Photoinactivation of Photosystem II in Prochlorococcus and Synechococcus

Cole D Murphy et al. PLoS One. .

Abstract

The marine picocyanobacteria Synechococcus and Prochlorococcus numerically dominate open ocean phytoplankton. Although evolutionarily related they are ecologically distinct, with different strategies to harvest, manage and exploit light. We grew representative strains of Synechococcus and Prochlorococcus and tracked their susceptibility to photoinactivation of Photosystem II under a range of light levels. As expected blue light provoked more rapid photoinactivation than did an equivalent level of red light. The previous growth light level altered the susceptibility of Synechococcus, but not Prochlorococcus, to this photoinactivation. We resolved a simple linear pattern when we expressed the yield of photoinactivation on the basis of photons delivered to Photosystem II photochemistry, plotted versus excitation pressure upon Photosystem II, the balance between excitation and downstream metabolism. A high excitation pressure increases the generation of reactive oxygen species, and thus increases the yield of photoinactivation of Photosystem II. Blue photons, however, retained a higher baseline photoinactivation across a wide range of excitation pressures. Our experiments thus uncovered the relative influences of the direct photoinactivation of Photosystem II by blue photons which dominates under low to moderate blue light, and photoinactivation as a side effect of reactive oxygen species which dominates under higher excitation pressure. Synechococcus enjoyed a positive metabolic return upon the repair or the synthesis of a Photosystem II, across the range of light levels we tested. In contrast Prochlorococcus only enjoyed a positive return upon synthesis of a Photosystem II up to 400 μmol photons m-2 s-1. These differential cost-benefits probably underlie the distinct photoacclimation strategies of the species.

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Conflict of interest statement

Open source (www.sourceforge.net) PSIWORX-R scripts for extracting and analyzing chlorophyll fluorescence induction and relaxation parameters from data generated by PSI Fluorometers were generated (EA & AB) with funding from an NSERC Canada Engage grant with sponsorship from QuBit Systems, Kingston, Ontario. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The scripts are freely available via www.sourceforge.net. We have no other competing interests.

