Journal of Applied Phycology

https://doi.org/10.1007/s10811-021-02527-8

Weakened growth, cell division, and energy metabolism,

but enhanced resistance, signaling, and anabolism: responses of Ulva
prolifera to copper elucidated by omics
Chuner Cai1,2,3 · Xuanhong Liu1 · Hui Zhao1 · Ting Jiang1 · Rui Jia1,2 · Peimin He1,2,3

Received: 26 January 2021 / Revised and accepted: 9 June 2021

© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
Ulva spp. have outstanding survivability in copper-rich environments, but research into the interactions between these algae
and copper is required. This study combined transcriptomics, proteomics, and metabolomics to investigate the expression of
various metabolites in Ulva prolifera after exposure to 10 mg L ­ −1 of copper sulfate for 12 h. The results showed that copper
stress in U. prolifera mainly manifested as a reduction in transcription and translation, which was related to gene expression,
protein activity, and metabolite content in cell division and energy metabolism. However, the resistance, signal transduction,
and metabolism of U. prolifera were enhanced to respond actively to acute copper stress in many aspects. These findings
demonstrate the impact of 12 h of 10 mg ­L−1 copper sulfate exposure on U. prolifera.

Keywords Ulva prolifera · Copper · Transcriptomics · Proteomics · Metabolomics

Introduction

The genus Ulva comprises large marine benthic algae of the

family Ulvaceae, a typical representative of Chlorophyta
(Wichard et al. 2015). Many countries, such as the USA
(Nelson et al. 2003), France (Charlier et al. 2008), Finland
Chuner Cai and Xuanhong Liu contributed equally to this paper
(Gubelit et al. 2015), the Philippines (Largo et al. 2004), and
* Chuner Cai Japan (Yabe et al. 2009), have experienced ecological disas-
cecai@shou.edu.cn ters caused by the coastal blooming of Ulva spp., but the most
Xuanhong Liu famous is the green tide disaster that broke out in Qingdao on
m200401084@st.shou.edu.cn the eve of the 2008 Beijing Olympic Games (Sun et al. 2008a;
Hui Zhao Liu et al. 2009). At the same time, reports on the relationship
m190400974@st.shou.edu.cn between Ulva and the environment continued to emerge, and
Ting Jiang the characteristics of the genus, such as resistance to high
961352471@qq.com temperature, intense light, drying out, and high salt, have
Rui Jia been partly attributed to the formation of green tides (Zhang
rjia@shou.edu.cn et al. 2019). In its natural state, Ulva has a strong morphologi-
Peimin He cal plasticity (Tan et al. 1999). Its morphology is even more
pmhe@shou.edu.cn diverse in spontaneous or artificial mutants (Bryhni 1974;
1 Wichard 2015). Consequently, Ulva is considered a model
College of Marine Ecology and Environment, Shanghai
Ocean University, Shanghai 201306, China organism for studying morphogenesis (Loevlie 1964; Wichard
2 et al. 2015), and knowledge will continue to deepen with the
National Demonstration Center for Experimental Fisheries
Science Education (Shanghai, Ocean University), Shanghai, publication of the Ulva genome (Clerck et al. 2018).
China Between 1975 and 1990 the waters of Caleta Palito in
3
Co‑Innovation Center, Jiangsu Marine Bio-Industry northern Chile were contaminated by copper mines and all
Technology, Lianyungang, China invertebrates and most of the algae species disappeared.

