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My master's thesis project presentation on Transcriptomics of Iron Limitation in Phaeocystis antarctica supervised by Assist. Prof. Ahmed Moustafa (who surprised me with slide 2 :)

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  1. 1. Transcriptomics of Iron Limitation in Phaeocystis antarctica Mariam Reyad Rizkallah Supervisors: Assist. Prof. Ahmed Moustafa Dr. Sára Beszteri
  2. 2. 2004 – 2010 BSc Pharmacy Cairo University 2010 – 211 Diploma Software Engineering Information Technology Institute (ITI) 2011 – 2014 MSc Biotechnology American University in Cairo (AUC) & Alfred Wegener Institute (AWI) 2014 – PhD Max Planck Institute (MPI) & Alfred Wegener Institute (AWI)
  3. 3. Transcriptomics of Iron Limitation in Phaeocystis antarctica Mariam Reyad Rizkallah Supervisors: Assist. Prof. Ahmed Moustafa Dr. Sára Beszteri
  4. 4. Outline Introduction  Phytoplankton: The Hidden Trees of the Ocean  The Iron Hypothesis  The Antarctic (Southern) Ocean: The Most Important High-Nitrate Low- Chlorophyll Region  Phaeocystis antarctica as a Model Organism for Adaptation to Iron Limitation Study Objectives and Design Materials and Methods  Culturing and Physiological Assessments  Computational Analysis of P. antarctica Transcriptome Results and Discussion  P. antarctica Transcriptome Characterization  P. antarctica under Changing Iron Conditions: Growth and Physiology and Differential Gene Expression Conclusions and Future Directions
  5. 5. Alarming Climate Changes… Source:
  6. 6. … Ocean Contributes to CO2 Export Source: Average global chlorophyll a concentration in 2010 - NASA’s satellite sensor, SeaWiFS image
  7. 7. Phytoplankton: The Trees of the Ocean Photosynthetic prokaryotes and unicellular eukaryotes Responsible for half of the global net primary production Various size classes and ecological distributions Biotic (e.g., competition and grazing) and abiotic (e.g., nutrients and light intensity) limit their productivity Phytoplankton bloom off Denmark in 2004 (NASA): ocean-color-hurricanes-environment-weather-science-global- warming/ Antarctic phytoplankton samples:
  8. 8. Phytoplankton in an Iron-Limited World Ocean Equatorial Pacific Ocean Subarctic Pacific Ocean The Southern Ocean After Boyd et al., 2008 and de Baar et al., 2002 12 3 4 5 1 EIFEX 2004 Maximum solitary 2 EisenEx 2000 16-fold colonial and solitary 3 SOFEX-N 2002 Colonies 4 SOFEX-S 2002 N/A 5 SOIREE 1999 Sulfur gas precursors detected
  9. 9. The Southern Ocean The National Oceanic and Atmospheric Administration (NOAA)  Extends 60° South circulating Antarctica  Accounts for 20% of the world ocean area  Comprises water masses from the Atlantic, Indian and Pacific Oceans  Its surface water temperature is of 5 °C to -1.86 °C southwards  Variable low iron contents (< 1 nM; low dust deposition)  Strong deep circulation of nutrients (Antarctic Circumpolar Current (ACC))  Drives the world ocean’s circulation  Regulates the Earth system  Its paleo-records supports the iron hypothesis  Thus, its phytoplankton assemblage is important
  10. 10. Haptophyta (Phaeocystis) After Schoemann et al., 2005 Flagellates (haploid motile), macroflagellates and/or attached aggregates (diploid nonmotile and colonial cells (diploid nonmotile) Colonies up to 2000 µm in diameter Blooms in the Arctic and Antarctic Oceans and North Sea Major dimethylsulfoniopropionate (DMSP) producers Zingone et al., 2011
  11. 11. Keeling et al., 2005 PhaeocystisintheTreeofLife
  12. 12. Iron Utilization in Phaeocystis antarctica Parameter Under iron limitation Under iron repletion Growth rate (day-1) 0.28 0.52 Cell size 16.9 µL/cell 2-fold increase Morphology Only solitary form Equal mixture of solitary and colonial forms Photopigments 0.5-fold decrease in 19’-hexanoyloxyfucoxanthin:Chl a 8-fold increase in fucoxanthin:Chl a C content (mol/L cell volume) ~15.3 (1.4-fold increase) 11.1 C:N 5-8 6 Fe content (µmol/L cell volume) 31 63.9 Fe uptake Uptake significantly increases with increasing limited conditions, and increases with increasing Fe. Irradiance Uptake rates increases in light conditions. Non-ligand specificity Extracellular reduction of Desferroxamine B-bound Fe(III) significantly greater under iron limitation with no effect of dissolved iron concentration. Strzepek et al., 2011; DiTullio et al., 2007; Schoemann et al., 2005
  13. 13. Problem Statement What are the genetic basis of P. antarctica adaptation to iron limitation and behavior under iron enrichment? Challenge 1: No published genome. Challenge 2: Previous iron limitation transcriptomic and proteomic profiling have been employed extensively only in diatoms. P. antarctica’s instant response to iron addition recommends it as an ideal model organism for studying the response in a time-series manner.
