T RANSCRIPTOMICS AND P ROTEOMICS
OF A S ECONDARY G REEN A LGA
1,2,3
P EREZ
,
2
L APAILLE ,
4
D EGAND ,
2
C ILIBRASI ,
Emilie
Marie
Hervé
Laura
1
4
5
2,3
Maxime G ILSOUL , Pierre M ORSOMME , Mark C. F IELD , Claire R EMACLE ,
Pierre CARDOL2,3, Denis B AURAIN1,3
1
Eukaryotic Phylogenomics, University of Liège, Belgium
2
Genetics and Physiology of Microalgae, University of Liège, Belgium
3
PhytoSYSTEMS, University of Liège, Belgium
4
SST/ISV, Institut des Sciences de la Vie, Université Catholique de Louvain, Belgium
5
Division of Biological Chemistry and Drug Discovery, University of Dundee, Scotland, UK
Euglena gracilis is a secondary green alga related to Trypanosomatidae. It derives from a secondary endosymbiosis between
a phagotrophic ancestor and a prasinophycean green alga.
Our general objective is to study the interactions established between the chloroplast and the mitochondrion following the endosymbiotic event and to determine the phylogenetic origins of
the genes encoding the proteins involved in these interactions.
We sequenced the complete transcriptome of E. gracilis, assembled the transcripts and predicted the corresponding proteins.
We analysed the mitochrondrial respiratory chain composition
of E. gracilis and assessed its similitude with the chain described
in Trypanosomatidae. Finally, we performed a high-throughput
analysis of the mitochondrial proteome of E. gracilis.
Taxonomic affiliation of
Euglena gracilis transcripts
11361
14897
29256
28649
100
80
demonstrate that the secondary green alga
shares many additional subunits with Trypanosomatidae. In addition, our transcriptome allows us to
identify 30% more proteins than public ESTs (missing
proteins were identified using genomic reads).
W
intermembrane space
respiration
& ATP biosynthesis
36
matrix
amino acid
metabolism
100
90
80
pyruvate
metabolism
Krebs &
glyoxylate
cycles
7
cellular
protection
mechanisms
14
70
9
fatty acid
& lipid
metabolisms
indeterminate
pathways
60
18
translation &
transcription
12
50
8
transport
10
40
outer membrane
inner membrane
30
identify a total of 131 mitochondrial proteins involved in about 10 different metabolic pathways,
including energy producing pathways.
W
20
60
E
40
1
1e-03
1e-20
1e-50
1e-100
0
Relative abundance of mitochondrial
proteins across different conditions
E-value
20
1e-100: E-value <= 1e-100, 1e-50: 1e-100 <= E-value <= 1e50, 1e-20: 1e-50 < E-value <= 1e-20, 1e-03: 1e-20 < E-value <=
1e-03, and 1: 1e-03 < E-value <= 1.
SOO
SKO
olor7Key
and7Histogram
XKO
j
Plantae
SAR
CCTH
Excavata
B
K
x
Value
g
v
I
BBSXj||respiration||epsilon
jvxxS||)TP_biosynthesis||)TPTwB
BKxIx||)TP_biosynthesis||)TPTwXS
xXISS||amino_acid_metabolism||sulfate_adenylyltransferase
Bxxvj||nFiF||hypothetical_protein
xBOgI||nFiF||hypothetical_protein
KxggI||)TP_biosynthesis||OS P
xKOjO||nFiF||co)_binding_protein
xSSBv||)TP_biosynthesis||)TPTwX
KIgjv||)TP_biosynthesis||beta
xjSjj||)TP_biosynthesis||alpha
KxKIg||respiration||pXv
xSSOj||amino_acid_metabolism||glycine_dehydrogenase
KxIvI||transport||carrier_protein
KSIvO||)TP_biosynthesis||gamma
BvBjg||)TP_biosynthesis||)TPTwx
KIxgv||peptide_synthesis||peptide_synthetase
KKgXK||respiration||NDUFSS
KBgOS||respiration||NDUF)I
KjvgK||respiration|| )G
BKvSK||carotenoid_metabolism||geranylgeranyl_pyrophosphate_synthase
xSvOO||transport||transporter
BvvBK||nFiF||unknown_protein
xXSXK||fatty_acid_metabolism||sterol_XB−demethylase
