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Laboratory of Plant Molecular Biology, Rockefeller University, New York, NY 10065, USA; 2Institut de Biologie de lEcole Normale Superieure (IBENS), Centre National de la Recherche
Scientifique (CNRS) UMR 8197 INSERM U1024, 46 Rue dUlm, 75005 Paris, France
Summary
Author for correspondence:
Nam-Hai Chua
Tel: +1 212 327 8126
Email: chua@rockefeller.edu
Received: 29 April 2015
Accepted: 27 October 2015
Phosphorus (P) is an essential element to all living cells, yet fluctuations in P concentrations
are recurrent in the marine environment. Diatoms are amongst the most successful phytoplankton groups, adapting to and surviving periods of suboptimal conditions and resuming
growth as soon as nutrient concentrations permit. A knowledge of the molecular underpinnings of diatom ecological success is, however, still very incomplete.
By strand-specific RNA sequencing, we analyzed the global transcriptome changes of the
diatom Phaeodactylum tricornutum in response to P fluctuations over a course of 8 d, defining five distinct physiological states.
This study reports previously unidentified genes highly responsive to P stress in
P. tricornutum. Our data also uncover the complexity of the P. tricornutum P-responsive sensory and signaling system that combines bacterial two-component systems with more complex pathways reminiscent of metazoans. Finally, we identify a multitude of novel long
intergenic nonprotein coding RNAs (lincRNAs) specifically responsive to P depletion, suggesting putative regulatory roles in the regulation of P homeostasis.
Our work provides additional molecular insights into the resilience of diatoms and their ecological success, and opens up novel routes to address and explore the function and regulatory
roles of P. tricornutum lincRNAs in the context of nutrient stress.
Introduction
As a fundamental element to all living cells, phosphorus (P) is
a building block of nucleic acids and membrane phospholipids.
Through its water-soluble and inorganic active forms (phosphate esters and orthophosphate, respectively), P is also
involved in cellular energy transfers, metabolic pathways and
protein activation. Life in the oceans can be subject to extreme
environments, depleted of the essential elements that sustain
phytoplankton growth, especially in the open ocean far from
land (Morel, 2003). Moreover, in coastal areas, P is often considered to be the potentially limiting element (Bauerfield et al.,
1990; Woodward & Owens, 1990). It is currently accepted
that P, like nitrogen (N), iron or light, may drive marine
microbial evolution and niche adaptation (Dyhrman et al.,
2007).
Diatoms are amongst the most ecologically successful phytoplankton groups living in the oceans. Their ecological relevance
is paramount, sustaining both marine and terrestrial ecosystems
by their role in the global carbon cycle, producing each year
the same amount of organic carbon as all of the terrestrial
rainforests combined (Nelson et al., 1995; Field et al., 1998).
The marine environment is subjected to recurrent fluctuations
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primer-blast/) defining a PCR amplicon size of < 180 bp and
Phaeodactylum tricornutum CCAP 1055/1 (taxid: 556484) as the
reference organism to check for primer pair specificity. Quantitative PCR conditions were set as follows: 95C for 10 s, followed
by 40 cycles of 95C for 5 s and 60C for 30 s, and a final cycle
of 95C for 10 s and 60C for 5 s. Data were collected and analyzed by a Bio-Rad CFX96 real-time system (BioRad, Hercules,
CA, USA). CDKA and HISTONE 4 mRNA levels were used for
normalization (Siaut et al., 2007).
Correlation analysis between transcription factors (TFs) and
putative target genes
To predict the gene targets of selected transiently expressed heat
shock factors (HSFs), the PCCs were calculated using FPKMs vs
all Pi-responsive protein coding genes as well as noncoding genes.
Only the positively correlated gene targets (PCC > 0 and
r2 0.6) were retained. As HSFs bind to heat shock elements
within promoter regions of their target genes (Nover et al.,
2001), only targets with the conserved sequence 50 GAAnnTTC30
in their promoter sequences (broadly defined as sequences 3 kb
upstream of the transcriptional start site) were considered. The
results of the correlation analysis were visualized using Cytoscape
(v2.8.3) (Smoot et al., 2011).
