Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation Andrew E. Allen*†, Julie LaRoche‡§, Uma Maheswari*, Markus Lommer‡, Nicolas Schauer¶, Pascal J. Lopez*, Giovanni Finazziʈ, Alisdair R. Fernie¶, and Chris Bowler*§** *Centre National de la Recherche Scientifique Unite Mixte de Recherche 8186, Dept of Biology, Ecole Normale Supe´rieure, 46 rue d’Ulm, 75005 Paris, France; ‡Leibniz-Institut fu¨r Meereswissenschaften, 24105 Kiel, Germany; ¶Max Planck Institute of Molecular Plant Physiology, Am Muhlenberg 1, 14476 Potsdam-Golm, Germany; ʈCentre National de la Recherche Scientifique Unite Mixte de Recherche 7141, Universite´Paris 6 Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France; and **Stazione Zoologica, Villa Comunale, I 80121 Naples, Italy Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved May 3, 2008 (received for review December 4, 2007) Marine primary productivity is iron (Fe)-limited in vast regions of Table 1. General cellular, physiological, and biochemical the contemporary oceans, most notably the high nutrient low characteristics of Fe-limited P. tricornutum cells and cultures chlorophyll (HNLC) regions. Diatoms often form large blooms upon Fe-limited/ the relief of Fe limitation in HNLC regions despite their prebloom Parameter Fe-limited Fe-replete Fe-replete low cell density. Although Fe plays an important role in controlling Ϯ Ϯ Ϯ diatom distribution, the mechanisms of Fe uptake and adaptation Growth rate 0.18 0.05 0.88 0.01 0.2 0.06 Ferric reductase assay, 0.717 Ϯ 0.03 0.020 Ϯ 0.004 35.9 Ϯ 7.3 to low iron availability are largely unknown. Through a combina- AU per cell tion of nontargeted transcriptomic and metabolomic approaches, Ferric reductase assay, 0.01 0.0002 50 we have explored the biochemical strategies preferred by Phaeo- AU/␮m3 dactylum tricornutum at growth-limiting levels of dissolved Fe. Carbon fixation, 0.010 Ϯ 0.006 0.141 Ϯ 0.022 0.07 Ϯ 0.04 Processes carried out by components rich in Fe, such as photosyn- pmol C per thesis, mitochondrial electron transport, and nitrate assimilation, cell ϫ hour ␮ were down-regulated. Our results show that this retrenchment is Cell diameter, m 2.5 3.5 0.71 Cell volume, ␮m3 60 100 0.60 compensated by nitrogen (N) and carbon (C) reallocation from Chl per cell, pg per cell 0.22 0.5 0.44 protein and carbohydrate degradation, adaptations to chlorophyll Chl per volume, fg/␮m3 3.67 5.0 0.73 biosynthesis and pigment metabolism, removal of excess electrons Fv/Fm 0.18 Ϯ 0.1 0.5 Ϯ 0.05 0.36 Ϯ 0.2 by mitochondrial alternative oxidase (AOX) and non-photochem- PSII 1.2 Ϯ 0.14 1 1.2 Ϯ 0.14 ical quenching (NPQ), and augmented Fe-independent oxidative PSI 0.43 Ϯ 0.1 1.0 0.43 Ϯ 0.10 Ϯ Ϯ stress responses. Iron limitation leads to the elevated expression Cytochrome b6f 0.75 0.13 1.0 0.75 0.13 Cytochrome c 0.7 Ϯ 0.05 1.0 0.70 Ϯ 0.05 of at least three gene clusters absent from the Thalassiosira 6 NPQ 4.8 Ϯ 0.8 3.1 Ϯ 0.51 1.5 Ϯ 0.4 pseudonana genome that encode for components of iron capture AOX activity 0.6 Ϯ 0.15 0.25 Ϯ 0.18 2.4 Ϯ 1.83 and uptake mechanisms. Photosystem and electron carrier concentrations are given in relative units normalized to Fe-replete levels. AOX activity is given as a percentage of total genome ͉ metabalomics ͉ photosynthesis ͉ transcriptomics ͉ nutrients respiration (see SI Methods for details). Fe levels in Fe-limited and Fe-replete cultures corresponded to 13.4 pmol⅐literϪ1 FeЈ and 2.6 nmol⅐literϪ1 FeЈ, he oxidizing conditions of contemporary marine ecosystems respectively. Tresult in exceedingly low levels of dissolved Fe (1), and the cellular Fe demand of modern phytoplankton is frequently in excess of Fe availability (1, 2). This paradox reflects the likelihood that The recently completed genome sequence of P. tricornutum their biochemical machinery evolved in the Fe-replete, reducing (http://genome.jgi-psf.org/Phatr2/Phatr2.home.html) sheds light on Proterozoic oceans. Fe limitation is a major factor controlling some important differences between P. tricornutum and T. phytoplankton growth in the large, perennially high nutrient low pseudonana, which may account for their respective thresholds for chlorophyll (HNLC) regions in the subarctic Pacific, equatorial Fe limitation. We evaluated the whole-cell response of P. tricornu- Pacific, and Southern Oceans (3–5) and may be important in the tum to Fe limitation with multiple approaches combining gene North Atlantic (6). expression profiling and comparative genomics with gas chroma- Marine diatoms are the most important eukaryotic phytoplank- tography-mass spectroscopy (GC-MS)-aided nontargeted metabo- Ϸ ton for carbon sequestration, contributing 40% of global oceanic lomic analysis and a range of physiological measurements. Genes organic carbon production per year. More than 70% of blooms stimulated