Effect of elevated <i>p</i>CO₂ on gene regulation.

<p>Number of readings found for genes associated to ion transport and C_i acquisition in the 150 µatm and 1400 µatm CO₂ treatments relative to the present-day (380 µatm) CO₂ treatment.</p

Conceptual model of regulated proteins in a <i>T. heimii</i> cell.

<p>The regulated proteins involved in ion transport and C_i acquisition are shown on their putative locations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065987#pone.0065987-Mackinder1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065987#pone.0065987-Reinfelder1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065987#pone.0065987-Taylor2" target="_blank">[57]</a><b>.</b> Proteins involved in vacuolar Ca²⁺ and H⁺ transport include P-type Ca²⁺ ATPases (P-ATPase), Ca²⁺/Na⁺ exchangers (NCX), Ca²⁺/H⁺ antiporters (VCX), and vacuolar H⁺ ATPases (V-ATPase). Active uptake of HCO₃⁻ may occur via a SLC4 family anion exchanger (AE) or an SLC26 family SO₄^3−/HCO₃^−/C₂O₄²⁻ anion exchanger (SAT-1). Carbonic anhydrases (CA) are located intracellularly or extracellularly and enhance the interconversion between CO₂ and HCO₃⁻.</p

Effect of elevated <i>p</i>CO₂ on cyst morphology.

<p>Cells grown under (A–C) 150 µatm CO₂ and (D–H) 1400 µatm CO₂. Black arrows indicate cysts that are shown in detailed images, white arrows show collapsed cysts.</p

Effect of increasing CO₂ concentrations on the stable isotope composition.

<p>(A) ¹³C fractionation of organic carbon (ε_p) and calcite (ε_k), (B) ¹⁸O composition of calcite (δ¹⁸O_calcite) and DIC (δ¹⁸O_DIC), and (C) relationship between the oxygen isotopic composition of calcite (δ¹⁸O_{calcite-water}) in <i>Thoracosphaera</i> from this study (open diamonds) and from Ziveri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065987#pone.0065987-Ziveri1" target="_blank">[9]</a> (grey diamonds). Horizontal lines in (B) indicate δ¹⁸O values for HCO₃⁻ and CO₃²⁻, and dashed line indicates trend of curve. Solid lines indicate linear regressions (<i>n</i> = 12) with (A) ε_p: R² = 0.75, P<0.001, and ε_k: R² = 0.90, P<0.001, (B) δ¹⁸O_DIC: R² = 0.76, P<0.001, and (C) This study: R² = 0.99, P<0.001, and Ziveri et al. (2012), (<i>n</i> = 7): R² = 0.95, P<0.001.</p

Combined effect of elevated pCO₂ and nitrogen-limitation.

<p>(A, B) POC production, (C, D) Chl-a:POC ratios and (E, F) ε_p versus CO₂ of <i>A</i>. <i>fundyense</i> (left) and <i>S</i>. <i>trochoidea</i> (right) cultured under nitrogen-replete conditions (HN; filled symbols) and nitrogen-limited conditions (LN; open symbols). Linear trend lines, R² and P-values represent statistically significant relationships. Symbols indicate means of technical replicates. Means ± SD for all treatments are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154370#pone.0154370.t002" target="_blank">Table 2</a>. POC production and Chl-a:POC ratios have previously been published in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154370#pone.0154370.ref015" target="_blank">15</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154370#pone.0154370.ref018" target="_blank">18</a>], and ε_p in the HN treatments in Hoins et al. 2015.</p

Conceptual model of a dinoflagellate cell and processes at the thylakoid membrane of the chloroplasts.

<p>(A) high-light (HL) and nitrogen-replete (HN) conditions, (B) low-light conditions (LL) and (C) nitrogen-limitation (LN). Processes potentially influencing ¹³C fractionation ([1]–[8]) are highlighted in red, while + and − refer to an increase or decrease in ¹³C fractionation, respectively. (A) Saturating light and nutrient-replete conditions: Light provides the energy (= photons) needed for Photosystem II (PSII; in thylakoid membrane) to oxidize water to O₂, thereby producing electrons (e^-) and protons (H⁺). Electrons are transported by plastohydroquinone (PQ), thereby pumping more protons into the lumen. The cytochrome b₆f complex oxidizes PQ molecules, thereby producing electrons, which are then transported to Photosystem I (PSI) where they reduce NADP⁺ to NADPH. Protons are transported to F-ATPase to synthesize ATP. (B) Under light-limitation, the overall decreased amount of energy arriving at PSII causes a decrease in water oxidation, thereby producing less electrons and protons, and thus also less ATP and NADPH. (C) Under nitrogen-limitation, less NADPH is needed for NO₃^- reduction, thus the excess electrons are transported back to PSII by cyclic energy flow. Protons are still pumped by F-ATPase, thereby increasing the amount of ATP synthesized.</p

Combined effect of elevated pCO₂ and light-limitation.

