Diatom Proteomics Reveals Unique Acclimation Strategies to Mitigate Fe Limitation

Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when T. pseudonana is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies.This work was supported by National Science Foundation grants OCE1233014 (BLN) and the Office of Polar Programs Postdoctoral Fellowship grant 0444148 (BLN). DRG was supported by National Institutes of Health 5P30ES007033-10. AH and MTM were supported by Natural Sciences and Engineering Research Council of Canada. RFS and PWB were supported by the New Zealand Royal Society Marsden Fund and the Ministry of Science. This work is supported in part by the University of Washington's Proteomics Computer Resource Centre (UWPR95794). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Κρητικό ιατροσόφιον του 19ου αιώνα / έκδοση, εισαγωγή, σημειώσεις Νικόλαος Ε. Παπαδογιαννάκης.

There is an intricate interaction between iron (Fe) and copper (Cu) physiology in diatoms. However, strategies to cope with low Cu are largely unknown. This study unveils the comprehensive restructuring of the photosynthetic apparatus in the diatom Thalassiosira oceanica (CCMP1003) in response to low Cu, at the physiological and proteomic level. The restructuring results in a shift from light harvesting for photochemistry-and ultimately for carbon fixation-to photoprotection, reducing carbon fixation and oxygen evolution. The observed decreases in the physiological parameters Fv/Fm, carbon fixation, and oxygen evolution, concomitant with increases in the antennae absorption cross section (σPSII), non-photochemical quenching (NPQ) and the conversion factor (φe:C/ηPSII) are in agreement with well documented cellular responses to low Fe. However, the underlying proteomic changes due to low Cu are very different from those elicited by low Fe. Low Cu induces a significant four-fold reduction in the Cu-containing photosynthetic electron carrier plastocyanin. The decrease in plastocyanin causes a bottleneck within the photosynthetic electron transport chain (ETC), ultimately leading to substantial stoichiometric changes. Namely, 2-fold reduction in both cytochrome b6f complex (cytb6f) and photosystem II (PSII), no change in the Fe-rich PSI and a 40- and 2-fold increase in proteins potentially involved in detoxification of reactive oxygen species (ferredoxin and ferredoxin:NADP+ reductase, respectively). Furthermore, we identify 48 light harvesting complex (LHC) proteins in the publicly available genome of T. oceanica and provide proteomic evidence for 33 of these. The change in the LHC composition within the antennae in response to low Cu underlines the shift from photochemistry to photoprotection in T. oceanica (CCMP1003). Interestingly, we also reveal very significant intra-specific strain differences. Another strain of T. oceanica (CCMP 1005) requires significantly higher Cu concentrations to sustain both its maximal and minimal growth rate compared to CCMP 1003. Under low Cu, CCMP 1005 decreases its growth rate, cell size, Chla and total protein per cell. We argue that the reduction in protein per cell is the main strategy to decrease its cellular Cu requirement, as none of the other parameters tested are affected. Differences between the two strains, as well as differences between the well documented responses to low Fe and those presented here in response to low Cu are discussed

Diatom Proteomics Reveals Unique Acclimation Strategies to Mitigate Fe Limitation

<div><p>Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (F_v/F_m). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, <i>Thalassiosira pseudonana</i>, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, <i>T. pseudonana</i> up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when <i>T. pseudonana</i> is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies.</p></div

Copper dependent growth rates of <i>Thalassiosira oceanica</i> TO03 and TO05.

<p>Mean values ± standard error are shown; numbers of biological replicates (n) are indicated in brackets. Note that TO05 was not able to grow under 1.96 nM Cu in the medium (indicated by X in the graph). Given the scope of this study, TO03 was not grown under 1.96 nM or 6.08 nM, as it was able to grow under Cu concentrations <1 nM.</p

Comparison between EST-mapped proteomics datasets of TO03 and TO05, emphasizing the similarity in their genomes.

<p><b>A) Results for TO03 (plusEST). B) Results for TO05 (plusEST)</b>. The left side of the panel shows the proportion of identified predicted proteins coming from the TO05 genome <i>vs</i>. the TO03 transcriptome (ESTs). The right side of the panel shows the proportion of the different types of ESTs that have been mapped by the peptides coming from the LC-MS/MS. Even though we used the combined database including all known TO05 and TO03 predicted proteins, neither strain shows bias towards its own subset of proteins.</p

The effects of Cu limitation on growth rate, cell diameter, protein content and a series of photophysiological parameters in two strains of <i>T</i>. <i>oceanica</i>.

<p><b>2A-C</b>, growthrate, cell diameter, and Chl<i>a</i> per cell, respectively; <b>2D-F</b>, gross oxygen production, Fv/Fm, and the absorption cross section of PS II antennae, respectively; <b>2G-I</b>, ¹⁴C PE curve parameters α, P_max, and E_K, respectively; <b>2J-L</b>, ETR_PSII PE curve parameters α, P_max, and E_K, respectively; <b>2M-O</b>, conversion factor, NPQ_NSV, and cellular protein content, respectively. The values are mean ± std. error of three biological replicates. Differing letters above bars represent statistically significant changes (p < 0.05) using a 2-way ANOVA with post-hoc interaction analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181753#sec002" target="_blank">Methods</a> for details). Note that both strains have the same physiological responses under metal replete conditions. Under low Cu conditions, only growth rate and cell size are significantly reduced in the same manner in both strains.</p

Overview of expression of all putative LHCs in both strains of <i>T</i>. <i>oceanica</i>.

<p>Overview of expression of all putative LHCs in both strains of <i>T</i>. <i>oceanica</i>.</p

Venn diagram of number of proteins identified in Fe-replete and Fe-limited <i>T. pseudonana</i>.

<p>Proteins results presented were confidently identified from triplicate PAcIFIC analyses on the LTQ-VELOS. Fe-replete (blue) and Fe-limited (red) conditions were harvested at mid-exponential growth phase after acclimation. Numbers parenthetically annotated indicate homologous protein identifications.</p

Total peptide spectral counts from photosystem complex subunits.

<p>Spectral counts result from quadruplicate analyses on Fe-replete (blue) and Fe-limited (red) cultures. Photosystem II requires 2–3 atoms of Fe per complex, Cytochrome (Cyt) <i>b₆f</i> complex requires 6 Fe atoms per complex, and photosystem I requires 12 Fe atoms per complex. “*” indicates that the protein was determined to be significantly up- or down-regulated by QSpec (i.e. Bayes Factor >10 and log₂ fold change >0.5).</p

Differential expression of proteins involved in the photosynthetic electron transport chain (ETC) in response to chronic Cu limitation in <i>T</i>. <i>oceanica</i>.

<p>Differential expression of proteins involved in the photosynthetic electron transport chain (ETC) in response to chronic Cu limitation in <i>T</i>. <i>oceanica</i>.</p