Figures

Fig 1
Fig 1. Conceptual overview of fates of photons in Photosystem II.
Incident photons (downward waves) are differentially absorbed (ā*) depending upon the particular absorbance spectra of the antenna pigment protein complexes serving PSII, indicated as a green trapezoid to reflect the diversity of antenna pigments and protein structures across taxa. A variable fraction of the excitons is dissipated as heat (yellow-orange arrow, NPQ); a further small fraction of excitons are dissipated as fluorescence emitted from the antenna (red arrow). The remaining excitons are transferred into the inner antenna of the PSII reaction centre (downward red arrows). Of these excitons a small but variable fraction are again dissipated as fluorescence (red arrow) from the pigments of the reaction centre. The remaining excitons provoke PSII photochemistry (σPSII; A2 quanta-1) leading to charge separation and electron transport with a variable yield (ΦPSII), or to fast recombinations resulting in heat emission (yellow-orange arrow, NPQ). If excitation is high relative to downstream electron transport excited intermediates can accumulate. This provokes production of singlet oxygen (1O2), which leads to an increase in the probability of PSII inactivation, herein parameterized Φi PSII, a dimensionless yield for inactivation of a PSII by a photon delivered through the PSII antenna. Short wavelength photons can also be directly absorbed by the manganese cluster of the oxygen evolving complex (thin violet wave), leading to direct photoinactivation. This figure is based upon concepts reviewed in [18,44,52,62,68].
Fig 2
Fig 2. Cellular absorbance, light treatment color and protocol.
(A) Whole Cell Absorbance Spectra of Synechococcus sp. WH8102 and Prochlorococcus marinus MED4. The relative emission spectra of the actinic blue and red LED used to induce photoinactivation (Fig 2B) in Synechococcus and Prochlorococcus are overlaid on the absorbance spectra, normalized to their red chlorophyll a peak. (B) Light intensity and measurement timing through the duration of a representative light treatment experiment. The pre-zero dotted line indicates the growth light for the culture; 75 in this representative figure; 30 or 260 μmol photons m-2 s-1 in other experiments. Each treatment time course then consisted of 10 sequential periods of 327 s, shown by the solid black line. The first 327 s period was in the dark, the second period was under the growth irradiance for the particular sample. The third to ninth treatment periods, shown here at 700 μmol photons m-2 s-1, were at irradiance and colour combinations shown in Table 1. The tenth period was a low light recovery phase of 20 μmol photons m-2 s-1. The black triangle indicates a Fast Repetition chlorophyll fluorescence induction measurement taken after the initial dark period, using 40 flashlets of 1.2 μs duration, spaced by 2 μs darkness, which cumulatively delivered a single turnover saturating flash over 128 μs. From this induction curve we used PSIWORX-R script to extract estimates for F0, FM and σPSII. Each green triangle thereafter indicates the timing of a chlorophyll fluorescence induction measurement taken after an illuminated period, represented in the inset above the treatment trace. At each measurement point we captured an induction applied in the presence of continuing actinic irradiance to extract estimates for FS, FM′ and σPSII′. We then interrupted the actinic irradiance for 2 s of darkness to allow PSII centres to re-open, followed by another fluorescence induction to extract estimates for F0′, FM′ (2s) and σPSII′(2s). The post-induction fluorescence relaxation phase of the full Fast Repetition and Relaxation fluorescence profile is omitted from this schematic diagram for clarity.
Fig 3
Fig 3. Representative photoinactivation treatment data.
Prochlorococcus marinus MED4 was grown under 260 μmol photons m-2 s-1 and then treated under 1200 μmol photons m-2 s-1 of red light (red squares) or 1200 μmol photons m-2 s-1 of blue light (blue squares) following the protocol outlined in Fig 2A. For non-least squares non-linear modelling of the data (nlsLM, R) [104] each FV′ 2s/FM′ 2s derived from an individual Fast Repetition and Relaxation chlorophyll fluorescence induction after 2 s of darkness following actinic light conditions (Fig 2A, inset) was weighted by the inverse of its 95% confidence interval (plotted as error bars on the points) to account for variability in the precision of individual estimates of FV′ 2s/FM′ 2s. (A) The decay of the quantum yield of PSII (FV′ 2s/FM′ 2s) (Fig 2A, inset), plotted versus time and cumulative incident photons since the start of the treatment. The decay of FV′ 2s/FM′ 2s was fit (solid lines) to the annotated equation to extract σi, a target size parameterization of the probability of an incident photon inducing photoinactivation of PSII. In these examples the σi was 1.65 × 10−5 Å2 PSII-1 under red light treatment (red diamonds) and 1.23 × 10−4 Å2 PSII-1 under blue light treatment (blue squares). 95% C.I. on the fit plotted as dotted lines. (B) The decay of FV′ 2s/FM′ 2s (solid lines) against cumulative photons delivered to PSII photochemistry, estimated as cumulative incident photons multiplied by the effective absorption cross section of the sample, σPSII′ 2s. The decay is fit to the annotated equation to extract Φi PSII, the probability of photoinactivation by a photon delivered to PSII through the antenna. The fitted values of Φi PSII were 2.8 × 10−7 PSII photon -1 under red light treatment (red diamonds) and 7.2 × 10−7 PSII photon -1 under blue light treatment (blue squares). 95% C.I. on the fit plotted as dotted lines.
Fig 4
Fig 4. σi plotted against treatment light intensities.
