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Transcriptome analysis reveals regulation of gene expression during

photoacclimation to high irradiance levels in Dunaliella salina
(Chlorophyceae)

Article in Phycological Research · April 2019

DOI: 10.1111/pre.12379

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Phycological Research 2019
...........................................................................................................................................................................................
Transcriptome analysis reveals regulation of gene expression
during photoacclimation to high irradiance levels in
Dunaliella salina (Chlorophyceae)
Yuanxiang Li,1,2,3† Wenhui Gu,1,2† Aiyou Huang,1,2 Xiujun Xie,1,2 Songcui Wu1,2 and Guangce Wang
1
Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese
Academy of Sciences, Qingdao, China, 2Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for
Marine Science and Technology, Qingdao, China and 3College of Earth Sciences, University of Chinese Academy of
Sciences, Beijing, China
........................................................................................
SUMMARY
The unicellular green alga Dunaliella salina is an attractive
organism for studying photoacclimation responses. Changes
in irradiance level during cell growth affect the organization
and structure of the photosystem and the composition of pig-
ments. In this study, D. salina was exposed to different irradi-
ance levels, and cell growth, photosynthetic activity,
pigments, and neutral lipids were analyzed. Photosystem II
activity and chlorophyll content were reduced, carotenoids
content and neutral lipids increased as light intensity
increased, suggesting that photosynthetic apparatus was
reduced and photoprotection mechanisms were activated. The
RNA of D. salina was sequenced to investigate the trans-
criptomic response of the organism after transitioning from
normal light conditions to higher light intensity. Genes
encoding for enzymes involved in photosynthesis were down-
regulated, whereas genes involved in the metabolism of carot-
enoid and triacylglycerol were upregulated. Genes encoding
for photoprotective enzymes related to reactive oxygen species
scavenging and to the xanthophyll cycle were also upregulated
at higher irradiance levels. The present transcriptomic study
would assist in the comprehensive understanding of pho-
toacclimation mechanisms of D. salina.
Key words: carotenoid, light treatment, microalgae, photosyn-
thesis, photosynthetic activity.
........................................................................................
INTRODUCTION
Plants and algae need light for photosynthesis, but excessive
light can cause damage to photosynthetic organisms. Mecha-
nisms for avoiding damage from light include short-term and
long-term acclimation mechanisms (Erickson et al. 2015).
Short-term mechanisms are induced within minutes or hours,
and comprise at least two major processes, non-
photochemical quenching via the xanthophyll cycle and state
transition (Allorent et al. 2013). Long-term acclimation mech-
anisms involve reduction in the abundance of Lhc transcripts
which decrease the size of light-harvesting complex (LHC),
and moving LHC II antenna from photosystem (PS) II to PS I
to balance the excitation rate (Fujita et al. 1989; Allen 1992;
Durnford et al. 2003).
© 2019 Japanese Society of Phycology
doi: 10.1111/pre.12379
1,2
*
In Chlamydomonas reinhardtii, expression of genes
encoding polypeptides of the light-harvesting antenna, Lhcb,
Lhcb4, and Lhca was reduced by 65 to 80% under high
intensity light stress (Durnford et al. 2003); while the expres-
sion of the stress-related LHC gene, LHCSR, was upregulated
under irradiance stress (Maruyama et al. 2014).
The unicellular green algae Dunaliella salina accumulates
β-carotene under high irradiance conditions. D. salina lacks a
rigid cell wall, which facilitates extraction of lipids and
genetic manipulation (Oren 2005). These physiological and
structural traits make D. salina an attractive model for study-
ing the response to high light stress.
Accumulation of photosynthetic pigments, increased LHC
and PS II reaction centers were observed in Dunaliella
tertiolecta after light transition from 700 to 70 μmol photo-
ns
m−2s−1 (Sukenik et al. 1990). After 48 h of high light
exposure，photoacclimation was accompanied by an increase
in the chlorophyll a/b ratio, and reduction of the antenna size
(Berner et al. 1989; Webb, and Melis 1995). The response to
high light stress in D. salina was similar to that of
C. reinhardtii and other higher plants.
Several genes related to photoacclimation have been ana-
lyzed through randomly selected expressed sequence tags
(Park et al. 2006). The development of next generation
sequencing technology has made high-throughput short-read
sequencing a useful tool for genome-wide profiling of trans-
criptomes (Grabherr et al. 2011). Hence, by carrying out a
genome-wide transcriptional profiling of D. salina, it could be
determined whether transcription plays a role in pho-
toacclimation mechanisms resulting from irradiance changes.
In the photosynthetic apparatus, an increase of irradiance
causes a reduction in the number of reaction centers and a
decrease in the size of the light-harvesting antenna (Fujita
et al. 1989; Sukenik et al. 1990). The reduction of the pro-
teins of the photosynthetic machinery involves the reduction
of the total amount of protein (Gu et al. 2014), which is prob-
ably caused by the degradation of damaged proteins during
.........................................................................................
*To whom correspondence should be addressed.
Email: gcwang@qdio.ac.cn
†
These authors contributed equally to this work.
Communicating Editor: Rei Narikawa
Received 12 October 2018; accepted 12 March 2019.
2 Y. Li et al.
the repair process (Virgin et al. 1990), or by regulation at the
