Gene Expression Profile during short-term heat stress in the Red Sea Coral Stylophora pistillata
Keren Maor-Landaw1#; Sarit Karako-Lampert1#; Hiba Waldman Ben-Asher1; Stefano Goffredo2;
Giuseppe Falini3; Zvy Dubinsky1; Oren Levy1*
1
The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan,
Israel.
2
Marine Science Group, Department of Biological, Geological and Environmental Sciences,
Alma Mater Studiorum–University of Bologna, Bologna, Italy.
3
Dipartimento di Chimica ‘G. Ciamician,’ Alma Mater Studiorum Universita` di Bologna,
Bologna, Italy.
# These authors contributed equally to this work.
SI Materials and methods
Coral sampling and experimental design
During May 2012 a colony of the scleractinian Stylophora pistillata was collected by SCUBA
diving at a depth of 10 m in the Gulf of Eilat (Red Sea) (latitude: 29.5, longitude: 34.9). The top
of the colony was split into 42 fragments approximately 5 cm in size. By fragmenting a single
colony, we established clonal “micro-colonies,” eliminating unwanted sources of biological
variability that are associated with corals derived from colonies of different sizes, shapes and
thermal/light life histories (Brown et al. 2002; Tambutté et al. , 1995)
The fragments were acclimated for three weeks in a 600 L aquarium with circulating artificial
seawater (Brightwell Aquatics, Pennsylvania, USA) under controlled conditions of 24°C and 35%
salinity at Bar-Ilan University, mimicking the average annual ambient temperature in Eilat. Light
periodicity was achieved using an Advanced Control Lighting System (ACLS, Sfiligoi, Italy) with
HQI (hydrargyrum quartz iodide) light bulbs (400 w, 14,000 Kelvin) configured to simulate a
year-long diurnal-dimming light regime. The fragments were fed once per day with a microvore
microdiet (Brightwell Aquatics). Following the one-month acclimation period, the fragments
were placed into two 300 L aquariums prior to the experiment; the control aquarium was
1
maintained at 24°C with continuous water flow using a computer-controlled closed circulation
system, which can compensate for salinity fluctuations and water level changes (constant
salinity level of 35‰). The experimental aquarium was subjected to an increase of 1°C per day
from 24°C to 34°C. Three fragments were sampled at the same time of day from both the
control and heat treatment aquariums at time points corresponding to 28°C, 32°C and 34°C,
i.e., days 5, 10 and 13, respectively (sampling was conducted 24h after temperature elevation)
(see figure S1). The sampled fragments were snap-frozen in liquid nitrogen and kept at -80°C
until the RNA extractions.
Figure S1 – Graphical illustration of the experimental design.
PAM Fluorometry
An imaging pulse amplitude modulation (IPAM) fluorometer (Heinz Waltz GmbH, Germany) was
used to evaluate the photosynthetic efficiency of photosystem II of the algal symbionts. The
fluorescence of three S. pistillata fragments was measured following 30 min of dark acclimation
at each sampling point; 28°C, 32°C and 34°C, corresponding to days 0, 5, 10 and 13,
respectively. The effective quantum yield (Fv/Fm) was calculated for each sample by
determining the dark-level fluorescence yield (F0) and the maximum fluorescence yield (Fm)
when all PSII reaction centers were photochemically reduced (Fv/Fm=((Fm-F0)/Fm). The
effective quantum yield helped in monitoring the photosynthetic performance during the
2
experiment, which is putative indicator of thermal stress (Ainsworth et al. 2008; Fitt et al.
2001).
RNA extraction and microarray hybridization
Total RNA was extracted from each of the fragments using TRIzol (Invitrogen), according to the
methods of (Levy et al., 2007, 2011), and the samples were further purified using an RNA Clean
and Concentrator kit (Zymo Research). The RNA concentrations were measured using a
NanoDrop spectrophotometer (ND-1000), and the sample quality was checked using a
Bioanalyzer (Agilent). A 200 ng sample of total RNA was labeled and hybridized against the
microarray. Microarray experiments were performed using a custom Agilent two-color gene
expression microarray platform with 8 × 15 K probes per slide. Oligonucleotide probes (60mers) were designed based on approximately 12,000 genes predicted to encode proteins
retrieved from a recent de novo assembly of 454-sequenced EST libraries of S. pistillata
(Lampert-Karako S In press). Labeling and hybridization were conducted using an Agilent LowInput Quick Amp Labeling Kit according to the manufacturer’s instructions. The intensity of the
emitted fluorescence from each target spot on the array was detected using an Agilent
G2565BA microarray scanner. The raw data as well as the processed data of the microarray
were deposited under accession number GSE47779 [NCBI GEO]. Stylophora pistillata EST data is
also
stored
at
the
Cnidarian
Database
of
Centre
Scientifique
de
Monaco:
http://data.centrescientifique.mc/CSMdata-home.html
Microarray data analysis
The data from all arrays were first subjected to background correction and LOESS within-array
normalization using Agilent Feature Extraction software (version 9.5.1.1 Agilent Technologies,
Santa Clara, CA). The remaining analyses were performed in Partek® Genomics Suite software
(version 6.6, Copyright ©2012, Partek Inc., St. Louis, MO, USA). The log expression ratios
produced during the normalization step were analyzed. Data from three biological replicates
and two to four technical replicates were used to perform a one-way ANOVA. The normalized
data were analyzed to identify genes with significantly up- or down-regulated expression (FDR
(false discovery rate) p<0.05) with an arbitrary cutoff of at least a two-fold change.