For regressions each σi derived from an individual treatment time course (ex. Fig 3A) was weighted by the inverse of its 95% confidence interval (error bars on the points) to account for variability in the precision of individual estimates of σi. (A) σi from treatments of Synechococcus sp. WH8102 under a range of light levels. σi (Å2 photon-1) estimates derived from red light treatment (open red symbols) fell on a single regression against treatment light intensity (μmol photons m-2 s-1) for cultures grown under both low (red open inverted triangles) or high (red open diamonds) light; (solid red line, slope = 5.620 × 10−8 ± 1.212 × 10−8, intercept = 4.850 × 10−6 ± 1.027 × 10−5 (not significantly different from zero, p>0.05), R2 = 0.6616, dotted red lines denote 95% confidence intervals on the regression). σi estimates derived from blue light treatment (open blue symbols) fell on different regressions for cultures grown under low light (blue open triangles) (solid blue line, slope = 5.200 × 10−8 ± 1.814 × 10−8, intercept = 1.586 × 10−4 ± 1.146 × 10−5, R2 = 0.2401, dotted lines show 95% confidence intervals on the regression), or for cultures grown under high light (blue open squares) (slope = 1.008 × 10−7 ± 1.626 × 10−8, intercept = 3.749 × 10−5 ± 1.011 × 10−5, R2 = 0.846) (S1 Statistics). (B) σi from treatments of Prochlorococcus marinus MED4 measured under a range of treatment irradiances. σi estimates derived from red light treatment fell on a single regression (solid red line, slope not significantly different from zero, p>0.05; intercept = 2.130 × 10−5 ± 9.023 × 10−6, R2 = 0.05971, dotted red lines denote 95% confidence intervals) for cultures grown under either low (red inverted triangles) or high (red diamonds) light. σi estimates derived from blue light treatment fell on a single regression (solid blue line, slope = 4.013 × 10−8 ± 1.714 × 10−8, intercept = 1.191 × 10−4 ± 1.214 × 10−5, R2 = 0.2437, dotted blue lines denote 95% confidence intervals) for cultures grown under low (blue triangles) or high (blue squares) growth light (S2 Statistics).
Fig 5
Fig 5. σi versus excitation pressure on Photosystem II.
σi plotted against excitation pressure, measured as 1—qP after 300 s of treatment light exposure. Under red or blue treatment light, qP showed remained nearly steady from 327 s onwards (data not shown) so the plotted values for 1—qP were taken after the first period of 327 exposure to treatment light. For regressions each σi derived from an individual treatment timecourse (Fig 3A) was weighted by the inverse of its 95% confidence interval (error bars on the points) to account for variability in the precision of individual estimates of σi. (A) σi from Synechococcus sp. WH8102. σi estimates derived from red light treatments fell on a single regression (solid red line, slope = 1.103 × 10−4 ± 2.670 × 10−5, intercept not significantly different from zero, p>0.05; R2 = 0.6079, dotted red lines denote 95% confidence intervals on the regression). for Synechococcus cultures grown under low (red open inverted triangles) or high (red open diamonds) growth light. For σi estimates derived from blue light treatment, low light grown cultures (blue open triangles) fell upon a regression (solid blue line, slope = 2.349 × 10−4 ± 4.437 × 10−5, intercept = 9.742 × 10−6 ± 1.540 × 10−5, R2 = 0.3813, dotted lines show 95 % confidence intervals), while the high light grown cultures (open blue squares) fell upon a different regression (solid blue line, slope = 1.332 × 10−4 ± 3.326 × 10−5, intercept not significantly different from zero, p>0.05; R2 = 0.8001, dotted lines show 95 % confidence intervals on the regression) (S3 Statistics). (B) σi from Prochlorococcus marinus MED4. σi estimates derived from red light treatment (low light grown cultures, red inverted triangles; high light grown cultures, red diamonds) fell on a single regression (solid red line, slope = 3.484 × 10−5 ± 1.222 × 10−5, intercept not significantly different from zero, p>0.05;, R2 = 0.5755, dotted red lines denote 95 % confidence intervals). σi estimates derived from blue light treatment (low light grown cultures, blue triangles; high light grown cultures, blue squares) fell on a single regression (solid blue line, slope = 1.262 × 10−4 ± 5.667 × 10−5, intercept not significantly different from zero, p>0.05; R2 = 0.2259, dotted blue lines denote 95 % confidence intervals) (S4 Statistics).
Fig 6
Fig 6. Φi PSII versus excitation pressure on Photosystem II.
Synechococcus sp. WH8102 (open symbols) and Prochlorococcus marinus MED4 (closed symbols) fell on a common regression for Φi PSII (PSII photon-1) measured under red light treatment (solid red line, slope = 3.142 × 10−7 ± 1.110 × 10−7, intercept = 1.968 × 10−7 ± 7.308 × 10−8, R2 = 0.2968, dotted red lines denote 95% confidence intervals). Synechococcus sp. WH8102 and Prochlorococcus marinus MED4 also fell on a common regression Φi PSII measured under blue light treatment (solid blue line, slope = 3.731 × 10−7 ± 4.921 × 10−8, intercept = 4.046 × 10−7 ± 3.458 × 10−8, R2 = 0.5156, dotted red lines denote 95% confidence intervals on the regressions). Species had no statistically significant effect on the regressions of Φi PSII versus excitatation pressure, nor did low (circles) versus high (squares) growth light when either species or growth light was including in a linear model of the data as a binary interaction term, using ‘lm’ in R [128] (S5 Statistics).
Fig 7
Fig 7. Rates of electron transfer through Photosystem II versus Treatment Light intensity, compared to electron equivalent cost for Photosystem II synthesis or recycling.
Photochemical events (larger circles) were estimated as σPSII′ 2s × qP × E. Photoinactivation eventswere estimated as σi × E and multiplied by the e- equivalent cost for biosynthesis of PSII (smaller black circles; shaded grey segment) or by the e- equivalent cost for turnover of PsbA/PsbD (+ or ×; black segment along X axes). (A) Synechococcus under blue light. (B) Prochlorococcus under blue light. (C) Synechococcus under red light. (D) Prochlorococcus under red light.
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Grants and funding

This work was funded by the Canada Research Chair program (DC) and the Natural Sciences and Engineering Research Council of Canada (DC), using equipment funded by the Canada Foundation for Innovation and the New Brunswick Foundation for Innovation (DC). CM and MR were supported by summer fellowships from Mount Allison University. EA & AB were supported by NSERC Canada Engage funding to write PSIWORX-R scripts for extracting and analyzing chlorophyll fluorescence induction and relaxation parameters from data generated by PSI Fluorometers, with sponsorship from QuBit Systems, Kingston, Ontario. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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