transcriptional level. The changes in protein content caused
by protein degradation or by transcriptional regulation need to
be further studied.
In order to investigate changes in D. salina during pho-
toacclimation to high light stress, pigments, neutral lipids and
photosynthetic parameters were measured at increasing irradi-
ance levels (150, 300, 450, 600, 750, 900, 1200,
1500 μmol photons
m−2s−1). At different irradiance
(150, 600, 1500 μmol photons
m−2s−1), changes in mRNA
abundance were examined and transcriptome results were
analyzed. The effects of medium intensity light (600 μmol
photons
m−2s−1) and high intensity light (1500 μmol photo-
ns
m−2s−1) on expression of genes encoding LHC and reaction
centers of the photosystem were examined.
MATERIALS AND METHODS
Growth of D. salina
D. salina HG-01 was isolated from salterns in Hangu, Tianjin,
China and cultivated in DM medium (Pick et al. 1986). To
confirm the phylogenetic relationship of the strain, a phyloge-
netic tree was constructed (Fig. S1). Algal cells were culti-
vated in a Multi-Cultivator MC 1000-OD (Photon Systems
Instruments, Czech Republic) at 22  1 C with 150  5
μmol photons
m−2s−1 provided by cold white fluorescent
lamps with 16:8 h light–dark cycles. D. salina cultures were
aerated at a flow rate of 0.5 L
min−1. Cells in mid-exponential
growth phase were harvested by centrifugation for 4 min at
2000 g at room temperature, and inoculated into fresh
medium for light treatment. Irradiance was shifted in a single
step from 150 μmol photons
m−2s−1 to a range of light inten-
sities (150, 300, 450, 600, 750, 900, 1200, 1500 μmol
photons
m−2s−1). Cell growth was monitored using a Z2 Coul-
ter Particle Count and Size Analyzer (Beckman Coulter Co.,
Ltd., USA).
Pigment extraction and quantification
D. salina cells grown under different light intensities on the
seventh day were harvested by centrifugation for 4 min at
2000 g at room temperature and immediately frozen in liquid
nitrogen. Pigments were extracted using a precooled mixture
of methanol and acetone (1:1, v: v) for 1 h. The extracts were
centrifuged at 8000 g for 4 min and the supernatants were fil-
tered through 0.22 μm nylon filters. Pigments were separated
and identified using an Essentia LC-16 apparatus (Shimadzu
Corporation, Tokyo, Japan) equipped with an Rx-C18 analyti-
cal column (4.6 × 250 mm) (Agilent Technologies Inc., CA,
USA) as described by Xie et al. (2015).
Initial solvent (water: methanol: acetonitrile: acetyl ace-
tate = 5:30:65:0, v: v: v: v) was used for balancing the col-
umn for 10 min at flow rate of 0.8 mL/min. Separation of
pigments was started by a 5-min linear gradient from the ini-
tial solvent to water: methanol: acetonitrile: acetyl acetate
(0:15:85:0, v: v: v: v), and then continued for 7 min. Eluents
then was transited to water: methanol: acetonitrile: acetyl ace-
tate (0:45:35:20, v: v: v: v) within 2 min, followed by 16 min
linear gradient to water: methanol: acetonitrile: acetyl acetate
(0:45:0:55, v: v: v: v). Temperature of the column oven was
set as 20 C. Chlorophyll a, chlorophyll b, α-carotene, all trans
β-carotene, 9-cis β-carotene, lutein, and zeaxanthin
(Zx) standards were obtained from Sigma-Aldrich (St. Louis,
MO, USA). Violaxanthin (Vx) and antheraxanthin
(Ax) standards were obtained from the International Labora-
tory USA (South San Francisco, CA, USA).
Analysis of chlorophyll fluorescence
Chlorophyll fluorescence measurements were performed using
a Dual-PAM-100 fluorometer (Walz, Effeltrich, Germany) con-
nected to a computer equipped with WinControl software.
Cells grown under different light intensities on the seventh
day were harvested by centrifugation for 4 min at 2000 g at
room temperature, and optical density was adjusted to 1.5 at
680 nm using an UV-1800 spectrophotometer (Shimadzu
Corporation, Tokyo, Japan). Cells were acclimated to darkness
for 10 min before measuring chlorophyll fluorescence. After
acclimation to darkness, cultures were exposed to an irradi-
ance level lower than 1 μmol photons
m−2s−1 and intrinsic
fluorescence (F0) was determined immediately. A saturating
flash was then applied to the culture to detect the maximum
fluorescence (Fm). The maximum quantum yield of PS II,
Fv/fm, was calculated as (Fm-F0)/fm; F0 represents the mini-
mum and Fm represents the maximum fluorescence after
acclimation to darkness (Kramer et al. 2004; Schreiber
2004). The maximum fluorescence yield of algal cells was
recorded as Fm0 . The effective photochemical quantum yield
of PS II (YII) was calculated using the formula: YII = (Fm0 - F)/
Fm0 (Kramer et al. 2004).
Determination of nile red (NR) fluorescence
Determination of NR fluorescence was modified according to
Wu et al. (2014). NR (Sigma-Aldrich, USA) was prepared as a
stock solution of 0.1 mg/ mL in acetone and stored at 4 C.
Cells illuminated by different light intensities at 168 h were
diluted to a cell concentration of 1 × 106 cells/ mL before
staining. Cells were stained by 0.1 μg/ mL NR-acetone solu-
tion, and incubated in darkness for 6 min. The suspensions
were analyzed using a F4500 fluorescence spectrophotometer
(HITACHI, Japan) with excitation and emission wavelengths of
480 and 570 nm, respectively.
RNA extraction, construction of gene library
and sequencing
Total RNA was extracted from D. salina cells grown at differ-
ent light intensities for 168 h using an RNAprep Pure Plant
kit (Tiangen Biotech, Beijing, China). RNA was then treated
with RNAase-free DNase I (Takara, Japan) to remove any con-
taminating genomic DNA. mRNA-seq libraries were generated
according to the manufacturer recommendations for Illumina®
systems (NEB, USA). Subsequently, the libraries were
sequenced on an Illumina Hiseq platform and paired-end
reads were generated. Three libraries were generated and
named LL (control,150 μmol photons
m−2s−1), ML (600 μmol
photons
m−2s−1), HL (1500 μmol photons