13
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However, the rocks were occupied by large patches of
Ulva compressa. Research indicated that the U. compressa,
which survived in the area and became the dominant spe-
cies, was not genetically resistant to copper, but more likely
had adapted ecologically (Correa et al. 1996). Subsequently,
studies showed that Ulva spp. were significantly enriched in
copper (Andrade et al. 2004; Scoullos et al. 2004; Valdés
et al. 2018; Zúñiga et al. 2020) and their selective adsorp-
tion capacity for copper enabled them to become potential
environmental indicator organisms (Seeliger and Cordazzo
1982; Barraza and Carballeira 1999; Navarrete et al. 2019;
Siddiqui and Bielmyer-Fraser, 2019).
The mechanisms behind the response of Ulva cells to
copper ions are complex. Depending on the ATP produced
by light and photosynthesis, U. compressa activates several
transient receptor potential channels (TRP: A1/C5/M8/C4/
V1) in succession within 0–2 min after exposure to 10 μM
copper ion solution. This leads to calcium ions in the cul-
ture medium entering the cell and activating calmodulin-
and calcium-dependent protein kinases, which then leads to
the extracellular copper ions entering the cell through the
TRP channel (Gómez et al. 2015; 2016; 2017). The cop-
per entering the cell participates in the activation of L-type
voltage-dependent calcium channels, thereby inducing a
large amount of calcium ions in the medium to enter the
cell at over 12 h (Gonzalez et al. 2010; 2012; Gomez et al.
2016). These calcium ions, which converge from the culture
medium and endoplasmic reticulum to the cytoplasm, chlo-
roplast, and mitochondria, both promote the synthesis of
water and nitric oxide, which in turn induces the production
of ascorbate peroxidase, glutathione reductase, glutathione-
S-transferase, and other antioxidant enzymes (Gonzalez
et al. 2010; 2012).
The permeability of Ulva cells increases after the influx
of copper (Webster and Gadd 1996; Lee et al. 2005) and
alteration of the cytoplasmic composition (Andrade et al.
2004; Kováčik et al. 2018; Laporte et al. 2020). A high
concentration of copper can even severely affect the pho-
tosynthetic physiology (Andrade et al. 2004; Siddiqui
and Bielmyer-Fraser, 2019), growth (Lee et al. 2005), and
asexual reproduction (Han et al. 2009; Lee et al. 2019) of
Ulva. To resist copper damage, Ulva spp. relieves oxida-
tive damage via inherent antioxidants (Wu and Lee 2008;
Wu et al. 2009; Contreras-Porcia et al. 2011; Mellado et al.
2012; Kováčik et al. 2018; Navarrete et al. 2019) followed
by structural repair (Laporte et al. 2016; 2020; Rodriguez
et al. 2018). Excess copper is chelated by intracellular met-
allothionein (Laporte et al. 2016; Navarrete et al. 2019;
Zúñiga et al. 2020) and phytochelatin (Rijstenbil et al. 1998;
Mellado et al. 2012; Navarrete et al. 2019), isolated to the
vacuole and cell wall (Andrade et al. 2004; Lee et al. 2005;
Kováčik et al. 2018; Zeroual et al. 2020), excreted from
the cells (Navarrete et al. 2019; Celis-Plá et al., 2019), and
13
Journal of Applied Phycology
adsorbed by epiphytic bacteria (Andrade et al. 2004). The
basic outline of the interaction between Ulva and copper and
related mechanisms, has been gradually elucidated. How-
ever, a large number of important molecules that specifically
participate in this action, and their functions, remain to be
identified.
In some plants, 10 mg ­L−1 of copper is a critical threshold
value regarding impact. For example, the growth of maize
seedlings is promoted below this concentration and inhibited
above it (Huang 2006). Similarly, the germination rate, root
length, and bud length of chicory seeds decrease quickly
when the copper concentration increases from 4 to 10 mg
­L−1 and then slowly when increased above 10 mg L ­ −1 (Yang
2011). This concentration also applies to Microcystis copper
absorption (Yu et al. 1994). Regarding Ulva prolifera, when
the copper concentration increases from 1 to 10 mg L ­ −1, the
algae increase their copper adsorption, and content of solu-
ble sugar and protein then decreases when the concentration
is elevated from 10 to 20 mg ­L−1 (Guo et al. 2011). They
eventually become light green in 24 h (Sun et al. 2008b).
Therefore, 10 mg L ­ −1 of copper is a suitable concentration
for investigating molecular changes in U. prolifera.
Omics is currently the mainstream method for system-
atically studying the metabolites (metabolomics), transcrip-
tion factors (transcriptomics), and various stress-inducing
proteins (proteomics), among others, of plants under heavy
metal stress (Singh et al. 2016). This study focused on elu-
cidating the different expressions of various products in U.
prolifera after 12 h of copper sulfate stress, via transcrip-
tomics, proteomics, and metabolomics, for a comprehensive
understanding of the interactions among them.
Materials and methods
Samples and experimental treatment
Ulva prolifera was collected from the Qingdao sea area,
China (120°19’E, 36°04’N), in July 2008. The gametophyte
was characterized and sub-cultured as a pure-line progeny.
Samples of U. prolifera were primarily cultured in VSE
medium (Ott 1965) at 20 °C under a 12 h photoperiod and
photon flux of 120 μmol photons ­m−2 ­s−1. After 15 days,
240 healthy strains of U. prolifera thalli with similar lengths
were taken and divided into two groups, one without copper
addition (control) and another with 10 mg ­L−1 of copper sul-
fate for 12 h. Samples were dried with paper towels, frozen
in liquid nitrogen, and stored at − 80 °C. They were then
