Measuring growth rate and Photosystem II Photoinactivation.

<p>A) Turbidostat culture cell density was maintained at set-point by automatic dilutions activated by a sensor tracking optical density at 680 nm. The last 10 cycles of dilution before starting the light-shift experiment were used to calculate the growth rate (μ, d⁻¹) by fitting the increase in OD₆₈₀ versus time with an exponential curve for each dilution cycle. This example growth curve was taken from a culture growing at 240 µmol photons·m⁻²·s⁻¹, under bubbling with elevated pCO₂ of 750 ppmv. B) PSII photochemical yield during and after an upward light shift. Cells were shifted from the culture growth light (240 µmol photons·m⁻²·s⁻¹ in this example; 30, 80, 160, 240 or 380 µmol photons·m⁻²·s⁻¹ in other experiments) upward to 450 µmol photons·m⁻²·s⁻¹ and then back to the culture growth light again. Susceptibility to photoinactivation (σ_i, A²) was obtained by fitting an exponential decay curve to the decrease in F_V/F_M versus cumulative photons, in the sub-culture in which PSII repair was blocked by lincomycin.</p

Effects of growth light and pCO2 on functional absorption cross-section for PSII photochemistry.

<p>A) Functional absorption cross-section for Photosystem II measured under culture light level (σ′_PSII, A²·quanta⁻¹) under ambient or elevated pCO₂ plotted against the culture growth light (µmol photons·m⁻²·s⁻¹). Solid line: Polynomial curve for growth light response of σ′_PSII under ambient pCO₂. Dashed line: Polynomial curve for light response of σ′_PSII under elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curves. B) Photochemical quenching (qP) versus growth light under ambient or elevated pCO₂. Solid line: Polynomial curve for pooled growth light response for cultures from the two CO₂ treatments. Thin dotted lines: 95% confidence intervals on the fitted curve. C) Functional absorption cross-section (σ′_PSII) of the samples measured after 2 min of acclimation to culture-light, as a function of σ_PSII measured after 5 min dark-acclimation. Solid line: Linear regression of pooled response of cultures from ambient and elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curve. Dotted diagonal line indicates 1∶1 ratio. D) Functional absorption cross-section (σ″_PSII) of the samples measured after 2 min of acclimation to high-light of 450 µmol photons·m⁻²·s⁻¹, as a function of σ_PSII measured after 5 min dark-acclimation. Solid line: Linear regression of pooled response of cultures from ambient and elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curve. Dotted diagonal line indicates 1∶1 ratio.</p

Parameters of the seawater carbonate system of the cultures.

<p>Dissolved inorganic carbon and pH were measured upon terminal sampling of turbidostat cultures grown under ambient- (390 ppm) and elevated- (750 ppm) CO₂ treatments. Total alkalinity, pCO₂, carbonate (CO₃²⁻), bicarbonate (HCO₃⁻) and free CO₂ were calculated on the base of the temperature (18°C), salinity (35 g L⁻¹), pH and concentrations of dissolved inorganic carbon, phosphate (21 µmol L⁻¹) and silica (52.5 µmol L⁻¹) using the software CO2SYS. n = 3, mean ±S.D.; n = 2 in treatments of 390, 750 pCO₂ ppmv at the growth light of 31 µmol photons·m⁻²·s⁻¹; for these estimates we present mean ±1/2 range.</p

Malondialdehyde product of lipid peroxidation, growth rate and PSII photoinactivation.

<p>A) Malondialdehyde as a function of growth light. Solid line: Polynomial curve for malondialdehyde of cultures from ambient pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curve. B) Ratio of malondialdehyde measured at time 90 (T90) of a high light treatment to time 0 (T0) of control levels, versus growth light. Dotted horizontal line shows 1∶1 ratio of no change during the high light treatment period compared to the growth level. C) Malondialdehyde versus growth rate. Solid line: Linear regression of growth response of cultures from ambient pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curve. D) Functional cross section for photoinactivation of Photosystem II versus malondialdehyde. Solid line: Linear regression of σ_i versus malondialdehyde under ambient pCO₂. Dashed line: Linear regression of σ_i versus malondialdehyde under elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curves.</p

Photophysiology of cells grown under ambient and elevated CO2 versus growth light or growth rate.