m−2s−1). Each
library consisted of three biological replicates.
© 2019 Japanese Society of Phycology
Gene expression during photoacclimation
Transcriptome assembly and gene functional
annotation
After sequencing, clean reads were generated by removing
reads containing adapters, ploy-N, and low quality reads of
raw reads. Transcriptome assembly was performed using Trin-
ity (Grabherr et al. 2011). For functional annotation, unigenes
were defined as the longest transcript of each subcomponent.
Gene function was annotated based on the following data-
bases: NCBI non-redundant protein sequences (with an E-
value cut-off of 10−5), NCBI non-redundant nucleotide
sequences (with an E-value cut-off of 10−5), and Swiss-Prot.
PFAM protein family alignments were carried out using
HMMER 3.0 software. Gene ontology (GO) classification was
performed using Blast2GOv2.5 (Götz et al. 2008). Kyoto
Encyclopedia of Genes and Genomes (KEGG) classification
was performed using KASS and the KEGG Automatic Annota-
tion Server (http://www.genome.jp/kegg/).
Differential expression analysis
The abundance of transcripts is expressed as expected num-
ber of fragments per kilobase of transcript sequence per mil-
lions base pairs sequenced, which is the most commonly
used estimation method for gene expression (Trapnell et al.
2010). Differential expression analysis of two samples was
performed using DESeq R software (1.10.1) with a threshold
adjusted P-value (padj) < 0.05. The padj was corrected
P-value by multiple hypothesis tests to reduce false positive
cases. Differentially-expressed genes (DEGs) were generated
after Differential expression analysis. GO enrichment analy-
sis was carried out using the GOseq R software based on
Wallenius non-central hyper-geometric distribution (Young
et al. 2010). KEGG pathway analysis was performed using
KOBAS software to test the statistical enrichment of DEGs
(Mao et al. 2005).
Quantitative real-time PCR
Total RNA was extracted using the Plant RNA kit (OMEGA,
Norcross, USA). RNA concentration was measured using a
NanoPhotometer® spectrophotometer (IMPLEN, CA, USA).
The PrimeScript RT regent kit with gDNA Eraser (TaKaRa,
Dalian, China) was used to generate cDNA. All procedures
were performed on ice. Transcriptomic sequenced genes were
randomly chosen, and primers (Table S1) were designed
based on the randomly chosen genes using the Primer Pre-
mier 5.0 software. Transcript levels were quantified using
reagents from the Fast-Essential DNA Green Master (Roche,
Germany) and a Bio-Rad iQ5 Multicolor Real-Time PCR Reac-
tion system (Bio-Rad, Hercules, CA, USA). The cycling param-
eters for quantitative real-time PCR (qPCR) were 95 C for
10 min, followed by 40 cycles at 94 C for 10 s, 54 C for
20 s, and 72 C for 10 s. To ensure that only a single specific
DNA fragment was amplified, melting curve analyses were
performed on all PCR products. Triplicate qPCRs were per-
formed for each sample. Data were analyzed using the Bio-
Rad optical system software.
© 2019 Japanese Society of Phycology
3
Statistical analysis
Two-tailed student t-tests and one-way analysis of variance
were used to assess the significance of differences between
samples using SPSS software (IBM Co., Armonk, NY, USA).
The statistical significance level was set at P < 0.05. Differen-
tial expression analysis between samples was performed with
a threshold of padj < 0.05.
RESULTS
Characteristics of D. salina growth
To investigate the optimal light level and the light tolerance
range, D. salina was cultivated at different light intensities. As
shown in Figure 1a, the biomass of D. salina increased as
light intensity increased from 150 to 600 μmol photo-
ns
m−2s−1, and the optimal intensity for biomass was
600 μmol photons
m−2s−1 (ML). Under normal growth condi-
tions (150 μmol photons
m−2s−1, LL), biomass was lower than
the maximum yield obtained under ML. As the light intensity
increased above 600 μmol photons
m−2s−1, the growth rate
slowed down, yet growth was higher than that of algae grown
under LL.
Increased irradiance reduced the content of cellular Chl
a and b, and increased the Chl a/b ratio (Table 1). Under
1500 μmol photons
m−2s−1, the content of total carotene
increased 153.64% compared with that under 150 μmol pho-
tons
m−2s−1. The content of all-trans and 9-cis β-carotene
under 1500 μmol photons
m−2s−1 increased 192.39% and
57.13%, respectively, compared to 150 μmol photo-
ns
m−2s−1. The content of xanthophyll cycle-related caroten-
oid, Zx, increased from 150 to 1500 μmol photons
m−2s−1.
The content of Vx decreased from 366.6  4.8 to 86.6  8
mg/g dry weight with the increase of light intensity. The de-
epoxidation state (DEPS) was used to describe the content of
Ax and Zx in the pool of xanthophyll pigments, and was calcu-
lated according to the equation (Ax +Zx) / (Vx + Ax +Zx). As
shown in Table 1, DEPS increased 5.10-fold at 1500 μmol
photons
m−2s−1 (P < 0.05). A lower amount of lutein was
observed in D. salina under ML and HL.
The values of maximum quantum yield of PS II (Fv/Fm)
and the effective photochemical quantum yield of PS II (YII)
of the algal cells decreased significantly, as light intensity
increased from 150 to 1500 μmol photons
m−2s−1 (Fig. 1b).
As shown in Figure 1c, the fluorescence emitted by Nile red
significantly increased as light intensity increased. At
600 μmol photons
m−2s−1, nile red fluorescence was
5.15-fold higher than fluorescence at 150 μmol
photons
m−2s−1.
Annotation of D. salina transcriptome
To investigate the response of D. salina to changes in light
intensity, we analyzed the transcriptome of D. salina exposed
to 150, 600 and 1500 μmol photons
m−2s−1. There were
440 151 742 clean reads generated from D. salina; 95.36%
of the clean reads bases had a Q-value ≥20. The raw data and
processed data have been submitted to NCBI GEO database
(GSE120965). De novo assembly generated 76 875 unigenes
4 Y. Li et al.