xBxIS||stress_response||HSPgO
KIKII||amino_acid_metabolism||ketol−acid_reductoisomerase
xSKOj||amino_acid_metabolism||ornithine_aminotransferase
xXIvK||nFiF||clavaminate_synthase
BKIIS||amino_acid_metabolism||glycine_decarboxylase
xBxXK||amino_acid_metabolism||acetolactate_synthase
xjxxB||fatty_acid_metabolism||hydroxymethylglutaryl− o)_synthase
KxjSB||amino_acid_metabolism||S−isopropylmalate_synthase
KXjjv||post−translation_process||glycosyltransferase
xjxSg||amino_acid_metabolism||isopropylmalate_dehydratase
xjOgK||translation||factor_EfG
KKgjS||)TP_biosynthesis||delta
xBSSI||malate_metabolism||malic_enzyme
xjxvK||fatty_acid_metabolism||propionyl− o)_carboxylase
xBOBg||glyoxylate_cycle||malate_synthase−isocitrate_lyase
xBSjO||Krebs_cycle||aconitate_hydratase_S
KvBOg||stress_response||superoxide_dismutase
xSSvj||fatty_acid_metabolism||acyl− o)_dehydrogenase
KIIgS||malate_metabolism||malic_enzyme
xSxIX||fatty_acid_metabolism||acetyl− o)_acetyltransferase
xBKBI||fatty_acid_metabolism||propionyl− o)_carboxylase
KxSIX||Krebs_cycle||malate_dehydrogenase
xOBgg||Krebs_cycle||isocitrate_dehydrogenase
KxjgB||Krebs_cycle||succinyl− o)_synthetase
BvvjI||stress_response||peroxiredoxin
xSjvB||Krebs_cycle||citrate_synthase
KvgSX||fatty_acid_metabolism||Propionyl−co)_carboxylase
KxgKK||stress_response||peptidylprolyl_isomerase
Kggjg||nucleotide_metabolism||adenylate_kinase
KjIxx||amino_acid_metabolism||succinate−semialdehyde_dehydrogenase
KISgx||amino_acid_metabolism||succinate−semialdehyde_dehdyrogenase
xBgxx||fatty_acid_metabolism||acyl− o)_dehydrogenase
KxOxK||fatty_acid_metabolism||L−j−hydroxyacyl− o)_dehydrogenase
xBOOI||amino_acid_metabolism||glutamine_synthetase_III
KSBBK||transport||carnitine_carrier
KxvOx||fatty_acid_metabolism||Hydroxyacyl−co)_dehydrogenase
KXOBj||fatty_acid_metabolism||acetyl− o)_acetyltransferase
xXXvj||transport||porin
xXxgv||transport||N)D5PR_transhydrogenase
KSKvj||transcription||regulator_LysR
xSjgx||fatty_acid_metabolism||acetyl− o)_acetyltransferase
KISXB||respiration||ETFw
Kvvxx||fatty_acid_metabolism||enoyl− o)_hydratase
KOXSO||RN)_silencing||RN)_dependent_RN)_polymerase
KjKBj||alcohol_metabolism||dehydrogenaseVreductase_SDR
BvXgx||respiration||Q Rx
xjXSK||fatty_acid_metabolism||trans−S−enoyl− o)_reductase
KgKOx||nucleotide_metabolism||thymidylate_kinase
BIjgj||respiration|| YTX
KKXOS||stress_response||peroxiredoxin
KBjxB||nFiF||uncharacterized_protein
KgIIx||respiration||RIPX
xXIjI||respiration||SDHX
KXIXg||transcription||pSS
xjxjX||pyruvate_metabolism||pyruvate_decarboxylase
xjSgx||respiration||NDTwXS
xBjKS||alcohol_metabolism||alcohol_dehydrogenase
KIvOB||Krebs_cycle||fumarate_hydratase
xBIvS||nFiF||hypothetical_protein
KIvBS||respiration|| OXK
xSOgK||Pentose−P_metabolism||x−phosphogluconate_dehydrogenase
BgXOS||signal||histidine_kinase
xOvKB||respiration||Q RS
BggXx||fatty_acid_metabolism||carnitine_O−acetyltransferase
KgxxO||)TP_biosynthesis||)FGj
xKOxK||pyruvate_metabolism||pyruvateQ_N)DPu_oxidoreductase
KKIKS||respiration||SDHS
xXIgK||respiration||NDUF)x
KXKXg||respiration||Q Rx
xjjXj||pyruvate_metabolism||pyruvate_dehydrogenase_EX
KIxBX||stress_response||DnaK
xjxBx||respiration||Q RX
xOOgj||pyruvate_metabolism||pyruvate_dehydrogenase_Ej
KxxSg||amino_acid_metabolism||betaine−aldehyde_dehydrogenase
xSvIO||alcohol_metabolism||)LDH
xSggx||pyruvate_metabolism||pyruvate_dehydrogenase_ES
BIOjB||respiration||SDHT j