  14. 14. Study Objectives 1. Assessing the physiological, morphological and elemental changes of P. antarctica under iron-limited and iron-enriched condition 2. Reporting the preliminary de novo assembled and functionally characterized transcriptome of P. antarctica 3. Inferring the statistically and biologically differentially expressed genes and their expression patterns in P. antarctica in a time-dependent manner before and after iron supplementation
  15. 15. Culturing Fe supplementation Physiology • Photosynthetic fitness • Cell count • Chlorophyll a • Nutrients (C and N) • RNA extraction Time-series transcriptomics: Behavior under limitation and enrichment • Total reads assembly • Functional annotation • Structural analysis • Abundance estimate • Differential expression Study Design
  16. 16. -Fe +Fe Day 3 + FeCl3 Microscopy Photosynthesis pam/basic_version.html Chromatographic nutrients measurement Fluoremetric Chlorophyll a measurement Total RNA extraction Illumina RNA-sequencing (16 samples, Day 0 – Day 5) Materials and Methods (1/2)
  17. 17. Materials and Methods (2/2) De novo total reads assembly using Trinity Haas et al., 2013 Functional annotation Source: Differential gene expression using DESeq
  18. 18. P. antarctica Transcriptome Sequenced samples 16 samples (3-4 replicates per each time-point + T0) Total number of reads 389, 846, 414 No. of components (genes) 88,630 No. of isoforms (transcripts) 162,436 Contig N50 of transcripts (bases) 1,190 GC% 63.36%
  19. 19. P. antarctica Transcriptome 77.6% Largest gene family of 241 isoforms encode for zinc-dependent DNA polymerase and/or quinone oxidoreductase
  20. 20. Transcriptome Functional Annotation
  21. 21. Transcriptome Functional Annotation No. of ORFs 113,563 Unknown genes in UniProt 64,822 (73.1%) Known genes in Uniprot 2,923 (33%) Assigned to eggNOG 17,729 (20%; 2,932 groups) Assigned to GO 25,836 (29.2%) Assigned to Pfam 23,809 (26.9%)
  22. 22. Transcriptome Functional Annotation
  23. 23. Nuclear-Encoded Plastid-Targeted Genes
  24. 24. ** P. antarctica Photosynthetic Fitness *Insignificant (p > 0.05) **Significant (p < 0.05) *** ** ** **
  25. 25. P. antarctica Growth
  26. 26. P. antarctica Cell Size Day 0 (-Fe) Day 1 (+Fe) Day 3 (+Fe) Day 5 (+Fe) Day 8 (+Fe) Day 8 (+Fe)
  27. 27. P. antarctica Nutrients *Insignificant (p > 0.05) **Significant (p < 0.05) **
  28. 28. P. antarctica Growth and Physiology 1. Significant recovery of photosynthetic fitness one day following iron addition (from 0.36 to 0.51) 2. Significant increase in Chl a contents (2-fold) 3. Shift towards colonization following iron supplementation (from 4% to 12%) 4. Observed increase in cell size 5. Increase in growth rate (from 0.1 day-1 to 0.46 day-1) 6. Significant increase in N utilization resembling healthy exponentially growing cells (1.4-fold drop in C:N ratio)
  29. 29. Up-regulated at Day 2 (112 components) Up-regulated at Day 5 (48 components) - Fe-independent oxidative stress defense: Glutathione peroxidase, 2- oxoglutarate Fe(II)-dependent oxygenase, and iron-stress response protein - Signaling: Ca++-dependent - Apoptosis: Helicases, histone H4, and proteins involved in nucleic acid phosphodiester bond hydrolysis, nucleophagy - Reductive iron uptake: Ferric reduction oxidase - Oxidative stress: Vanadium-dependent bromoperoxidase - Aerobic respiration: Pyruvate carrier 3 and cytochrome c oxidase Day 2 vs. Day 5 Differential Expression