Fig. 1. Growth of D. salina under different light intensities. (a) Growth curve of D. salina. (b) Photosynthetic characteristics of D. salina at
168 h. (c) Nile red fluorescence of D. salina at 168 h. [Color figure can be viewed at wileyonlinelibrary.com]

with an average length of 1103 bp and N50 of 1675 bp. The
length distribution of unigenes is illustrated in
Figure 2a. After transcriptome assembly, the significant differ-
ent expression genes (DEGs) were then identified (Fig. 2b-c).
We have acquired 9374 DEGs from ML (600 μmol photo-
ns
m−2s−1) and 11 297 from HL (1500 μmol photo-
ns
m−2s−1), with 4446 shared DEGs. Under ML, 4194 genes
were found to be significantly upregulated, whereas 5180
genes were downregulated.
After annotating the acquired unigenes using the NCBI
non-redundant protein sequences database, we obtained
sequence similarity between D. salina and its related species.
Of the sequences with similarity to unigenes, Volvox carteri
(31.1%), C. reinhardtii (24.9%), Monoraphidium neglectum
(10.6%), Coccomyxa subellipsoidea (5.9%) and Chlorella
variabilis (4.8%) were the top five algae, in descending order.
Of all the unigenes assembled, 38.16% were annotated
using GO terms through the GO site (http://www.geneontology.
org/). Three main functional categories were classified
according to GO terms: molecular function, biological process,
and cellular component (Fig. 3). The sequences were divided
into 10 subcategories, being the dominant subcategories
‘binding’ (45.23%) and ‘catalytic activity’ (37.23%) in the
molecular function category. For sequences assigned to bio-
logical process, 24 subcategories were classified and the first
two clusters were cellular (21.01%) and metabolic processes
(18.91%). In cellular components there were 21 subcate-
gories, being ‘cell’ (19.15%) and ‘cell part’ (19.12%) the
most abundant.
There were 8872 sequences that matched KOG/COG
(Clusters of Orthologous Groups of proteins) database
(Fig. S2); categories containing the largest number of
unigenes were ‘general functional prediction’, ‘posttransla-
tional modification, protein turnover and chaperons’, and
‘translation, ribosomal structure, and biogenesis’.
The transcriptome results were validated by qPCR, through
analysis of 10 randomly selected genes. Eight genes out of
the selected ten genes showed a positive correlation between
mRNA-Seq and qPCR (Fig. S3). Assembled unique sequences
were then searched against the KEGG database. A total of

© 2019 Japanese Society of Phycology

 Gene expression during photoacclimation 5

0.062  0.00045

0.0058  0.00092
9001 unique sequences were assigned to KEGG biochemical

0.13  0.0015
4.3  0.086

5.9  0.019

2.6  0.019

0.37  0.036
pathways (Fig. 4). The pathways were mainly involved in cel-

69.5  0.87

26.4  0.36

34.9  0.36

13.9  0.66
lular processes, environmental information processing,

86.6  8
genetic information processing, metabolism and organic sys-
tems. Metabolic pathways occupied the largest number of
1500

genes (58.47%), which included the following most abundant

processes: carbohydrate metabolism (9.65%), energy metabo-
lism (9.57%), overview (8.90%), cofactors and vitamins

0.13  0.0016
0.35  0.0019
5  0.027
0.063  0.001

5.1  0.052

2.3  0.011

13.6  0.063
79.3  0.86

22.8  0.13

30.2  0.17
87.3  0.59
metabolism (5.77%), lipid metabolism (5.48%). Of all the

0.0055  0
global biochemical pathways, we focused on photosynthesis,
carotenoid biosynthesis, and lipid metabolism, to investigate
1200

the mechanisms of photoadaption and photoprotection.

Changes in the photosynthetic apparatus

0.37  0.00085

0.006  0.00046
0.063  0.0011

0.14  0.0043
22.8  0.082
2.4  0.041
5.2  0.19

5.4  0.31

30.5  0.35

17.6  0.64

The relative expression of genes of the photosynthetic appara-

82.2  1.6

84.2  5.9

tus, including light-harvesting proteins, reaction center pro-

teins, and electron transfer related proteins, changed
900

significantly during exposure to high irradiance levels and dur-

ing the acclimation process.
After changing light from 150 to 600 μmol photo-
2.2  0.00057

0.0057  0.00035
0.077  0.0053

0.13  0.0046
0.36  0.0025

ns
m−2s−1, 18 genes of the PS II were significantly down-

5.3  0.081

5.5  0.27
20  0.18

27.6  0.45

17.6  0.12

regulated. Changing light from 150 to 600 μmol

69  5.7

86  6.6

photons
m−2s−1, genes of light-harvesting chlorophyll a/b

binding protein and PS I were downregulated as well
750

(Table 2). The expression of cytochrome b6f complex related

genes were upregulated at 600 μmol photons
m−2s−1.
Table 1. Effects of different irradiance levels (μmol photons
m−2s−1) on pigments content  SD in D. salina

Irradiance at 1500 μmol photons
m−2s−1 significantly

0.12  0.00025

0.005  0.00026
0.078  0.0055

0.31  0.0055

downregulated the expression of genes encoding proteins of

5.1  0.042

1.9  0.014
4.9  0.54
16.3  0.16

23.2  0.69

18.4  0.26
65.5  5.2

85.7  3.5

LHC and photosynthetic electron transport chain. As shown in

Table S2, 21 unigenes that encode chlorophyll a/b-binding
proteins of PS I and PS II were downregulated at 1500 μmol
600

photons
m−2s−1. At 1500 μmol photons
m−2s−1, The expres-

sion of cytochrome b6f complex proteins were also
0.12  0.00011

upregulated approximately 1.26–1.6-fold.

0.11  0.0012

5.2  0.0024

0.32  0.0094

0.0035  0.0003
16.5  0.034
6.7  0.22

23.6  0.16
126.1  13.4

26.8  0.04
60.3  1.3

1.9  0.2

Carotenoid biosynthesis and regulation

At 600 μmol photons
m−2s−1, we detected expression of the
450

following carotenoid biosynthesis genes: phytoene synthase

(PSY), phytoene desaturase (PDS), ζ-carotene desaturase
0.12  0.00052

(ZDS) and lycopene beta cyclase (LCYB). PSY, which cata-

1.6  0.0045

0.12  0.0026

0.0031  0.0001
0.28  0.019

lyzes the first step of the carotenoid biosynthesis pathway, is

6.8  0.17

4.3  0.35
12  0.17

17.9  0.52

28  0.42
58.6  1.2

127.1  4.5

regarded as an important regulatory step of carotenoid biosyn-

thesis (Sandmann et al. 2006). As shown in Table 2, PSY was
upregulated 1.56-fold, and LCYB was upregulated approxi-
300

mately 1.46–3.29-fold. PDS and ZDS were downregulated

approximately 0.69–0.77-fold.
0.0011  0.00031

The regulation of carotenogenesis pathway requires sen-

0.28  0.0012

13.8  0.0034

0.22  0.0019
3.1  0.026
9  0.018
1.7  0.011

sors and transduction elements (Jin and Polle 2009). An early

12.9  0.29

46.8  0.86

0.19  0.11
366.6  4.8

54.9  1.8

light-induced-like protein, carotene biosynthesis-related pro-

tein (Cbr) (Banet et al. 2000), is known to be induced expres-
sion during high-light treatment. The Cbr is highly expressed
150

at 600 and 1500 μmol photons
m−2s−1. Six genes identified

as carotene globule proteins were upregulated approximately
De-epoxidation state.