Identification of long intergenic nonprotein coding RNAs
(lincRNAs) and characterization of their genomic features
All assembled intergenic transcription units were collected as
lincRNA candidates. Candidates with a length of 200
nucleotides and a predicted open reading frame (ORF) of 100
amino acids were defined as lincRNAs (Liu et al., 2012). When
considering gene models of protein coding genes, the best gene
models obtained from the Phatr2 annotation were used; for
those of lincRNAs, the gene models with maximum intron numbers were used. The lengths of entire unspliced transcripts (including introns) were used to compare transcript length
distribution. For ORF predictions, the spliced transcripts were
used and sent to GenScan (Burge & Karlin, 1997). To compare
the length of stop codon-free sequences, the distances between
each stop codon of each gene in three reading frames were calculated, as described previously (Niazi & Valadkhan, 2012). Mfold
was used to calculate the free energy of the spliced transcripts
(Zuker, 2003). As longer transcripts have lower free energy, to
better compare the free energy of lincRNAs and mRNAs from
protein coding genes, we classified them into different groups
based on sequence length. This was also performed when comparing the free energy with the GC contents of lincRNAs and
mRNAs.
To find putative cis-regulation of lincRNAs on their neighboring genes, the PCCs were calculated using FPKMs of all the
responsive lincRNAs vs mRNAs encoded by their neighboring
upstream and downstream genes. Only those Pi-responsive
neighboring genes with r2 0.6 (positive or negative correlation)
were considered as putative gene targets of cis-regulatory
lincRNAs.
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DIC
(a)
(c)
C 4d
-Pi
+Pi
5
4
3
2
**
**
Pi 4d
0
0
Pi 8d
Time (d)
(b)
2.5
+Pi 4d
2
1.5
*
1
0.5
C 8d
NR + Chl
0
C 4d
Pi 4d
+Pi 4d
define genes that were present in one species were those published previously (Armbrust et al., 2004; Bowler et al., 2008):
30% sequence positives; 30% alignment coverage of either
the query or subject sequences; and BLAST e-value of < 1e-5. The
larger fraction of Pi differentially expressed genes (30%) corresponded to proteins common to plants and animals (Eukaryotes),
20% were core proteins and 19.4% were exclusively of plant
origin (Fig. 2c). Shared proteins between P. tricornutum and
Thalassiosira pseudonana constituted < 2% of the differentially
expressed genes, and those found exclusively in P. tricornutum
accounted for c. 16% (Fig. 2c). The latter are probable de novo
genes that have evolved since the two diatom lineages diverged c.
90 million yr ago (Bowler et al., 2008). Seventy-seven differentially expressed genes were exclusively found in bacteria and were
absent in all other organisms (Fig. 2c). Many of the bacterial
genes that responded to Pi fluctuation were related to metabolism
and redox processes. Interestingly, included in the Pi differentially expressed genes were 123 genes also present in viruses, some
of which were simultaneously present in plants (four), in plants
and bacteria (19), in animals and bacteria (five) and in all the
groups (62), suggesting a possible horizontal viralhost gene
exchange. These consisted largely of membrane proteins, including several putative signaling protein kinases and Pi transporters.
The presence of Pi transporter genes in several virus genomes
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(a)
(b)
(c)
Fig. 2 Genome-wide transcriptome analysis of Phaeodactylum tricornutum response to inorganic phosphate (Pi) fluctuations. (a) Venn diagrams
representing the up- and downregulated genes in response to progressive Pi depletion and resupply. Cutoff of two-fold difference in fragments per
kilobase of exons per million fragments mapped, P < 0.05, when compared with 4 d control cultures. Pi 4d, 4 d Pi depleted; Pi 8d, 8 d Pi depleted; +Pi
4d, 4 d Pi resupply after 4 d Pi depletion. (b) Correlation between strand-specific RNA-sequencing (ssRNA-Seq) and quantitative reverse transcription
polymerase chain reaction (RT-qPCR) in the detection of Pi-responsive Phaeodactylum tricornutum genes. The x-axis gives the log2 value of the fold
change detected by ssRNA-Seq and the y-axis gives the same value detected by RT-qPCR. Green dots represent differentially expressed (DE) genes
detected by both platforms; gray dots represent genes that do not change by more than two-fold in expression under Pi fluctuation. (c) Gene origins of the
6436 Pi DE protein coding genes. Phaeodactylum tricornutum protein sequences were compared with the National Center for Biotechnology Information
nonredundant (NCBI nr) database using BLASTp. The criteria used to define genes that were present in one species were as follows: 30% sequence
positives; 30% alignment coverage of either the query or subject sequences; and a BLAST e-value of < 1e-5. Core, proteins present in animals, plants,
bacteria; Unique, proteins only found in P. tricornutum; Diatom, proteins common to Thalassiosira pseudonana and P. tricornutum.