<p>(A, B) POC production, (C, D) Chl-a:POC ratios and (E, F) ε_p versus CO₂ of <i>G</i>. <i>spinifera</i> (left) and <i>P</i>. <i>reticulatum</i> (right). Linear trend lines, R² and P-values represent statistically significant relationships. Symbols indicate means of technical replicates. Means ± SD for all treatments are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154370#pone.0154370.t001" target="_blank">Table 1</a>. Note that the trend line for <i>G</i>. <i>spinifera</i> under HL excludes the highest <i>p</i>CO₂ treatment (see also [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154370#pone.0154370.ref016" target="_blank">16</a>]). ε_p in the HL treatments have previously been published in Hoins et al. 2015.</p

Overview of the growth parameters in the HL and LL treatments.

<p>Growth rate (μ, d^-1), POC quota (pg C cell^-1), Chl-a content (pg cell^-1) and ¹³C fractionation ε_p (‰) of <i>G</i>. <i>spinifera</i> and <i>P</i>. <i>reticulatum</i> grown under high-light and low-light conditions. Values represent the mean of triplicate incubations (n = 3 ±SD). Superscript letters indicate significant differences between <i>p</i>CO₂ treatments (P<0.05). Superscript symbols refer to earlier published data in Hoins et al. 2015 (*).</p

Combined Effects of Ocean Acidification and Light or Nitrogen Availabilities on ¹³C Fractionation in Marine Dinoflagellates

<div><p>Along with increasing oceanic CO₂ concentrations, enhanced stratification constrains phytoplankton to shallower upper mixed layers with altered light regimes and nutrient concentrations. Here, we investigate the effects of elevated <i>p</i>CO₂ in combination with light or nitrogen-limitation on ¹³C fractionation (ε_p) in four dinoflagellate species. We cultured <i>Gonyaulax spinifera</i> and <i>Protoceratium reticulatum</i> in dilute batches under low-light (‘LL’) and high-light (‘HL’) conditions, and grew <i>Alexandrium fundyense</i> and <i>Scrippsiella trochoidea</i> in nitrogen-limited continuous cultures (‘LN’) and nitrogen-replete batches (‘HN’). The observed CO₂-dependency of ε_p remained unaffected by the availability of light for both <i>G</i>. <i>spinifera</i> and <i>P</i>. <i>reticulatum</i>, though at HL ε_p was consistently lower by about 2.7‰ over the tested CO₂ range for <i>P</i>. <i>reticulatum</i>. This may reflect increased uptake of (¹³C-enriched) bicarbonate fueled by increased ATP production under HL conditions. The observed CO₂-dependency of ε_p disappeared under LN conditions in both <i>A</i>. <i>fundyense</i> and <i>S</i>. <i>trochoidea</i>. The generally higher ε_p under LN may be associated with lower organic carbon production rates and/or higher ATP:NADPH ratios. CO₂-dependent ε_p under non-limiting conditions has been observed in several dinoflagellate species, showing potential for a new CO₂-proxy. Our results however demonstrate that light- and nitrogen-limitation also affect ε_p, thereby illustrating the need to carefully consider prevailing environmental conditions.</p></div

Overview of the growth parameters in the HN and LN treatments.

<p>Growth rate (μ, d^-1), POC quota (pg C cell^-1), Chl-a (pg cell^-1), POC:PON ratios (molar) and ε_p (‰) of <i>A</i>. <i>fundyense</i> and <i>S</i>. <i>trochoidea</i> grown under nitrogen-replete conditions and nitrogen-limitation. Values represent the mean of duplicate incubations (n = 2 ±SD). Superscript letters indicate significant differences between <i>p</i>CO₂ treatments (ANOVA; P<0.05; only applied when n>2). Superscript symbols refer to earlier published data in Hoins et al. 2015 (*), and Eberlein et al. 2014 (†) and 2016 (‡).</p