1.4–2.07-fold at 600 μmol photons
m−2s−1. Beta-carotene

All trans β-carotene
9-Cis β-carotene

isomerase D27 catalyzes all-trans/9-cis β-carotene isomeriza-

Antheraxanthin
Total carotene
Concentration

Chlorophyll.
Violaxanthin

tion (Lin et al. 2009). Genes identified as carotene isomerase

Dry weight.
Chl a/ chl b
(mg/g DW§)

Zeaxanthin
α-Carotene

D27 were upregulated approximately 2.55–3.72-fold at

DEPS‡
Lutein

600 μmol photons
m−2s−1, which was also observed under

Chl a
Chl b
†

high light stress in D. bardawil (Davidi and Pick 2017).

†
‡
§

© 2019 Japanese Society of Phycology

6 Y. Li et al.

Fig. 2. Length distributions of

D. salina unigenes and differen-
tially expressed genes (DEGs) at
600 (ML) and 1500 μmol photo-
ns
m−2s−1 (HL). (a) length distri-
butions of D. salina unigenes. (b)
Venn diagram of the number of
shared or unique DEGs under ML
and HL. (c) comparison of expres-
sion patterns of DEGs identified
between ML and LL (150 μmol
photons
m−2s−1). The red (right)
and green (left) dots represent up-
or downregulated DEGs, and the
blue dots represent non-DEGs.
[Color figure can be viewed at
wileyonlinelibrary.com]

Fig. 3. Gene ontology classification analysis of unigenes. The unigenes were classified into three functional categories: biological process,
cellular component, and molecular function. [Color figure can be viewed at wileyonlinelibrary.com]

Lipid metabolism (ACC), and was significantly upregulated approximately

1.24–2.62-fold (Fig. 5). The second step of fatty acid biosyn-
Genes of the fatty acid biosynthesis pathway were upregulated thesis pathway is the transfer of a malonyl group from
at 600 μmol photons
m−2s−1. At 600 μmol photons
m−2s−1, malonyl-CoA to acyl carrier protein (ACP), catalyzed by
five genes were identified as acetyl-coenzyme A carboxylase malonyl-CoA-acyl carrier protein transacylase (MCAT). At

© 2019 Japanese Society of Phycology

Gene expression during photoacclimation
Fig. 4. KEGG classification analysis
of unigenes. The unigenes were clas-
sified into five main categories:
(a) cellular processes; (b) environ-
mental information processing; (c)
genetic information processing; (d)
metabolism; (e) organic systems.
[Color figure can be viewed at
wileyonlinelibrary.com]
600 μmol photons
m−2s−1, two genes identified as MCAT
were upregulated 1.42–1.46-fold. Single genes for
3-ketoacyl-ACP-reductase and 3-hydroxyacyl-ACP dehydratase
related to fatty acid condensation reactions were upregulated.
At 600 μmol photons
m−2s−1, genes of triacylglycerol
(TAG) biosynthesis pathway were also detected: glycerol-
3-phosphate acyl-transferase, diacylglycerol acyltransferase,
and phospholipid: diacylglycerol acyltransferase (PDAT). Two
genes encoding for glycerol-3-phosphate acyl-transferase were
upregulated over 2-fold at 600 μmol photons
m−2s−1 (Fig. 5),
and the upregulation of diacylglycerol acyltransferase genes
were approximately 1.34–4.96-fold. Besides, a single gene
for PDAT was upregulated 1.36-fold.
DISCUSSION
Low to high light shift: adaptation of the
photosynthetic apparatus
In order to adapt to higher irradiance, both LHC and PS genes
on the thylakoid membrane were correspondingly down-
regulated to reduce energy absorption and over-reduction of
PS. Generally, it is believed that acclimation of PS increases
growth rate under suboptimal conditions (Geider et al. 1998),
which is consistent with the growth curve shown in Figure 1a.
In the present study, a decrease in cellular Chl a and
b content, with a concomitant increase in the Chl a/b ratio, was
observed. Through evolution, algal cells devised a series of
acclimation mechanisms for adapting to irradiance changes;
these mechanisms include a reduction of PS antenna size and
lower amounts of Chl (Jin et al. 2003). By observing changes
in pigmentation after changing irradiance from 70 to 700 μmol
photons
m−2s−1, Berner et al. (1989) found a similar increase
in the Chl a/b ratio. Since different Chl a/b ratios in cells grown
at different light intensities are due to changes in the levels of
© 2019 Japanese Society of Phycology
7
LHC, changes in the Chl a/b ratio could be used to assess the
size of the antenna (Fujita et al. 1989). The increase of the Chl
a/b ratio that we observed shows that LHC became smaller at
600 and 1500 μmol photons
m−2s−1.
Previous studies reported a reduction in the amount of pro-
teins of the reaction center and LHC under high irradiance
conditions (Melis 1991; Gu et al. 2014). The decrease in
amount of protein, induced by high irradiance, may be due to
degradation, or to changes in gene expression at the transcrip-
tional level. The repair process induced by high irradiance
involves removal of damaged proteins and assembly of newly
synthesized proteins, which changes protein content. Fujita
et al. (1989) found that the reduction in LHC levels is not
attributable to accelerated degradation of LHC under high
irradiance levels, indicating that the changes in gene expres-
sion may play a key role in the control of photosynthetic pro-
teins. In our study, the expression of subunits of PS reaction
center and LHC were downregulated at 600 and 1500 μmol
photons
m−2s−1. Similar results were reported by Jin et al.
(2001). Thus, the regulation of transcription seems necessary
for long-term adaptation of photosynthetic elements.
The cytochrome b6f complex serves as an intermediate in the
electron transport steps. At high irradiance levels, the un-limited
light absorption by PS facilitates the increase in the content of
the cytochrome b6f complex (Melis 1991). In our transcriptome
analysis, genes that encode components of the cytochrome b6f
complex were upregulated at 600 and 1500 μmol photo-
ns
m−2s−1. We have reported increased proteins of the cyto-
chrome b6f complex during short-term adaptation of D. salina to
1000 μmol photons
m−2s−1 (Gu et al. 2014).
Low to high light shift: modulation of
photodamage and repair process
At the transcriptional level, repair and protection mechanisms
are activated to relieve the damage and maintain normal
8 Y. Li et al.