3
We generated networks of highly interconnected proteins using the STRING (Search Tool for
the Retrieval of Interacting Genes, Heidelberg, Germany) 9.0 database [83]. The clustering of
protein interactions with high confidence scores (at least 0.7) were examined and subsequently
exported to Cytoscape software 2.8 [84] for graphical editing. We focused on main stressrelevant clustering protein groups that consist of multi-interactions connections, as provided by
the STRING database. The Gene Ontology (GO) cellular component and biological processes
associated with the up- and down-regulated proteins were retrieved from the STRING database
and compared between the controls and heat stress treatments.
4
Figure S2 - Principle component analysis (PCA). All the samples and replicates are plotted in a
three-dimensional space using three principal components (PC) capturing 65.3% of total
variability. This image was constructed using Partek Genomics SuiteTM software.
5
Figure S3 – Venn diagram of all differentially expressed genes. The Venn diagram presents
differentially expressed genes in the 28°C (t1 vs. c1), 32°C (t2vs. c2) and 34°C (t3 vs. c3) trials.
The number of genes is indicated within each field.
6
Table S1 – A list of 163 differentially expressed genes - “Common genes” - either up- or downregulated, at all three sampled temperatures. -1 and 1 indicate significant (FDR p<0.05) downand up-regulation, respectively.
Probeset ID
Probe ID
swissprot T1 vs. C1 T2 vs. C2 T3 vs. C3
isotig14260
CUST_2825_PI426075177
-1
-1
-1
isotig13853
CUST_5489_PI426075177
-1
-1
-1
isotig13537
CUST_3587_PI426075177
-1
-1
-1
isotig09554
CUST_4579_PI426075177
-1
-1
-1
isotig06129
?
-1
-1
-1
isotig04307
CUST_2656_PI426075177
-1
-1
-1
isotig03953
CUST_1973_PI426075177
-1
-1
-1
isotig03525
CUST_2626_PI426075177
-1
-1
-1
isotig01880
CUST_373_PI426075177
-1
-1
-1
fgenesh1_pg.scaffold_5441000001 CUST_3096_PI426075177
-1
-1
-1
fgenesh1_pg.scaffold_28000065
CUST_3334_PI426075177
-1
-1
-1
e_gw.222.19.1
CUST_3573_PI426075177
-1
-1
-1
e_gw.196.95.1
CUST_5381_PI426075177
-1
-1
-1
YRHG
CUST_4205_PI426075174
O05399
-1
-1
-1
Y1586
CUST_3230_PI426075174
P44263
-1
-1
-1
VPE1
CUST_4547_PI426075174
O24325
-1
-1
-1
SAHH_NOVAD
CUST_7349_PI426075174
Q2G6T1
-1
-1
-1
S22AF_MOUSE
CUST_5657_PI426075174
Q504N2
-1
-1
-1
PSBB
CUST_3619_PI426075174
P48103
-1
-1
-1
PSAE1
CUST_3981_PI426075174
Q9S831
-1
-1
-1
PGK
CUST_3707_PI426075174
P41759
-1
-1
-1
7
PDC2
CUST_3468_PI426075174
Q92345
-1
-1
-1
PCNA_ORYSJ
CUST_5497_PI426075174
P17070
-1
-1
-1
NRGA
CUST_317_PI426075174
Q07429
-1
-1
-1
NIA_VOLCA
CUST_8029_PI426075174
P36841
-1
-1
-1
MDGA1_HUMAN
CUST_7753_PI426075174
Q8NFP4
-1
-1
-1
LECB6
CUST_4867_PI426075174
B4XT05
-1
-1
-1
LCFA
CUST_3276_PI426075174
Q8ZES9
-1
-1
-1
HSP90
CUST_4086_PI426075174
O44001
-1
-1
-1
HERC2_MOUSE
CUST_8229_PI426075174
Q4U2R1
-1
-1
-1
HEBP2_MOUSE
CUST_8013_PI426075174
Q9WU63
-1
-1
-1
GUX1_PENJA
CUST_7345_PI426075174
Q06886
-1
-1
-1
GLC7A_CAEEL
CUST_6561_PI426075174
Q27497
-1
-1
-1
GCKDGN103HAFS0
CUST_1018_PI426075177
-1
-1
-1
GCKDGN103HACEY
CUST_1976_PI426075177
-1
-1
-1
GCKDGN103GIGTQ
CUST_4116_PI426075177
-1
-1
-1
GCKDGN103G9F3G
?
