7/29/2013. Molecular insights from diatoms living in iron-deprived oceans: individual genes to community transcriptomics

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1 7/29/203 Diatoms are young, important and beautiful! Molecular insights from diatoms living in iron-deprived oceans: individual genes to community transcriptomics Adrian Marchetti University of North Carolina at Chapel Hill Highly diverse (>00,000 species) Present in almost all environments Range in size from μm to mm Base of most aquatic food chains Some produce marine toxins Important to global biogeochemistry OCB Workshop July 203 Armbrust Nature 2009 Take home messages. There is a high amount of genetic diversity in diatoms which translates into physiological and biogeochemical diversity (i.e. not all diatoms are created equal!).. Metatranscriptomics provides a new way of elucidating plankton-environment interactions (but there are still limitations). Paul J. Harrison Why do pennate diatoms bloom when iron is added to HNLC regions? Diatom phylogeny Pennates Iron limitation and iron fertilization Fragilariopsis million years ago Pseudo-nitzschia Phaeodactylum Bowler et al. Nature 2008 T. pseudonana (coastal) Armbrust et al. Science 2004 Centrics Thalassiosira T. oceanica (oceanic) Lommer et al. Genome Biology 202 Alaska 0 million years ago SRIS July 29 th, 2002 Day 20 Patch size = 700 km 2 Station Papa Fe Grow-out (June 2008) 80 million years ago Kooistra et al., in volution of Primary Producers, 2007 Courtesy of Jim Gower, Institute of Ocean Sciences, Canada

2 7/29/203 Oceanic versus coastal diatoms Pennate versus centric oceanic diatoms Growth Rate (d - ) Pseudo-nitzschia granii (HNLC waters of the N Pacific) μ / μ max P. granii T. oceanica Pseudo-nitzschia granii (HNLC waters of the N Pacific) 0.4 P. granii 0.2 P. multiseries Total Fe (nm) Pseudo-nitzschia multiseries (coastal waters of the NW Atlantic) Total Fe (nm) No growth Thalassiosira oceanica (oligotrophic waters of the N Atlantic) Diatom acclimation to variable iron conditions When dealing with low iron, diatoms will: change their cell morphology Iron requirements in coastal versus oceanic diatoms High Fe quota reduce their iron requirements increase their iron uptake affinity have an extensive iron storage capacity Low Fe quota Oceanic Pseudo-nitzschia spp. Oceanic Thalassiosira spp. Coastal Pseudo-nitzschia spp. Coastal Thalassiosira spp. Marchetti et al. L&O 2006 Using molecular approaches to study eukaryotic plankton is used for iron storage in pennate diatoms amino acid residue P.m. P. granii (oceanic) Single genes and pathways C.m. monomer H Q multimer (24mer) H Q SynCC93 R.c. T.m. H H H Q Q D Q P. multiseries (coastal) Molecule Organism cosystem Courtesy of Jim Gower, Institute of Ocean Sciences, Canada Marchetti, Parker et al. Nature

3 7/29/203 P. granii and T. oceanica continuous cultures diversity in eukaryotes P. granii (high iron pre-acclimated) Continuous culture No added iron healthy contains ferritin gene Pseudo-nitzschia multiseries 9.2 stressed Pseudo-nitzschia granii Fragilariopsis cylindrus T. oceanica (high iron pre-acclimated) Phaeodactylum tricornutum.3 Thalassiosira oceanica Thalassiosira pseudonana Marchetti et al. Nature 2009 An updated FTN phylogeny Animals Using molecular approaches to study eukaryotic plankton Green and red algae, land plants Bacteria and archaea Cyanobacteria Diatoms. All examined pennates have FTN 2. Some, but not all, examined centrics have FTN Molecule Organism cosystem Courtesy of Jim Gower, Institute of Ocean Sciences, Canada Community genomics and transcriptomics ( meta-omics ) centric pennate Metatransciptomics in the marine environment gene expression in microorganisms not easily cultured and in their natural environment previously there were few metatranscriptomic studies in marine eukayotes due to: a) poorly established methods b) few reference genomes However, we are making progress... ukaryotic phytoplankton reference organisms Haptophytes (includes coccolithophores) miliana huxleyi Isochrysis galbana Phaeocystis antarctica and globosa Chlorophytes (includes green algae) Ostreococcus tauri Volvox carteri Ostreococcus lucimarinus Chlorella sp. Chlamydomonas reinhardii Micromonas pusilla Heterokontophyta (includes diatoms) Thalassiosira pseudonana Phaeodactylum tricornutum Pseudo-nitzschia multiseries Pseudo-nitzschia granii Fragilariopsis cylindrus Aureococcus anophagefferens Chaetoceros sp. Pseudo-nitzschia australis Dinoflagellata (includes dinoflagellates) multiple species whole genomes xpressed Sequence Tags (STS) Cryptophyta Guillardia theta multiple species Now available!!! 3