Table 2. mRNA-Seq data for the genes involved in photosystems, carotene biosynthesis, fatty acid biosynthesis, TAG biosynthesis, ROS-
scavenging enzymes and xanthophyll cycle at 600 μmol photons
m−2s−1 in D. salina

Gene name Abbreviation Gene_id Fold change padj

Photosystem and antenna

Photosystem II protein D1 psbA Cluster-5009.342 81 0.73 3.38E-04
Photosystem II protein D1 psbA Cluster-5009.338 33 0.64 3.66E-04
Photosystem II protein D1 psbA Cluster-5009.336 11 0.51 1.52E-15
Photosystem II CP47 reaction center protein psbB Cluster-5009.336 57 0.62 3.77E-18
Photosystem II CP43 reaction center protein psbC Cluster-5009.358 67 0.38 9.38E-39
Photosystem II CP43 reaction center protein psbC Cluster-5009.266 52 0.52 1.76E-04
Photosystem II D2 protein psbD Cluster-5009.340 62 0.47 7.58E-58
Photosystem II D2 protein psbD Cluster-5009.336 56 0.44 6.53E-04
Cytochrome b559 subunit alpha psbE Cluster-5009.356 94 0.63 2.31E-03
Cytochrome b559 subunit beta psbF Cluster-5009.528 45 0.43 2.63E-04
Photosystem II reaction center protein psbH Cluster-5009.198 97 0.52 1.65E-05
Photosystem II reaction center protein L psbL Cluster-5009.9630 0.31 2.67E-14
Photosystem II reaction center protein L psbL Cluster-5009.9630 0.31 2.67E-14
Photosystem II reaction center protein M psbM Cluster-5009.397 35 0.54 1.39E-05
Photosystem II 10 kDa polypeptide, chloroplastic psbR Cluster-5009.337 24 0.81 7.61E-03
Photosystem II reaction center protein T psbT Cluster-5009.528 35 0.35 6.66E-06
Photosystem II reaction center W protein, chloroplastic psbW Cluster-5009.262 03 0.86 1.42E-02
Photosystem II D1 precursor processing protein PSB27-H2 PSB27-2 Cluster-5009.174 90 0.66 4.02E-03
Photosystem I P700 chlorophyll a apoprotein A1 psaA Cluster-5009.349 35 0.44 1.05E-02
Photosystem I P700 chlorophyll a apoprotein A1 psaA Cluster-5009.349 37 0.41 7.26E-05
Photosystem I P700 chlorophyll a apoprotein A2 psaB Cluster-5009.315 07 0.56 7.38E-12
Photosystem I P700 chlorophyll a apoprotein A2 psaB Cluster-5009.327 37 0.44 4.07E-03
Photosystem I P700 chlorophyll a apoprotein A2 psaB Cluster-5009.375 38 0.36 4.43E-02
Photosystem I iron–sulfur center psaC Cluster-5009.376 03 0.54 4.30E-04
Photosystem I reaction center subunit IV, chloroplastic PSAE Cluster-5009.347 70 0.64 9.04E-09
Photosystem I reaction center subunit III, chloroplastic PSAF Cluster-5009.326 42 0.73 3.42E-10
Photosystem I reaction center subunit XI, chloroplastic PSAL Cluster-5009.337 76 0.83 1.36E-03
Chlorophyll a-b binding protein of LHCII type I, chloroplastic N/A Cluster-5009.321 73 0.81 5.15E-05
Chlorophyll a-b binding protein CP29 lhcb4 Cluster-5009.332 23 0.80 2.83E-05
Chlorophyll a-b binding protein CP26, chloroplastic LHCB5 Cluster-5009.333 98 0.57 2.87E-32
Chlorophyll a-b binding protein CP26, chloroplastic LHCB5 Cluster-5009.549 68 0.23 2.51E-02
Chlorophyll a-b binding protein 6, chloroplastic LHCA1 Cluster-5009.331 26 0.69 1.51E-07
Chlorophyll a-b binding protein 4, chloroplastic LHCA4 Cluster-5009.319 77 0.71 2.49E-05
Chlorophyll a-b binding protein 4, chloroplastic LHCA4 Cluster-5009.334 08 0.75 1.22E-04
Chlorophyll a-b binding protein 8, chloroplastic CAB8 Cluster-5009.547 95 0.18 4.11E-02
Chlorophyll a-b binding protein type 1 member F3 CABF3 Cluster-5009.303 71 0.80 7.19E-05
Cytochrome b6-f complex iron–sulfur subunit, chloroplastic petC Cluster-5009.375 83 2.68 1.17E-52
Cytochrome b6-f complex iron–sulfur subunit 1, chloroplastic petC1 Cluster-5009.530 73 3.36 1.58E-02
Cytochrome b6-f complex iron–sulfur subunit 1, chloroplastic petC1 Cluster-5009.510 43 1.60 4.94E-02
Carotene biosynthesis
phytoene synthase PSY Cluster-5009.458 75 1.56 3.55E-02
phytoene desaturase PDS Cluster-5009.394 20 0.69 1.64E-03
PDS Cluster-5009.356 53 0.77 1.98E-03
Zeta-carotene desaturase ZDS Cluster-5009.344 71 0.71 4.50E-08
Lycopene beta cyclase LCY Cluster-5009.342 18 1.46 1.13E-10
LCY Cluster-5009.367 96 3.29 3.57E-06
Carotene biosynthesis-related protein CBR Cluster-5009.342 02 2.44 4.81E-55
CBR Cluster-5009.372 58 3.31 9.73E-17
CBR Cluster-5009.351 05 2.66 3.28E-11
CBR Cluster-5009.342 77 1.33 1.37E-07
CBR Cluster-5009.299 00 1.36 7.63E-05
CBR Cluster-5009.235 56 13.86 1.52E-02
Carotene globule protein N/A Cluster-5009.183 93 1.87 8.02E-15
N/A Cluster-5009.258 66 1.60 3.90E-02
Beta-carotene isomerase D27 D27 Cluster-5009.117 93 2.55 1.15E-04
D27 Cluster-5009.117 94 3.72 7.38E-03
Fatty acid biosynthesis
Acetyl-CoA carboxylase ACC Cluster-5009.351 90 2.62 1.88E-35
ACC Cluster-5009.383 01 1.45 1.61E-06
ACC Cluster-5009.337 60 2.17 1.58E-40
ACC Cluster-5009.341 11 1.27 1.19E-04
ACC Cluster-5009.302 40 1.24 1.30E-03
Malonyl-CoA-acyl carrier protein transacylase MCAT Cluster-5009.345 91 1.42 1.43E-11