by mesoscale in situ Fe fertilization of Fe-limited HNLC waters were dominated by diatoms (7), indicating that diatoms Author contributions: A.E.A., J.L., P.J.L., and C.B. designed research; A.E.A., J.L., U.M., M.L., persist in chronically Fe-limited environments and resume rapid N.S., and G.F. performed research; J.L., P.J.L., G.F., A.R.F., and C.B. contributed new reagents/analytic tools; A.E.A., J.L., U.M., M.L., N.S., G.F., and A.R.F. analyzed data; and growth when the limitation pressure is alleviated. The tolerance of A.E.A., J.L., and C.B. wrote the paper. diatoms to Fe limitation varies widely between species (8–11). The authors declare no conflict of interest. Phaeodactylum tricornutum is highly tolerant to Fe limita- This article is a PNAS Direct Submission. tion and can grow in steady-state laboratory cultures at iron Data deposition: The data reported in this paper have been deposited in the Gene levels 50 times lower than those tolerated by the centric diatom Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE8675). Thalassiosira pseudonana (12). Steady-state growth of P. tri- †Present address: J. Craig Venter Institute, 10355 Science Center Drive, San Diego, CA 92121. ⅐ Ϫ1 Ј cornutum is Fe limited in the range of 10–30 pmol liter Fe §To whom correspondence may be addressed. E-mail: [email protected] or (FeЈ is the sum of all unchelated Fe species) (Table 1), similar [email protected]. to T. oceanica and Pseudonitzschia spp., two open ocean This article contains supporting information online at www.pnas.org/cgi/content/full/ diatoms that are commonly found in the most severely Fe 0711370105/DCSupplemental. limited regions of the world’s oceans (13). © 2008 by The National Academy of Sciences of the USA 10438–10443 ͉ PNAS ͉ July 29, 2008 ͉ vol. 105 ͉ no. 30 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711370105 Downloaded by guest on October 1, 2021 Fig. 1. Hypothetical cellular pathways and processes in iron-limited pennate diatom cells. All roman green or red type depicts gene transcripts found to be up- or down-regulated, respectively. Italicized and underlined green type indicates metabolites found to be enriched relative to total protein in iron-limited cells. DF, diffusion factor; EPS, extracellular polymeric substances; FR, ferric reductase; HMA, heavy metal-associated; PCD, programmed cell death; ROS, reactive oxygen species. responsive to Fe limitation were identified through a statistically- carbon fluxes into the cell and toward RuBisCO are adjusted to verified quantitative comparison (14) of 8,669 expressed sequenced match reductant supply. In accordance, Calvin/Benson cycle- tags (ESTs) derived from Fe-limited P. tricornutum cells with related genes downstream of RuBisCO, such as plastid localized 104,783 ESTs derived from cells grown in 11 different (all iron- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plastid replete) culture conditions (www.biologie.ens.fr/diatomics/EST). A fructose bisphosphate aldolases (FBA) I and II were up-regulated partial genome microarray and qRT-PCR provided further assess- under Fe stress (Fig. 1 and Table S1), likely in response to ment and verification of differential regulation, leading to the reductions in substrate abundance. identification of 212 up-regulated and 26 down-regulated genes. Higher levels of expression of genes encoding galactokinase and The represented acclimation strategies were grouped into three endo-1,3-beta-glucanase under Fe limitation (Fig. 1 and Table S1) categories: down-regulation, compensation, and acquisition. point to increased degradation of carbohydrates relative to Fe- replete conditions. Significant increases in cellular glucose and in Results and Discussion the intermediate sugars maltose and trehalose (Fig. S2 and Table Down-Regulation of Photosynthesis. Low Fe supply leads to cellular S2), coupled with the elevated levels of genes encoding the cytosolic energy limitation in P. tricornutum (15) and causes significant enzymes phosphofructokinase, GAPDH, and phosphoglycerate changes in carbon metabolism. Carbon fixation rates per cell were mutase (Fig. 1 and Table S1) are strong indicators that the 14-fold lower in Fe-limited P. tricornutum cells compared with conversion of polysaccharides to glucose fuels increased glycolytic Fe-replete cells (Table 1). This difference remained significant activity under Fe limitation (Fig. 1). The altered cell size (Table 1 (8-fold) despite normalization to the smaller cell volume observed and Fig. S1) and the observed increase in expression of genes in Fe-limited conditions [Table 1 and supporting information (SI) putatively encoding polysaccharide-associated cell surface proteins Fig. S1]. Reductions in cell volume, chlorophyll (Chl) per cell, (e.g., integrins, fasciclins and mucins) (Fig.