subjected to transcriptomics (three biological replicates in
both the copper-treated group and control group), proteom-
ics (three replicates), and metabolomics (six replicates), with
Journal of Applied Phycology
10 healthy thalli of U. prolifera (~ 1.2 g) in each replicate,
detailed in the following text.
Transcriptomic operations
Quantitative samples were grinded with liquid nitrogen to
extract the total RNA using total an RNA extractor (Trizol) kit
(Sangon Biotech, China), followed by concentration measuring
with the Qubit RNA HS assay kit (Life Technologies, China),
and integrity evaluation with agarose gel and a bioanalyzer
(Agilent 2100, USA) (RNA integrity number value ≥ 8.0). Con-
struction of the mRNA library was performed using VAHTS
mRNA-seq V2 Library Prep Kit for the Illumina kit (Vazyme
Biotech, China), which included mRNA isolation and frag-
mentation, double-stranded cDNA synthesis, end repair and
dA-tailing, adaptor ligation, purification, fragment sorting, and
finally cDNA library amplification. After accurate quantifica-
tion using a DNA detection kit (Life Technologies), each group
of cDNA was mixed at a 1:1 ratio for sequencing (Illumina
Hiseq high-throughput, USA).
Transcriptomic analysis
The sequence reads were trimmed, and bacterial sequences
were eliminated by aligning the reads to bacterial reference
genomes from the NCBI-RefSeq database. They were then
evaluated by FastQC (Schmieder and Edwards 2011), mass
sheared by Trimmomatic (Bolger et al. 2014), and aligned
by Trinity (Haas et al. 2013), followed by annotation using
NCBI ­Blast+ (Altschul et al. 1997) with multiple databases,
including Gene Ontology (GO) function annotation from
Swissprot and TrEMBL, and Kyoto Encyclopedia of Genes
and Genomes (KEGG) annotation from KAAS (Moriya
et al. 2007), as well as coding sequence prediction using
TransDecoder. The RNAseq was then evaluated using Bow-
tie2 (Langmead and Salzberg 2012) for mapping, RSeQC
(Wang et al. 2012) for redundant sequence distribution, and
BEDTools (Quinlan and Hall 2010) for uniformity distri-
bution checking and gene coverage. Gene expression level
and co-expression were analyzed using Salmon (Patro et al.
2017). Finally, the gene expression difference was analyzed
using DESeq2, while gene enrichment was analyzed using
topGO for GO enrichment, and ClusterProfiler for KEGG
pathway and EuKaryotic Orthologous Group classification
enrichment. Only functional categories and pathways with
a |Fold change|> 2 and q-values under a threshold of 0.05
were considered significant.
Proteomics operations
Total protein from samples in each replicate was prepared by
tricarboxylic acid cycle/acetone precipitation and SDT lysis
(Jorrin-Novo 2014), followed by separation with sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and
filter aided sample preparation digestion (Wiśniewski et al.
2009). Each fraction was injected for nano liquid chroma-
tography with tandem mass spectrometry (MS) analysis. The
peptide mixture was loaded onto a reverse phase trap col-
umn (Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18)
(Thermo Scientific, USA) connected to the C18-reversed
phase analytical column (Thermo Scientific Easy Column,
10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A
(0.1% formic acid) and separated with a linear gradient of
buffer B (84% acetonitrile and 0.1% formic acid) at a flow
rate of 300 nL m ­ in−1 controlled by IntelliFlow technology.
The linear gradient occurred over 2 h as follows: 0–55%
buffer B for 110 min, 55–100% buffer B for 5 min, and held
in 100% buffer B for 5 min. Liquid chromatography with
tandem MS analysis was then performed on a Q Exactive
mass spectrometer (Thermo Scientific) that was coupled to
an Easy nLC Liquid Chromatograph (Thermo Scientific) for
120 min. The mass spectrometer was operated in positive ion
mode. MS data were acquired using a data-dependent top 10
method, dynamically choosing the most abundant precursor
ions from the survey scan (300–1800 m/z) for higher-energy
collisional dissociation fragmentation. Automatic gain con-
trol target was set to ­3e6 and maximum inject time to 10 ms.
The dynamic exclusion duration was 40.0 s. Survey scans
were acquired at a resolution of 70,000 at m/z 200, and reso-
lution for higher-energy collisional dissociation spectra was
set to 17,500 at m/z 200, and the isolation width was 2 m/z.
The normalized collision energy was 30 eV and the underfill
ratio, which specifies the minimum percentage of the target
value likely to be reached at maximum fill time, was defined
as 0.1%. The instrument was run with peptide recognition
mode enabled.
Proteomic analysis
The MS data were analyzed using MaxQuant software
v1.5.3.30 (Max Planck Institute of Biochemistry, Martin-
sried, Germany) (Cox and Mann 2008), the main parameters
were as follows: enzyme, trypsin; minimal peptide length,
7; peptide-spectrum match- and protein-level false discov-
ery rates, 0.01; fixed modifications, carbamidomethyl (C);
variable modifications, oxidation (M) and acetyl (protein
N-term); and database, UPR.maker.pep.fa (7089 sequences).
Based on Welch’s t test, proteins of Fold Change ≥1.5 or
Fold Change ≤ 0.6 and p values < 0.05 were considered sig-
nificant. The protein sequences of differentially expressed
proteins were in batches retrieved from UniProtKB database
followed by searching against the SwissProt database using
the NCBI BLAST + (Altschul et al. 1997) to find homolog
sequences in which the top 10 blast hits with E-value less
than ­1e−3 for each query sequence were retrieved and loaded
into Blast2GO (Götz et al. 2008) for GO mapping and
13