4 7/29/203 Comparative metatranscriptome at Station Papa (Papa-GO) Ocean Station Papa (P26: 4 W, 0 N) in June 2008 well-characterized iron-limited, HNLC region site of SRIS iron-enrichment experiment in July 2002 Diatoms exhibited the largest response to iron enrichment ~000L of seawater were filtered from ambient OSP (T0) PlusFe and Ctl Treatments 24 x 0L were filled with seawater and spiked with 4 nm FeCl3. 3 x 0L were left unamended After 98 h, seawater was filtered (T98) Metatranscriptomic annotation pipeline Functional Annotation Library No. of reads = 2.2 x 0 Ave. length = 98 b Control (T98) Taxonomic Identification PlusFe (T98) 44 GS-FLX nanoplankton picoplankton Library No. of reads =.4 x 07 Ave. length = 44 b e-value 0-3 diatom BWA TUB haptophyte GS Pseudo-nitzschia spp. SOLiD Nominal size & other ST db e-value 0-3 PlusFe (T98) Red fluorescence (cellular chlorophyll) Red fluorescence PlusFe Library No. of reads =.7 x 0 Ave. length = 99 b Nominal size PlusFe Library No. of reads =.9 x 07 Ave. reads = 4 b BWA chlorophyte PCY Marchetti, Schruth et al. PNAS 202 MANTA SOLiD: 3003 genes are significantly differentially expressed SOLiD Sequence Libraries Fold Change (log2[/ambient]) Transcripts are over-represented following iron enrichment MANTA Plots were created for taxonomic subsets Haptophytes Diatoms Chlorophytes Proportion of reads with taxonomic hits assigned to major phytoplankton groups (from 44 reads) Diatoms % 3% Haptophytes 30% 33% Chlorophytes 26% % Transcripts are under-represented following iron enrichment Count Average (log2[] + log2[ambient])/2 Marchetti, Schruth et al. PNAS 202 4

5 7/29/203 Photosynthetic architecture in diatoms differs depending on iron status Phytoplankton gene expression response to iron enrichment mitochondrion glutamine GS GOGAT glutamate GDH glutamate plas d flavodoxin UR NiR cytochrome c6 PSI urea FLD FDX ornithine OTC UTP NO2 Nature 996 urea PCY Cyt CYC PSII NR NRT citrulline Transcripts present ASS arginino succinate ASL Under-represented Over-represented Urea Cycle ARG arginine NiRT NO3 chlorophytes diatoms carbamoyl-p FNR c6 haptophytes uncps porphyrin and chlorophyll ATP FLD No transcripts polyamine biosynthesis fumarate NO2 urea AMT UTP TCA cycle Marchetti, Schruth et al. PNAS 202 SOLiD Transcripts ambient 00 0 FDX 2 FLD SOLiD Transcripts ambient 0 20 PCY /ND CYT2 c Proteorhodopsins in marine microbes 0 20 Beja et al. Science 2000

6 7/29/203 HGT of rhodopsins from bacteria to eukaryotic phytoplankton? 600 RHO is not present in all diatoms SOLiD Transcripts ambient ukaryotic phytoplankton fall within a xanthorhodopsin clade of eubacteria RHO Conclusions Pseudo-nitzschia granii transcriptome in response to iron More transcripts under Fe-replete conditions Immediately following iron enrichment, oceanic diatoms continue to express iron-free protein encoding genes (e.g flavodoxin, plastocyanin, non-fe SODs, etc.) Most highly expressed genes are characteristic of diatoms pathways (urea cycle, nitrate assimilation, Si transporters and polyamine synthesis, PUFA and chrysolaminarin biosynthesis) Oceanic diatoms contain ferritin and rhodopsin-type genes that may provide a competitive advantage in iron-limited environments Fold Change (log 2 [/ambient]) ferritin More transcripts under Fe-limiting conditions ISIP2A >2400 genes were differentially expressed! Count Average (log 2 [] + log 2 [ambient])/2 Future work: Transcriptome profiling ecologically important diatoms Fe-limitation N-limitation Light limitation High CO 2 6

7 7/29/203 Acknowledgements Collaborators: David Schruth Micaela Parker (UW) Colleen Durkin (WHOI) Rhonda Morales (UW) Robin Kodner (UWW) Chris Berthiaume (UW) Andy Allen (JCVI) Ginger Armbrust (UW) Maite Maldonado (UBC) Michael Murphy (UBC) Thanks to: Crew of CCGS J.P Tully Scientists at IOS Francois Ribalet (UW) Schuster Lab (Penn State) Robert Morris (UW) Shady Amin (UW) J. Hackett (ASU) J. Archibald (DalU) 7