  30. 30. Up-regulated at Day 2 (112 components) Up-regulated at Day 5 (48 components) - Photosynthesis Fucoxanthin- like, phototropin-2 and flavodoxin - Structural C Reallocation: Chitinase-like and mucins - Nitrate assimilation: Biogenic amine biosynthesis (sperimidine) - Carbon-fixation: Fructose-1,6- bisphosphatase - Iron transport to mitochondria: mitoferrin-1 - Photosynthesis: Fucoxanthin and PSI regulation proteins, PSI reaction center subunit XI, and ferrodoxin - Carbon fixation: Carbonic anhydrase, and fructose- bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase and ribulose- phosphate 3-epimerase - Proteome remodeling: Collagen iron-binding prolyl 4- hydroxylase subunit alpha Day 2 vs. Day 5 Differential Expression
  31. 31. Differential Expression
  32. 32. P.antarcticaDifferentialGeneExpression
  33. 33. P. antarctica Differential Gene Expression 432 comp. 1087comp. 664 comp. 188 comp.
  34. 34. Conclusions  Phaeocystis antarctica is one of the most ecologically important species endemic to the Southern Ocean (DMSP).  Its growth and productivity is limited by iron availability, however, it is well-adapted (non-ligand specific reductase).  It has been reported the first to after iron enrichment.  Transcriptome of P. antarctica revealed that it constitutes of 88,630 putative genes (162,436 transcripts).  The vast majority of the genes are of unknown function (64,822; 73.1%), while 17,729 (20%; 2,932 unique groups) were assigned to eggNOG.  It comprises 2,456 nuclear-encoded plastid-targeted ORFs, the majority of which are of green algal origin.
  35. 35. Conclusions  P. antarctica significantly recovered its photosynthetic fitness, colony-forming ability, and chlorophyll a, particulate organic carbon and nitrogen contents shortly after iron addition.  Transcriptomic data suggests a shift to the more efficient photopigment fucoxanthin production and PSI ferredoxin after iron enrichment.  Transcriptomic data supports P. antarctica ability to utilize bound iron in a reductive non-ligand-dependent mechanism.  Physiology and transcription suggests N and C reallocations under iron limitation.  Calvin cycle enzymes were overexpressed under iron enrichment in addition to carbonic anhydrase.  Iron requirements of P. antarctica are the lowest among all phytoplankton.
  36. 36. Future Directions Transcriptomics  Revisit the assembly (~88,000 genes vs. ~30,000 in Emiliania huxleyi  Polymorphism and paralogs (e.g., Serine/threonine protein kinase transcripts family of 1,864 members)  Data availability  Genes constitutively expressed (e.g., fucoxanthin-chlorophyll a-c binding protein B and mitochondrial gene expression regulation protein TAR1)  Small RNAs role in iron limitation gene regulation  Validation using real-time quantitative reverse transcription-PCR (qRT- PCR) Comparison to other algal classes from other HNLC regions Proteomics and iron utilization model
  37. 37. Dr. Ahmed Moustafa, Director Dr. Sara Beszteri and AWI Biosciences Division Dr. Rania Siam, Chair Hazem and Mustafa Sarah and Rehab Yasmeen and Hadeel AUC Biotechnology Faculty and Colleagues Amged and Mr. Osama Universität Bremen Faculty and Colleagues AUC Graduate Students Support Office AUC Laboratory Instructions Fellowship and Al-Alfi Foundation Fellowship Acknowledgements
  38. 38. Thank you!