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(a)
(b)
GO enrichments odds rao
Pi 8d
C 8d
+Pi 4d
Pi 8d
Phosphate transport
Nucleode-sugar metabolic process
Inorganic anion transport
Anion transport
Glycolysis
Glucose catabolic process
Monosaccharide catabolic process
Hexose catabolic process
Alcohol catabolic process
Carbohydrate catabolic process
Cellular carbohydrate catabolic process
Glucose metabolic process
Hexose metabolic process
Monosaccharide metabolic process
Cellular carbohydrate metabolic process
Alcohol metabolic process
Ubiquin cycle
Unsaturated fay acid biosynthec process
Unsaturated fay acid metabolic process
Phosphoenolpyruvate-dependent sugar phosphotransferase system
Regulaon of metabolic process
Intracellular signaling cascade
DNA replicaon iniaon
DNA packaging
Chroman assembly or disassembly
Chroman assembly
Chroman organizaon
Protein-DNA complex assembly
Nucleosome assembly
Nucleosome organizaon
Chromosome organizaon
Deoxyribonucleode metabolic process
Organelle organizaon
Regulaon of gene expression
Regulaon of macromolecule metabolic process
Establishement of localizaon
Localizaon
Gene expression
Transport
Nitrogen compound metabolic process
Nucleobase, nucleoside, nucleode and nucleic acid metabolic process
12.0
Pi 4d
2.0
12.0
Pi 4d
C 8d
2.0
Fig. 3 Gene ontology (GO) enrichment analysis of (a) upregulated and (b) downregulated genes in the transcriptome response of Phaeodactylum
tricornutum to inorganic phosphate (Pi) fluctuations. The top 15 (highest odds ratio) enriched GO terms of the biological process category in each sample
are shown. The odds ratio was defined as the ratio of the proportion of a GO term in (a) upregulated and (b) downregulated genes to the proportion of
this GO term in all diatom genes. The larger the odds ratio, the higher the relative abundance of this GO term compared with background. Multiple-test
adjusted P < 0.05 was used to define statistical significance. Pi 4d, 4 d Pi depleted; Pi 8d, 8 d Pi depleted; +Pi 4d, 4 d Pi resupply after 4 d Pi depletion; C
8d, 8 d control conditions.
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Na+/Pi co-transporter
Na+/Pi co-transporter
Pi transporter
Pi transporter
Na+/Pi co-transporter
Pi transporter
Na+/Pi co-transporter
Na+/Pi co-transporter
Na+/Pi co-transporter
Pi transporter
HYP
HYP
Pi transporter
Na+/tricarboxylate and Pi transporter
HYP
HYP
Na+/tricarboxylate and Pi transporter
Mito Pi carrier protein
Mito Pi carrier protein
Mito Pi carrier protein
Mito Pi carrier protein
Mito Pi carrier protein
HYP
Na+/Pi co-transporter
39432
49678
47869
45959
Alkaline phosphatase
Alkaline phosphatase
HYP
Alkaline phosphatase
+Pi 4d
Pi 8d
47239
47666
39515
23830
47667
22315
21441
33266
40433
46692
19030
22279
1277
14922
43646
17265
22453
47954
462
11414
37916
8987
11866
49842
3.0
42872
43665
50356
42467
9619
46400
49771
12431
46908
8860
1000
1611
40261
43116
11390
48445
54168
42683
12884
14125
3262
36877
34555
47576
43099
PolyP
metabolism
C 8d
+Pi 4d
3.0
Pi 8d
Pi 4d
C 8d
Pi transporters
AlkPhases
(b)
3.0
3.0
Pi 4d
(a)
50019
15281
48538
54257
VTC
VTC
VTC
VTC
Fig. 4 Heat maps of log2 fold expression changes under inorganic phosphate (Pi) fluctuation of putative (a) Pi transporters and alkaline phosphatase
(AlkPase) coding genes and (b) membrane lipid metabolism and polyphosphate metabolism (PolyP) coding genes in Phaeodactylum tricornutum. Numbers
correspond to Joint Genome Institute (JGI) protein identifiers. Pi 4d, 4 d Pi depleted; Pi 8d, 8 d Pi depleted; +Pi 4d, 4 d Pi resupply after 4 d Pi depletion;
C 8d, 8 d control conditions.