annotation. KEGG pathway annotation was performed using
the KEGG automatic annotation server (Moriya et al. 2007).
To further explore the impact of differentially expressed
protein in cell physiological processes and identify inter-
nal relationships between differentially expressed proteins,
enrichment analysis was performed. GO enrichment on three
ontologies (biological process, molecular function, and cel-
lular component) and KEGG pathway enrichment analyses
were applied based on Fisher’s exact test, considering the
whole quantified protein annotations as the background data-
set. Benjamini–Hochberg correction for multiple testing was
further applied to adjust derived p values. Only functional
categories and pathways with p values under a threshold of
0.05 were considered significant.
Metabolomic operations
Samples in each replicate was grinded with liquid nitrogen,
then added to 1 mL of pre-cooled methanol–acetonitrile
aqueous solution (v / v, 2: 2: 1) and vortexed. This was fol-
lowed by ultrasound twice at 30 min, storing at − 20 °C for
60 min and centrifugation at 13,000 × g for 15 min at 4 °C.
The supernatant was freeze dried before resolving with 100
μL acetonitrile aqueous solution (acetonitrile: water = 1:1,
v/v). The solution was centrifuged again for 15 min to retain
the supernatant.
The supernatant was then analyzed using an ultra-
high-pressure liquid chromatography (1290 Infinity liquid
chromatograph, Agilent Technologies) (Ivanisevic et al.,
2013) coupled to a quadrupole time-of-flight (AB Sciex
TripleTOF 5600). For hydrophilic interaction liquid chro-
matography separation, samples were analyzed using a
2.1 mm × 100 mm ACQUIY UPLC BEH 1.7 µm column
(Waters, USA). The column temperature was 25 °C, with
flow rate of 0.3 mL ­min−1 and injection volume of 2 μL.
In both ESI positive and negative modes, the mobile phase
contained buffer A (25 mM ammonium acetate and 25 mM
ammonium hydroxide in water) and buffer B (acetonitrile).
The gradient was 95% B for 1 min and was linearly reduced
to 65% in 14 min and then was reduced to 40% in 2 min and
held for 2 min and then increased to 95% in 0.1 min, with a
4.9 min re-equilibration period employed.
Samples dispersed from ultra-high-pressure liquid chro-
matography were then analyzed using a Triple TOF 5600
mass spectrometer (Applied Biosystems, USA). The ESI
source conditions were set as follows: ion source gas 1 as
60, ion source gas 2 as 60, curtain gas as 30, source tem-
perature at 600 °C, and IonSpray Voltage Floating ± 5500 V.
In MS only acquisition, the instrument was set to acquire
over the m/z range 60–1000 Da, product ion scan m/z range
25–1000 Da, TOF MS scan accumulation time 0.20 s/spec-
tra, and product ion scan accumulation time 0.05 s/spectra.
In auto tandem MS acquisition, the product ion scan was
13
Journal of Applied Phycology
acquired using information-dependent acquisition with high
sensitivity mode selected. The parameters were set as fol-
lows: collision energy was fixed at 35 ± 15 eV; declustering
potential ± 60 V; exclude isotopes within 4 Da; and candi-
date ions to monitor per cycle: 6.
Metabolomic analysis
The raw MS data (wiff.scan files) were converted to.mzXML
files using ProteoWizard MSConvert before importing into
freely available XCMS software. For peak picking, the fol-
lowing parameters were used: centWave m/z = 25 ppm,
peak width = c (10, 60), and prefilter = c (10, 100). For peak
grouping, bw = 5, mzwid = 0.025, and minfrac = 0.5 were
used. In the extracted ion features, only the variables with
more than 50% of the nonzero measurement values in at
least one group were retained. Compound identification of
metabolites was performed by comparing of accuracy m/z
value (< 25 ppm) and tandem MS spectra with an in-house
database established with available authentic standards
(Benton et al., 2015).
After normalization to total peak intensity, the processed
data were uploaded into SIMCA-P v.14.1 (Umetrics, Swe-