<p>A) Functional absorption cross-section for Photosystem II photochemistry (σ_PSII, A²·quanta⁻¹) versus growth light. Solid line: Polynomial curve for pooled growth light response of σ_PSII under both ambient and elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curve. B) Functional absorption cross-section for Photosystem II (σ_PSII) versus growth rate. C) Functional cross section for photoinactivation of Photosystem II (σ_i, A²·quanta⁻¹) versus growth light. Solid line: Polynomial curve for growth light response of σ_i under ambient pCO₂. Dashed line: Polynomial curve for growth light response of σ_i under elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curves. D) Functional cross section for photoinactivation of Photosystem II (σ_i) versus growth rate. Solid line: Linear regression for growth response of σ_i under ambient pCO₂. Dashed line: Linear regression for growth response of σ_i under elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curves. E) Ratio of σ_PSII:σ_i versus growth light. Solid line: Polynomial curve for light response of σ_PSII:σ_i under ambient pCO₂. Dash line: Polynomial curve for growth light response of σ_PSII:σ_i under elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curves. F) Ratio of σ_PSII:σ_i versus growth-rate. Solid line: Linear regression for growth response of σ_PSII:σ_i under ambient pCO₂. Dash line: Linear regression for growth response of σ_PSII:σ_i under elevated pCO₂. Thin dotted lines: 95% confidence intervals on the fitted curves.</p

Correction: Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy

<p>Correction: Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy</p

Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy

<div><p>Diatoms are marine primary producers that sink in part due to the density of their silica frustules. Sinking of these phytoplankters is crucial for both the biological pump that sequesters carbon to the deep ocean and for the life strategy of the organism. Sinking rates have been previously measured through settling columns, or with fluorimeters or video microscopy arranged perpendicularly to the direction of sinking. These side-view techniques require large volumes of culture, specialized equipment and are difficult to scale up to multiple simultaneous measures for screening. We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll <i>a</i> fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; <i>Thalassiosira pseudonana</i> CCMP1335, chain-forming <i>Skeletonema marinoi</i> RO5A and <i>Coscinodiscus radiatus</i> CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d^-1) using an RStudio script, SinkWORX. We thereby detected significant differences in sinking rates as larger diatom cells sank faster than smaller cells, and cultures in stationary phase sank faster than those in exponential phase. Our sinking rate estimates accord well with literature values from previously established methods. This well plate-based method can operate as a high throughput integrative phenotypic screen for factors that influence sinking rates including macromolecular allocations, nutrient availability or uptake rates, chain-length or cell size, degree of silification and progression through growth stages. Alternately the approach can be used to phenomically screen libraries of mutants.</p></div

Effect of cell or filament length and growth phase on sinking rate.

<p>Graph depicts the relationship of sinking rate (m day^-1) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185166#pone.0185166.t001" target="_blank">Table 1</a>) derived from the first phase of sinking (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185166#pone.0185166.g002" target="_blank">2</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185166#pone.0185166.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185166#pone.0185166.g004" target="_blank">4</a>) <i>versus</i> Cell or Filament Length (μm) with associated standard error bars. Three diatoms were studied; <i>Thalassiosira pseudonana</i> (square; <i>Skeletonema marinoi</i> (triangle) and <i>Coscinodiscus radiatus</i> (circle),. Sinking rates at different growth phases of each species were also studied, outlined grey shapes depict exponential phase while black filled shapes depict stationary phase.</p

Raw data sinking assays and scaled sinking assays of <i>Coscinodiscus radiatus</i> CCMP312 during exponential phase in a 24 well-plate.

<p><b>(A)</b> Data points from a triplicated sinking assay of <i>Coscinodiscus radiatus</i> in exponential phase plotted as RFU <i>versus</i> Elapsed Time (minutes). <b>(B)</b> Exponential growth phase triplicates with sqrt(Scaled RFU) x 3.8 <i>versus</i> Elapsed Time (minutes). Data was fit with a segmented linear regression after breakpoint analysis. The solid line is a linear regression of the first phase, prior to the break point, with a fixed y-intercept of 3.8. The dashed line represents a linear regression of the second phase after break point, while. The breakpoint is represented by the intersection of the lines.</p

Raw data sinking assays and scaled sinking assays of <i>Skeletonema marinoi</i> RO5A triplicates in exponential and stationary phase.

<p><b>(A)</b> Data points from a triplicated sinking assay <i>S</i>. <i>marinoi</i> in exponential phase plotted RFU <i>versus</i> Elapsed Time (minutes). <b>(B)</b> Triplicated sinking assay of <i>S</i>. <i>marinoi</i> in stationary phase plotted RFU <i>versus</i> Elapsed Time (minutes). <b>(C)</b> Exponential growth phase triplicates with sqrt(Scaled RFU) x 3.8 <i>versus</i> Elapsed Time (minutes). Data was fit in a segmented linear regression after breakpoint analysis. The long dashed line was the fitted linear regression of the first phase prior to the breakpoint, with no fixed intercept, while the nearly overlapping solid line is a linear regression prior to the breakpoint but using a fixed y-intercept of 3.8. The dashed line represents a linear regression of the second phase after the break point. The breakpoint is represented by the intersection of the dashed lines. <b>(D)</b> Stationary growth phase triplicates with sqrt(Scaled RFU) x 3.8 <i>versus</i> Elapsed Time (minutes). Data was fit in a segmented linear regression after breakpoint analysis. The long dashed line was the fitted linear regression of the first phase, with no fixed intercept, while the solid line is a fitted linear regression prior to the breakpoint but using a fixed y-intercept of 3.8. The dashed line represents a linear regression of the second phase following the breakpoint. The breakpoint is represented by the intersection of the dashed lines.</p