-1
-1
-1
GCKDGN103FYNNC
CUST_4862_PI426075177
-1
-1
-1
GCKDGN103FWKUB
CUST_896_PI426075177
-1
-1
-1
GCKDGN103F8AIU
CUST_1538_PI426075177
-1
-1
-1
GCKDGN102EPB8P
CUST_1040_PI426075177
-1
-1
-1
GCKDGN102EDPPV
CUST_3778_PI426075177
-1
-1
-1
GCKDGN102DY0KW
CUST_4871_PI426075177
-1
-1
-1
GCKDGN102C9ZYX
CUST_5060_PI426075177
-1
-1
-1
GCKDGN102C98VI
CUST_3473_PI426075177
-1
-1
-1
8
GCKDGN102C7I85
CUST_5025_PI426075177
-1
-1
-1
GCKDGN101CE342
CUST_4743_PI426075177
-1
-1
-1
GCKDGN101BW9JV
CUST_1809_PI426075177
-1
-1
-1
GCKDGN101BQ2FA
CUST_4059_PI426075177
-1
-1
-1
GCKDGN101BDSUE
CUST_4237_PI426075177
-1
-1
-1
GCKDGN101B1ZQV
?
-1
-1
-1
GCKDGN101A7HI4
?
-1
-1
-1
GCKDGN101A6MMV
CUST_3376_PI426075177
-1
-1
-1
G3PG
CUST_3632_PI426075174
P22513
-1
-1
-1
FER_PERBI
CUST_8298_PI426075174
P10770
-1
-1
-1
FENR
CUST_3062_PI426075174
Q55318
-1
-1
-1
FCPA
CUST_4297_PI426075174
Q40297
-1
-1
-1
FCL_MOUSE
CUST_5951_PI426075174
P23591
-1
-1
-1
ENOG
CUST_1865_PI426075174
P17183
-1
-1
-1
ENO2
CUST_3360_PI426075174
Q9BPL7
-1
-1
-1
EF2_RAT
CUST_5325_PI426075174
P05197
-1
-1
-1
DHSA_RICCN
CUST_6289_PI426075174
Q92J97
-1
-1
-1
DHAS
CUST_4255_PI426075174
O67716
-1
-1
-1
Contig_Sym_501
CUST_8942_PI426075174
-1
-1
-1
Contig_Sym_216
CUST_8923_PI426075174
-1
-1
-1
Contig_Sym_1455
CUST_8946_PI426075174
-1
-1
-1
CYF
CUST_2893_PI426075174
A0T0C9
-1
-1
-1
CLPAB
CUST_4442_PI426075174
P31542
-1
-1
-1
CCPR
CUST_4337_PI426075174
Q4PBY6
-1
-1
-1
9
CALM_PROMN
CUST_5709_PI426075174
A3E4D8
-1
-1
-1
BIP2
CUST_3284_PI426075174
P24067
-1
-1
-1
ARF_CANAL
CUST_5157_PI426075174
P22274
-1
-1
-1
AQP9
CUST_2693_PI426075174
Q9JJJ3
-1
-1
-1
AQP7
CUST_3592_PI426075174
O14520
-1
-1
-1
AQP3_RAT
CUST_5070_PI426075174
P47862
-1
-1
-1
ALF_DICDI
CUST_7791_PI426075174
Q86A67
-1
-1
-1
ALF_CAMJJ
CUST_7792_PI426075174
A1VYV7
-1
-1
-1
795_symbiodinum
?
-1
-1
-1
493_symbiodinum
CUST_8904_PI426075174
-1
-1
-1
422_symbiodinum
?
-1
-1
-1
301_symbiodinum
CUST_8869_PI426075174
-1
-1
-1
283_symbiodinum
CUST_8972_PI426075174
-1
-1
-1
210_symbiodinum
CUST_8919_PI426075174
-1
-1
-1
20_symbiodinum
?
-1
-1
-1
158_symbiodinum
?