den), where it was subjected to multivariate data analy-
sis, including pareto-scaled principal component analysis,
partial least-squares discriminant analysis (PLS-DA), and
orthogonal partial least-squares discriminant analysis
(OPLS-DA). The sevenfold cross-validation and response
permutation testing was applied to evaluate the robustness
of the model (Fan et al. 2021). The variable importance in
the projection value of each variable in the OPLS-DA model
was calculated to indicate its contribution to the classifica-
tion. Metabolites with |Fold change|> 2 and p values less
than 0.05 were considered statistically significant. KEGG
pathway annotation and enrichment analysis were performed
per analysis in the metabolome.
Results
General transcriptomic data
In the clean data from the raw data sheared by Trimmomatic,
the average read length of each sample was > 143 bps, with
a total read length > 44 Mb, a base amount > 6.4 Gb, and
a GC ratio > 59%. The Q30 (96.44–97.3%) and Q10 ratio
(mostly > 99.99%) indicated good data quality (Table S1).
Transcripts were de novo assembled by clean data using
Trinity, and the unigene was usually the longest transcript in
each redundant transcript cluster. A total of 28,526 unigenes
were obtained, with an average length of 1401 bp, wherein
the longest one was 30,777 bp (Table S2). After compari-
son, 1558 unigene sequences were annotated according to
Journal of Applied Phycology
databases of NR, KEGG, SwissProt, and EuKaryotic Orthol-
ogous Group, and the numbers of annotated genes were
8482, 1781, 8173, and 6093, respectively (Fig. S1). There
were 196 genes in U. prolifera that were significantly upreg-
ulated in expression (|Log2 |Fold Change| |>1, and p < 0.05)
under copper stress, and 199 genes that were downregulated.
General proteomic data
Mass spectrum data were identified using the Androm-
eda engine integrated by Max Quant. There were 11,842
identified peptides that assembled into 1844 proteins
(Table S3). Unique peptides, which usually characterize
protein, were mainly distributed among numbers 1–3. The
molecular weight of annotated proteins was mainly between
10 and 60 kDa, among which 20–30 kDa were dominant.
There were 82 proteins that were significant differentially
expressed (Fold Change ≥1.5 or Fold Change ≤ 0.6, and
p < 0.05) between the two groups, of which half were upreg-
ulated and half were downregulated (Fig. S2). Data from
the transcriptome and proteome were aligned and analyzed.
There were 941 genes (8.56% of the total annotated genes
in the transcriptome) responding to 860 proteins (38.63%
of the total annotated proteins in the proteome) separately,
which showed the same accession number by annotation in
the Swissprot database.
General metabolomic data
The chromatographic peak’s response intensity and retention
time in the positive and negative ion mode of the quality
control samples in the metabolome overlapped. SIMCA-P
software was used for principal component analysis revealed
that the clustering effect and separation trend between
groups of the positive ion model were more obvious than
that of the negative ion model (Figs. S3 and S4). All PLS-
DA and OPLS-DA models showed higher R ­ 2Y (> 0.9) and
­Q2 (> 0.8) values (Fig. S4). In addition, the Q2 intercepts of
the PLS-DA (cation) and OPLS-DA (anion) model replace-
ment test graphs were both below zero, confirming the effec-
tiveness of these two models in distinguishing different cate-
gories and predicting new samples (Figs. S4–S8). According
to the variable importance in the projection (VIP) obtained
by the OPLS-DA model, a single statistical analysis was
performed to screen and identify 339 different metabolites
(VIP > 1 and p < 0.01), including 330 significantly differ-
ent metabolites (VIP > 1 and p < 0.05) (238 upregulated, 92
downregulated).
Resistance of Ulva prolifera to copper stress
According to the transcriptome, many genes related to resist-