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recognition sequence, their binding does not lead to miR399assisted cleavage and remains stable, which results in the finetuning of Pi uptake (Franco-Zorrilla et al., 2007). We have thoroughly analyzed the P. tricornutum noncoding transcriptome and
have identified 1510 putative lincRNAs (Table S4), 202 of which
were specifically upregulated in response to Pi depletion and
downregulated when Pi was resupplied to the medium. The 1510
lincRNA sequences identified in this study were compared with
the 10 402 mRNAs obtained from Phatr2. Phaeodactylum
tricornutum lincRNAs are significantly shorter than mRNAs with
c. 90% at < 1000 nucleotides (Fig. 5a,b). Most of these diatom
lincRNAs (> 70%) lack ORFs with coding potential in any of the
three possible reading frames (Fig. 5c). Nonetheless, approximately 28% of the identified lincRNAs have the presence of a
very short putative ORF (< 100 amino acids) (Fig. 5c,d), which
could potentially be translated into a short peptide. The majority
of the P. tricornutum lincRNAs are intronless (90%) with only a
small fraction containing a single intron (Fig. 5e) of similar size
to the introns found in mRNAs (Fig. 5f). The exons of lincRNAs
are shorter than those of protein coding transcripts (Fig. S4a,c)
and the distance between stop codons and the longest stop
codon-free sequences are shorter in diatom lincRNAs than in
protein coding transcripts (Fig. S4d,e). LincRNAs also have
lower GC content and lower free energy compared with mRNAs
(Figs S4f, S5, S6). Interestingly, all of these genomic features have
also been detected in functional human lincRNAs (Niazi & Valadkhan, 2012), suggesting conserved features of these molecules
across kingdoms.
To search for lincRNAs with a similar function to that of IPS1
in higher plants, we analyzed whether the P. tricornutum Piresponsive lincRNAs had a predicted target mimicry function
(Wu et al., 2013). No target mimicry function could be predicted
in any of the Pi-responsive lincRNAs identified in this work.
Using recently publically available RNA-Seq reads (accession no.
SRP040703), we sought to investigate the expression changes of
the Pi-responsive lincRNAs under 48 h N depletion (Levitan
et al., 2015). Amongst the top 20 upregulated lincRNAs in Pi,
only two were significantly upregulated under N (two-fold difference and P < 0.05) (Fig. 6a), revealing very specific responses
of the noncoding transcriptome to stress conditions. The fact that
these diatom lincRNAs were specifically expressed in response to
Pi depletion (Figs 6a, S3) suggests that at least some of these transcripts could have regulatory roles in P. tricornutum.
LincRNAs have been shown to associate with chromatin
remodeling complexes and to affect gene expression by cis- and
trans-action (De Lucia & Dean, 2011; Nagano & Fraser, 2011;
Guttman & Rinn, 2012; Bonasio & Shiekhattar, 2014; Liu et al.,
2015). We investigated the coexpression correlation between the
Pi-responsive lincRNA genes and their neighboring genes to
explore putative cis-regulatory functions of the lincRNAs. Several
potential gene targets have been identified as having a correlation
(positive or negative) to lincRNAs (Fig. 6b; Table S5). These
results can be used to further investigate the function of diatom
lincRNAs in the context of Pi depletion, but also in the context
of other abiotic stresses that have putative common responsive
molecular modules, such as N stress.