© 2019 Japanese Society of Phycology

Gene expression during photoacclimation 9

Table 2. Continued

Gene name Abbreviation Gene_id Fold change padj

MCAT Cluster-5009.238 50 1.46 1.12E-07

3-Oxoacyl-[acyl-carrier-protein] reductase KAR Cluster-5009.184 11 2.28 6.70E-07
Hydroxyacyl-ACP dehydrogenase HD Cluster-5009.304 29 1.42 2.75E-05
Long chain acyl-CoA synthetase LACS Cluster-5009.334 51 2.47 1.27E-77
LACS Cluster-5009.393 13 2.15 2.03E-28
LACS Cluster-5009.323 29 2.04 1.52E-11
LACS Cluster-5009.323 30 1.71 1.78E-07
TAG biosynthesis
Glycerol-3-phosphate acyltransferase GPAT Cluster-5009.237 01 2.00 1.59E-04
GPAT Cluster-5009.405 86 2.73 2.11E-04
Diacylglycerol acyltransferase DGAT Cluster-5009.330 90 2.09 8.72E-08
DGAT Cluster-5009.310 48 1.34 1.18E-05
DGAT Cluster-5009.391 11 4.96 3.33E-03
Phospholipid: diacylglycerol acyltransferase PDAT Cluster-5009.438 74 1.36 3.37E-02
ROS- scavenging enzymes
Superoxide dismutase SOD Cluster-5009.374 37 2.59 1.90E-62
SOD Cluster-5009.349 76 1.49 9.26E-07
SOD Cluster-5009.328 09 7.51 3.29E-05
SOD Cluster-5009.350 43 2.95 2.62E-04
SOD Cluster-5009.369 47 1.85 7.70E-03
Catalase CAT Cluster-5009.322 43 1.70 5.80E-24
Glutathione reductase GR Cluster-5009.262 42 2.05 2.33E-05
Monodehydroascorbate reductase MDHAR Cluster-5009.312 40 3.98 2.41E-12
Dehydroascorbate reductase DHAR Cluster-5009.389 50 1.81 4.89E-06
DHAR Cluster-5009.323 17 1.34 2.82E-04
Glutathione peroxidase GPX Cluster-5009.348 87 1.31 3.74E-06
Xanthophyll cycle
Violaxanthin de-epoxidase VDE Cluster-5009.351 06 1.55 4.12E-09
Zeaxanthin epoxidase ZEP Cluster-5009.511 94 3.23 3.58E-08
ZEP Cluster-5009.417 90 1.31 2.67E-04

growth; involving upregulation of repairing and replacing of
reaction center proteins, and reactive oxygen species scaveng-
ing enzymes.
The D1 protein is one of the most important components
of the PS II complex, and it is also the direct target of singlet
oxygen. Specific proteases degraded the photodamaged D1
protein and the de novo synthesized D1 protein was inserted
into the reaction centers, after which the PS II complex was
reassembled and reactivated (Kim et al. 1993). We found
that PS II D1 and D2 genes were upregulated at 1500 μmol
photons
m−2s−1 (Table S2), suggesting a rapid turnover of
reaction center proteins under high irradiance conditions.
Early light-inducible proteins (ELIP) are some of the first
proteins to be induced under light stress (Harari-Steinberg
et al. 2001). In our study, expression of the Cbr protein, the
algal homolog of ELIP, was upregulated at 600 and
1500 μmol photons
m−2s−1.
Exposure to high irradiance would result in over-reduction
of the photosynthetic electron transport chain, which would
generate excess reactive oxygen species (ROS) and damage
the photosystem (Foyer and Mullineaux 1994). Algal cells had
developed defensive mechanisms to reduce oxidative stress
by inhibiting ROS production or improving activities of ROS
scavenging enzymes.
In our study, the expression of multiple genes coding for
ROS scavenging enzymes was detected at 600 μmol photo-
ns
m−2s−1: superoxide dismutase (SOD), catalase (CAT),
ascorbate peroxidase (APX), glutathione reductase (GR), mon-
odehydroascorbate reductase (MDHAR), dehydroascorbate
reductase (DHAR), and glutathione peroxidase (GPX). At
600 μmol photons
m−2s−1, we detected the upregulated
expression of a single gene for GPX and MDHAR. The
upregulated expression of five genes for SOD, four genes for
GR, DHAR and CAT were also detected (Table 2). At
1500 μmol photons
m−2s−1, eight genes of ROS scavenging
enzymes were detected, and were upregulated approximately
1.14–3.13-fold (Table S2). It seems that at high radiance
levels there is an increase in abundance of ROS scavenging
enzymes to cope with oxidative stress.
Low to high light shift: changes of carotenoid
biosynthesis
Carotenoids are anti-oxidative compounds that accumulate
under light stress in D. salina (Lamers et al. 2010). In our
study, the expression of genes of carotene biosynthesis
increased at 600 μmol photons
m−2s−1. Pick et al. (1986)
found that PSY and PDS were upregulated in response to high
light intensity; while Rabbani et al. reported that the expres-
sion PDS remained constant at high radiance levels (Pick
1998; Rabbani et al. 1998). These conflicting transcriptional
findings on carotene biosynthesis may be strain-dependent, or
caused by different working time (Zamani and Moradshahi
2016). Carotenoid biosynthesis related genes were all
upregulated in 24 h (Fig. S4), whereas the expression of PDS
and ZDS were downregulated at 168 h in transcriptome
results. This inconsistency of gene expression was also