xjjKO||stress_response||HSPxO
BIgjj||stress_response||HSPXO
BIgXx||respiration||NDUF)K
xSOXjFX||transport||)DPV)TP_translocase
KOgKv||transport||facilitator
KvBBj||respiration||SDHT x
KgxXg||transcription||regulator_IclR
xKOOK||pyruvate_metabolism||pyruvate_carboxylase
xSBXx||fatty_acid_metabolismVgluconeogenesis||pyruvate_carboxylase
xBvOI||Krebs_cycle||L−S−hydroxyglutarate_dehydrogenase
KgBSg||nFiF||dynamin
KBjOX||nFiF||hypothetical_protein
xSXSS||respiration|| )G
KjIvx|| ell−junction||Tw X
xXBxI||respiration||NDUFSX
KBSIB||transport||carrier_protein
KKjxj||transcription||factor_Tu
KIBOx||respiration||NDUF)Xj
KxKvj||nFiF||unnamed_protein
xXBIg||microtubule||)lpha−tubulin
KKSBS||translation||RPLXS
KBgjg||respiration|| OXj
xSIXS||respiration||NDUFVX
KgxKB||fatty_acid_metabolism||trans−S−enoyl− o)_reductase
xOIBK||stress_response||DnaJ
KjgBx||translation||RPLBj
xSOXjFS||transport||)DPV)TP_translocase
BvOvI||respiration|| )G
xOjgO||transport||carrier_protein
xjvBO||fatty_acid_metabolism||fatty−acyl− o)_synthase
44
38
15
1e-03
1
57
1e-20
94
1e-50
Taxonomic affiliation of
protein fragments
1e-100
Unikonta
Bacteria
Archaea
unclassified
O
E-value
ount
T
XOO
subcellular localization of the protein fragments is
mainly the mitochondrion. It indicates that our mitochondrial extracts are relatively pure.
HE
KO
1
1e-03
1e-20
1e-50
0
100
proportion of Euglenozoan origins increases with
E-value significance. However, we observe a large
fraction of hits corresponding to other phyla over the
whole E-value spectrum, which reflects the diverse
phylogenetic sources of E. gracilis genes.
HE
Mitochondrial respiratory chain
subunit composition
% identified protein fragments
1e-50: E-value <= 1e-50, 1e-20: 1e-50 < E-value <= 1e-20,
1e-03: 1e-20 < E-value <= 1e-03, and 1: 1e-03 < E-value <= 1.
T
other
pathways
5
12
cytoplasm
mitochondrion
n.i.
others
10
% transcripts
Coverage of the mitochondrial
metabolic pathways
E
Subcellular localization of
protein fragments
# identified protein fragments
Abstract
80
60
40
20
0
Plantae
SAR
CCTH
1e-100: E-value <= 1e-100, 1e-50: 1e-100 <= E-value <= 1e50, 1e-20: 1e-50 < E-value <= 1e-20, 1e-03: 1e-20 < E-value <=
1e-03, and 1: 1e-03 < E-value <= 1.
#,$: Not identified in bioinformatic analyses. Underlined: Found
in proteomic analyses. Bold and italics: Specific to Euglenozoa.
*: Found in diplonemids (D. papillatum). $: Not homologous
to the canonical QCR9. Red: Found in E. gracilis complete
transcriptome.
results are inconclusive, probably due to the reduced set of proteins examined (248 mitochondrial
proteins vs. 84,163 transcripts). However, the diverse
origins of E. gracilis mitochondrial proteins is obvious.
T
HE
Protist 2014, Banff Centre, Banff, Alberta, Canada, August 3rd - 8th, 2014
)DFX
)DFS
)LFS
)LFX
EDFS
EDFX
)HFX
Excavata
Unikonta
Bacteria
Archaea
)HFS
E-value
AD: Acetate 60mM Dark, AL: Acetate 60mM Low Light, AH: Acetate 60mM High Light, and ED: Ethanol 1% Dark.
patterns can be observed. Components of
Krebs and glyoxylate cycles are more abundant in
AH and ED conditions, components of fatty acid/lipid
metabolisms in ED and AL conditions, and components of the amino acid metabolism in ED, AD and AH
conditions. Finally, components of the respiration/ATP
synthesis are more abundant in AD condition, while
one half (ATP synthesis) is more abundant in ED condition and the other half (respiration) in AL condition.
S
OME
PhD student funded by