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LincRNA
0.5
Frequency (%)
P value = 4.12e243
(b)
0.6
Length of unspliced
transcripts (nt)
(a)
mRNA
0.4
0.3
0.2
0.1
3500
3000
2500
2000
1500
1000
500
<
50 500
0
10 100
00 0
1
15 500
00
20 200
00 0
25 250
00 0
3
30 000
00
35 350
00 0
40 400
00 0
4
45 500
00
5
00
0
>
50
00
Length of predicted
ORFs (aa)
LincRNA
80
Frequency (%)
P value = 1.63e-124
(d)
100
mRNA
60
40
20
1000
900
800
700
600
500
400
300
200
100
0
0
0
>1
(f)
P value 1
100
Frequency (%)
mRNA
60
40
20
200
150
100
50
0
0
LincRNA
80
>5
Number of introns
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+Pi 4d
Pi 8d
C 8d
Pi 4d
+Pi 4d
Pi 8d
C 8d
Pi 4d
+Pi 4d
C 8d
Pi 8d
(b)
Pi 4d
(a)
Log2 expression ratio
+Pi 4d
Pi 8d
XLOC_006307
XLOC_005436
XLOC_003159
XLOC_008284
XLOC_005700
XLOC_004740
XLOC_015130
XLOC_010279
XLOC_013710
XLOC_013110
XLOC_005097
XLOC_008283
XLOC_012004
XLOC_009323
XLOC_012001
XLOC_005083
XLOC_004568
XLOC_003589
XLOC_008370
XLOC_006663
D
D
U
Upstream
genes
LincRNAs
XLOC_003268
XLOC_000522
XLOC_014096
XLOC_012003
XLOC_005083
XLOC_012001
XLOC_009323
XLOC_007795
XLOC_008370
XLOC_012005
XLOC_004569
XLOC_003517
XLOC_003820
XLOC_003072
XLOC_006831
XLOC_001033
XLOC_015215
XLOC_013578
XLOC_012002
XLOC_011959
XLOC_000944
XLOC_009426
XLOC_010053
XLOC_013214
XLOC_014025
XLOC_002204
XLOC_014931
XLOC_002701
XLOC_010537
XLOC_003159
XLOC_015596
XLOC_001067
XLOC_010415
XLOC_009204
XLOC_011480
XLOC_015595
XLOC_006121
XLOC_002503
XLOC_010061
XLOC_014912
XLOC_000622
XLOC_010157
XLOC_012114
XLOC_009535
5.0
Pi 4d
C 8d
5.0
5.0
Downstream
genes
5.0
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(b)
Log2 expression rao
Pt_HSF1.1a
Pt_HSF4.3c
Pt_HSF4.3a
Pt_bZIP15
Pt_HSF3.1b
Pt_HSF4.4b
Pt_CCHH5
Pt_HSF1.2b
Pt_HSF4.7a
HSF, 18
ZnFinger, 6
+Pi 4d
other, 8
bHLH, 1
CXC, 2
3.0
C 8d
Hox, 2
3.0
Pi 8d
Pi 4d
(a)
Myb, 8
bZIP, 5
CBF/NF2,
1
Hox, 2
other
,6
bHLH, 3
CXC, 1
ZnFinger, 9
Myb, 6
bZIP, 6
3.1b 4.7a
HSF, 6
other, 10
E2F, 2
Myb, 12
bHLH, 1
CXC, 3
Zn-
(c)
HSF, 13
bZIP, 4
Acknowledgements
We thank Connie Zhao and Bin Zhang (Genomics Resource
Center, Rockefeller University), as well as Kaye Thomas, Pablo
Ariel and Tao Tong (Bio-imaging Resource Center, Rockefeller
University), for technical support. We also thank Jun Liu and
Huan Wang for help with the bioinformatic analysis, and the
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Author contributions
M.H.C.C., H-X.S. and N-H.C. planned and designed the
research; M.H.C.C. and H-X.S. performed the research;
M.H.C.C., H-X.S. and N-H.C. analyzed the data; M.H.C.C.,
H-X.S., C.B. and N-H.C. wrote the manuscript.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Phosphate content in the growth medium of
Phaeodactylum tricornutum on the sampling points for strandspecific RNA sequencing (ssRNA-Seq).
Fig. S2 Growth curves of Phaeodactylum tricornutum cell cultures
under different culture conditions.
Fig. S3 Validation of strand-specific RNA sequencing (ssRNASeq) expression fold changes by quantitative reverse transcription
14 Research
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Table S2 Primers designed for quantitative reverse transcription
polymerase chain reaction (RT-qPCR) analysis in Phaeodactylum
tricornutum
Table S3 Expression variation under phosphate fluctuations of
Phaeodactylum tricornutum micro-RNAs (miRNAs)
Table S4 Expression variation under phosphate fluctuations of
Phaeodactylum tricornutum long intergenic nonprotein coding
RNA (lincRNA) candidates
Table S5 Putative cis-targets of phosphate depletion-responsive
long intergenic nonprotein coding RNAs (lincRNAs) in
Phaeodactylum tricornutum
Table S6 Expression variation of Phaeodactylum tricornutum
transcription factors under phosphate fluctuations
Table S7 Correlation networks between HSF3.1b and HSF4.7a
and their putative target genes under early phosphate depletion
in Phaeodactylum tricornutum
Table S8 Expression variation of Phaeodactylum tricornutum
sensing- and signaling-related genes under phosphate fluctuations
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