-1
-1
-1
1125_symbiodinum
?
-1
-1
-1
108_symbiodinum
CUST_8968_PI426075174
-1
-1
-1
isotig10182
CUST_2700_PI426075177
1
-1
-1
fgenesh1_pg.scaffold_87000045
CUST_142_PI426075177
1
-1
-1
fgenesh1_pg.scaffold_582000006
CUST_11_PI426075177
1
-1
-1
GBRB2_MOUSE
CUST_8675_PI426075174
P63137
1
-1
-1
WNT7A
CUST_4991_PI426075174
P24383
1
-1
-1
AEQ2
CUST_4904_PI426075174
P02592
1
-1
-1
10
P68173
PDIA3_BOVIN
CUST_5646_PI426075174
786_symbiodinum
1
-1
-1
CUST_8858_PI426075174
-1
-1
1
isotig09988
CUST_2220_PI426075177
-1
1
1
DCP_20_7
?
1
-1
1
DCP_20_5
?
1
-1
1
DCP_20_3
?
1
-1
1
DCP_20_1
?
1
-1
1
DCP_20_0
?
1
-1
1
DCP_1_7
?
1
-1
1
DCP_1_2
?
1
-1
1
DCP_1_1
?
1
-1
1
DCP_1_0
?
1
-1
1
DCP_22_9
?
1
-1
1
DCP_22_7
?
1
-1
1
DCP_22_6
?
1
-1
1
DCP_22_4
?
1
-1
1
DCP_22_2
?
1
-1
1
DCP_22_0
?
1
-1
1
E1A_r60_n9
?
1
-1
1
E1A_r60_n11
?
1
-1
1
E1A_r60_a97
?
1
-1
1
E1A_r60_a22
?
1
-1
1
E1A_r60_a20
?
1
-1
1
E1A_r60_a135
?
1
-1
1
11
P38657
E1A_r60_a107
?
1
-1
1
E1A_r60_a104
?
1
-1
1
E1A_r60_3
?
1
-1
1
E1A_r60_1
?
1
-1
1
isotig15225
CUST_2639_PI426075177
1
1
1
PERP1
CUST_620_PI426075174
Q9D8I1
1
1
1
PDIA6_HUMAN
CUST_5529_PI426075174
Q15084
1
1
1
PDIA4_HUMAN
CUST_5860_PI426075174
P13667
1
1
1
CALR_CAEEL
CUST_8429_PI426075174
P27798
1
1
1
CALR_MOUSE
CUST_8428_PI426075174
P14211
1
1
1
824_symbiodinum
CUST_8928_PI426075174
1
1
1
CAT2_CLOK5
CUST_8523_PI426075174
P38942
1
1
1
PCKGM
CUST_3722_PI426075174
Q8BH04
1
1
1
PA2G4
CUST_1301_PI426075174
P50580
1
1
1
CALR_ONCVO
CUST_8427_PI426075174
P11012
1
1
1
ECSIT
CUST_1601_PI426075174
Q0V9C9
1
1
1
fgenesh1_pg.scaffold_735000001
CUST_3262_PI426075177
1
1
1
NAT13_DANRE
CUST_6840_PI426075174
Q6DBY2
1
1
1
202_symbiodinum
CUST_8833_PI426075174
Q5ZI13
1
1
1
HSP7C_CAEBR
CUST_8196_PI426075174
P19208
1
1
1
fgenesh1_pg.scaffold_161000010
CUST_5394_PI426075177
1
1
1
estExt_gwp.C_790178
CUST_4351_PI426075177
1
1
1
estExt_gwp.C_3070053
CUST_2958_PI426075177
1
1
1
estExt_fgenesh1_pg.C_180075
CUST_5181_PI426075177
1
1
1
12
estExt_GenewiseH_1.C_260219
CUST_4737_PI426075177
1
1
1
FGOP2
CUST_683_PI426075174
Q5ZKJ4
1
1
1
ARMET
CUST_831_PI426075174
B4NIN8
1
1
1
215_symbiodinum
CUST_8941_PI426075174
Q7PT10
1
1
1
HS90B_DANRE
CUST_7450_PI426075174
O57521
1
1
1
CREL2_MOUSE
CUST_5811_PI426075174
Q9CYA0
1
1
1
UD2B7
CUST_2282_PI426075174
P16662
1
1
1
SeqIndex65001
CUST_463_PI426075177
1
1
1
SUFS
CUST_4965_PI426075174
1
1
1
GCKDGN103HFVMV
CUST_1396_PI426075177
1
1
1
ERP29
CUST_4877_PI426075174
P81628
1
1
1
RS15_RAT
CUST_8565_PI426075174_rep P62845
1
1
1
RM19
CUST_1951_PI426075174
P49406
1
1
1
RDX
CUST_1630_PI426075174
Q9VFP2
1
1
1
QRFPR
CUST_1214_PI426075174
Q96P65
1
1
1
QCR9
CUST_4914_PI426075174
Q8R1I1
1
1
1
ENPL_PIG
CUST_5334_PI426075174
Q29092
1
1
1
PSB1B
CUST_2524_PI426075174
Q9IB83
1
1
1
ENPL_CHICK
CUST_5333_PI426075174
P08110
1
1
1
PRAF3
CUST_3610_PI426075174
Q5F433
1
1
1
PPIB_CHICK
CUST_6478_PI426075174
P24367
1
1
1
ENPL_CANFA
CUST_5336_PI426075174
P41148
1
1
1
13
A7MF59
Microarray validation using quantitative real-time PCR
Materials and methods
To validate the microarray results, quantitative real-time polymerase chain reaction (qRT-qPCR)
assays were performed for four selected genes, dnajc3 (co-chaperone involved in UPR during
ER stress), pdia6 (protein disulfide-isomerase A6, a chaperone that aids in the aggregation of