© 2019 Japanese Society of Phycology

10 Y. Li et al.

Fig. 5. Metabolic pathways of carotenoids, lipids, and triacylglycerol. Gray circles indicate significantly upregulated genes identified at
600 μmol photons
m−2s−1. PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, zeta-carotene desaturase; LCYB, lycopene beta-
cyclase; CHYB, carotenoid hydroxylase; ZED, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; ACC, acetyl-CoA carboxylase; MCAT,
malonyl-CoA: ACP transacylase; KAS, 3-ketoacyl-ACP-synthase; KAR, 3-ketoacyl- ACP-reductase; HD, 3-hydroxyacyl-ACP dehydratase;
ENR, enoyl-ACP reductase; GK, glycerol kinase; GPAT, glycerol-3-phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase;
PAP, phosphatidic acid phosphatase; DGAT, diacylglycerol acyltransferase; PDAT, phospholipid: diacylglycerol transferase.

observed by Zamani and Moradshahi (2016), that the expres-
sion of PDS was upregulated in 3–12 h, and downregulated at
48 h. The phenomenon of inconsistent carotene accumulation
and gene expression was observed in previous reports
(Rabbani et al. 1998; Sánchez-Estudillo et al. 2006),
suggesting that carotene accumulation may be regulated by
various factors.
It has been shown that the generation of carotenes is
related to ROS (Shaish et al. 1993), which might serve as a
signal that stimulates biosynthesis of β-carotene. We found a
positive correlation between ROS scavenging genes and caro-
tene biosynthesis genes under high-light stress, indicating a
relationship between ROS and carotenes.
Carotene and xanthophyll are two main classes of naturally
occurring carotenoids. Apart from being part of the LHC, xan-
thophyll, especially Zx, participates in non-photochemical
quenching process at high light level during photosynthesis
(Jin et al. 2003). The de-epoxidation of xanthophyll is an
important photoprotective mechanism induced by light
(Phillips et al. 1995). In our study, both zeaxanthin epoxidase
and violaxanthin de-epoxidase were found, and all genes iden-
tified as violaxanthin de-epoxidase were upregulated at
600 and 1500 μmol photons
m−2s−1 (Table 2 and S2). In
general, the xanthophyll cycle is a short-term adaptation
mechanism of plants. Evergreen plants also exhibit increased
de-epoxidation during long-term adaptation in winter (Gilmore
1997). Low temperature and strong light decrease electron
transfer efficiency (Maxwell et al. 1995). Similarly, to low
temperature, strong light generates large amounts of Zx.
Low to high light shift: changes in TAG
metabolism
Under stress conditions such as nitrogen (N) deprivation,
microalgae synthesized large amounts of TAG (Hu et al.
2008; Slocombe et al. 2015). Pick and Avidan found that
TAG accumulated during N deprivation in D. tertiolecta, and
around two-thirds of the carbon in total TAG originated from
starch, one-third was made de novo by CO2 assimilation
(Pick and Avidan 2017). In this study, the N in culture
medium was measured on the seventh day, and no N starva-
tion was observed (Table S3), indicating that the accumula-
tion of neutral lipid was induced by high light instead of N
deficiency.
In this study, the high light induced accumulation of TAG
in D. salina (Fig. 1c) was consistent with previous findings
(Roessler 1990). Increased levels of TAG under high light was
also observed in Nannochloropsis sp. and Scenedesmus
sp. (Sukenik et al. 1989; Liu et al. 2012). TAG is the main
composition of carotene-rich lipid droplets (Davidi et al.
2012) where accumulated β-carotene was stored in D. salina.
Expression of TAG-related genes indicated the
interdependence between TAG and carotene accumulation
under high light. This was confirmed by a positive correlation
between β-carotene accumulation and TAG biosynthesis in
this study (Figure 1c and Table 1) and previous results
(Rabbani et al. 1998). Consequently, the overproduction of
carotene required TAG-rich lipid droplets as a plastid-
localized sink.

© 2019 Japanese Society of Phycology

Gene expression during photoacclimation
In conclusion, under high light stress, there existed a com-
plicated long-term photoacclimation mechanisms in D. salina
including readjustment of size and pigment composition of
photosynthesis apparatus, changes in gene expression of pho-
tosystem and carotenoid biosynthesis, and activation of photo-
damage repair. The D. salina could cope with long-term
fluctuated light by readjusting the light-harvesting antenna,
altering electron transfer, and quenching excess light energy
to balance absorption and utilization of light. Additionally, the
algal flexibility in coping with short-term high light stress is
also helpful in understanding its photoacclimation. Therefore,
future studies on photoacclimation of D. salina should focus
on responses in short time scales, such as changes in the size
of light-harvesting antenna and excess energy dissipation
(non-photochemical quenching) mechanism.
ACKNOWLEDGMENTS
This work was financially supported by National Natural Sci-
ence Foundation of China (41506188), China Nantong
Municipal Applied Basic Research Program
(MS12017025-2), and Tianjin Demonstration Project for
Innovative Development of Marine Economy (BHSF2017-21).
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Table S1. List of primers used in the real-time quantitative
PCR analysis.
Table S2. mRNA-Seq data for the genes involved at 1500 μmol
photons
m−2s−1 in D. salina.
Table S3. Residual nitrate concentration in culture medium on
the seventh day.
Fig. S1. The internal transcribed spacer (ITS) rRNA phyloge-
netic tree for Dunaliella strains and a marine diatom outlier.
Fig. S2. KOG functional classification of all transcripts.
Fig. S3. Relative expression level of genes measured by quan-
titative real-time PCR.
Fig. S4. Relative expression level of carotenoid biosynthesis
genes in 24 h measured by quantitative real-time
PCR (qPCR).
© 2019 Japanese Society of Phycology