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 :)

1. Transcriptomics of Iron Limitation in
Phaeocystis antarctica
Mariam Reyad Rizkallah
Supervisors: Assist. Prof. Ahmed Moustafa
Dr. Sára Beszteri

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. Transcriptomics of Iron Limitation in
Phaeocystis antarctica
Mariam Reyad Rizkallah
Supervisors: Assist. Prof. Ahmed Moustafa
Dr. Sára Beszteri

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. Alarming Climate Changes…
Source:
http://www.telegraph.co.uk/earth/environment/climatechange/10733764/IPCC-report-No-one-will-be-untouched-by-climate-change.html
http://www.reuters.com/article/2014/05/26/climatechange-idUSL6N0OC24Z20140526

6. … Ocean Contributes to CO2 Export
Source: Average global chlorophyll a concentration in 2010 - NASA’s satellite sensor, SeaWiFS image

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):
http://news.nationalgeographic.com/news/2010/08/081013-
ocean-color-hurricanes-environment-weather-science-global-
warming/
Antarctic phytoplankton samples:
http://tawaclassgilberthorpeschool.wikispaces.com/Antarctica

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. 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. 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. Keeling et al., 2005
PhaeocystisintheTreeofLife

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. 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. 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. 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. -Fe +Fe
Day 3
+ FeCl3
Microscopy
Photosynthesis
http://www.walz.com/products/chl_p700/xe-
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. Materials and Methods (2/2)
De novo total reads assembly
using Trinity
Haas et al., 2013
Functional annotation
Source: http://trinotate.sourceforge.net/
Differential gene expression
using DESeq

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. P. antarctica Transcriptome
77.6%
Largest gene family of 241 isoforms encode for
zinc-dependent DNA polymerase
and/or quinone oxidoreductase

20. Transcriptome Functional Annotation

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. Transcriptome Functional Annotation

23. Nuclear-Encoded Plastid-Targeted Genes

24. **
P. antarctica Photosynthetic Fitness
*Insignificant (p > 0.05)
**Significant (p < 0.05)
***
**
**
**

25. P. antarctica Growth

26. P. antarctica Cell Size
Day 0 (-Fe) Day 1 (+Fe) Day 3 (+Fe)
Day 5 (+Fe) Day 8 (+Fe) Day 8 (+Fe)

27. P. antarctica Nutrients
*Insignificant (p > 0.05)
**Significant (p < 0.05)
**

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. 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. 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. Differential Expression

32. P.antarcticaDifferentialGeneExpression

33. P. antarctica Differential Gene Expression
432 comp.
1087comp. 664 comp.
188 comp.

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. 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. 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. 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. Thank you!