misfolded proteins), Collagen alpha and Carbonic anhydrase 6,in the 28°C and 34°C treatment
and control samples. Complementary DNAs were synthesized from total RNA using the
RevertAid First Strand cDNA Synthesis kit (Thermo), according to the manufacturer’s
instructions. Specific qRT-PCR primers were designed to amplify 100-200 bp PCR products.
cDNA aliquots were diluted 1:10 and used in triplicate for 10 μL qRT-PCRs with primers with
GoTaq qPCR Master Mix (Promega) for 50 cycles. The comparative delta CT method was
applied, and fold changes were calculated using the 2-ΔΔCt formula to estimate the relative
amounts of transcripts in each sample. Each dCT was normalized to the best-performing
housekeeping gene: 60S Ribosomal protein L22.
Results
Two relevant up-regulated genes were selected from the microarray data for qRT-PCR
validation: dnajc3, which was up-regulated only at 34°C; and pdia6, which exhibited increases in
expression (compared to the control samples) at both 28°C and 34°C. Additionally two relevant
down-regulated genes were selected: Collagen alpha, which was down-regulated only at 34°C;
and Carbonic anhydrase 6, which was up-regulated at 28°C and down-regulated at 34°C. The
fold changes calculated based on qRT-PCR were in the same direction as and consistent with
those in the microarray data, thus confirming the microarray results (Figure S3).
14
Figure S4 – The relative expression of selected genes assessed via quantitative real-time PCR
(ddCt). The expression levels of A) dnajc3 (DnaJ homolog subfamily C member 3), B) pdia6
(protein disulfide-isomerase A6) C) Collagen alpha and D) Carbonic anhydrase 6, in treatments 1
(28°C) and 3 (34°C) (indicted in black) and their corresponding controls (indicted in gray) are
shown. Each dCt was normalized to a housekeeping gene: 60S Ribosomal protein L22. Error
bars indicate the standard error.
15
Figure S5 – Fold change values of selected up-regulated (A) and down-regulated (B) genes.
The fold changes of genes for ER stress and protein folding in the ER and proteasomal ubiquitinmediated proteolysis (A), and genes for extracellular matrix organization, Wnt signaling, Notch
signaling and actin cytoskeleton organization (B) are shown with respect to temperature
treatments 1, 2 and 3. The color-scale legend indicating the relative fold change is shown on the
right. This figure was constructed using EXPANDER software.
16
Figure S6 – Venn diagram of total differentially expressed genes in preliminary 32°C and current
32°C and 34°C trials. The number of genes is indicated within each field.
17
Table S2 – Down-regulated genes from the skeletal organic matrix (SOM) predicted proteins
identified by Drake et al. 2013 proteomic analysis, in the 28°C (t1 vs. c1), 32°C (t2vs. c2) and
34°C (t3 vs. c3) trials. ↓ and ↑ indicate significant (FDR p<0.05) down-regulated and upregulated genes, respectively.
Name
Thrombospondin-1
Actin
Cadherin
sushi domain containing
Collagen - alpha
MAM domain containing protein
zona pellucida-like domain-containing
Vitellogenin
Myosin regulatory light polypeptide
Neurexin
Flagellar associated protein
Carbonic anhydrase
t1 vs c1
↑
↓
t2 vs c2
↓
↓
↓
↓
↓
↓
↑
18
t3 vs c3
↓
↓
↓
↓
↓
↓
↓
↓
↓
↓
↓
↓