ance were upregulated. The first group was involved in innate
immunity (e.g., NLRC5, scavenger receptor cysteine-rich
domain superfamily protein), enhancing defensive activ-
ity through calmodulin binding (e.g., Mildew Locus O-like
protein), general defense protein (e.g., ATP-binding cassette
[ABC] transporter G family member 31), and cell death
response (e.g., metacaspase-1 and cysteine-rich RLK). The
second group was related to repairing, including structural
reorganization (e.g., neurotrypsin, ATP-dependent zinc metal-
loprotease FtsH), RNA and DNA repairing (e.g., DNA exci-
sion repair protein ERCC-8, E3 ubiquitin ligase COP1, UV-
stimulated scaffold protein A homolog), and protein repairing
(e.g., protein-L-isoaspartate O-methyltransferase 2), while
those coding structural maintenance of chromosomes flexible
hinge domain-containing protein 1 (inhibiting homologous
recombination repair) were significantly downregulated. The
third group was associated with antioxidants, including those
involving peroxides (e.g., peroxide C1773.02c, protein anon-
37Cs, FAD dependent oxidoreductase), redox homeostasis
(e.g., photosystem II 22 kDa protein), and stress-mediated
response to hydrogen peroxide (e.g., glutamate–glyoxylate
aminotransferase 1). The final group was related to elimi-
nation reactions, e.g., protein phosphatase 2C 11 (metal ion
binding), and some of these were even extremely significantly
different, e.g., ariadne-like RING finger protein R811 (metal
ion binding) and protein VMS1 (involved in the endoplasmic
reticulum-associated degradation pathway) (Table1).
In the proteome, U. prolifera was resistant to copper
stress through protein enrichment, including heat shock pro-
tein 90.1, phospholipase D gamma 2 and obg-like ATPase
1 (involved in immune response), animal heme peroxidase
homolog, acylamino-acid-releasing enzyme and peroxire-
doxin Q (all in response to oxidative stress), persulfide diox-
ygenase ETHE1 homolog (detoxification), and glycine-rich
RNA-binding protein 2 (RNA transcription or processing
during stress). However, some proteins associated with anti-
oxidants (e.g., glutathione S-transferase DHAR2, mannose-
1-phosphate guanylyltransferase 2) and excretion (e.g., ABC
transporter C family member 2, transmembrane 9 superfam-
ily member 7) were downregulated.
It appears that U. prolifera adapts to acute copper stress
gradually according to its metabolome. Osmotic pressure
was regulated by an increase in proline (temporary response
of plants to stress via osmotic regulation, protein protec-
tion, membrane structure stability, ammonia enrichment, and
NAD and NADP providers), with its precursor of synthesis
(e.g., L-glutamic acid) and derivatives (e.g., L-pyroglutamic
acid), and betaine (long-term response of plants to stress via
osmotic regulation, protein protection, and membrane anti-
oxidants). However, oxidative damage occurred, which was
indicated by an increase in 2-hydroxyadenine (product from
DNA oxidation) and a decrease in docosahexaenoic acid
(highly unsaturated fatty acid). Therefore, myristic acid was
increased to participate in the biosynthesis of unsaturated
13
 Journal of Applied Phycology

Table 1  Fluctuation of molecules in Ulva prolifera related to resistance after 12 h of exposure to 10 mg ­L−1 copper sulfate
Transcript ­IDa Annotation Accession No Fold change

TRINITY_DN6409_c0_g2 NLRC5 sp|Q86WI3 2.7

TRINITY_DN6739_c1_g3 Scavenger receptor cysteine-rich domain Superfamily protein sp|F7J220 3.7
TRINITY_DN6228_c0_g2 MLO-like protein tr|A4S677 2.8
TRINITY_DN3532_c0_g1 ABC transporter G family member 31 sp|Q7PC88 7.7
TRINITY_DN6361_c1_g1 Metacaspase-1 sp|Q7XJE6 5.3
TRINITY_DN5732_c0_g1 Cysteine-rich RLK IPR001245 1.3
TRINITY_DN6665_c2_g11 Neurotrypsin sp|P56730 2.2
TRINITY_DN6064_c2_g4 ATP-dependent zinc metalloprotease FtsH sp|O82150 2.1
TRINITY_DN4689_c0_g1 DNA excision repair protein ERCC-8 sp|Q13216 2.4
TRINITY_DN6692_c4_g3 E3 ubiquitin ligase COP1 sp|P43254 2.0
TRINITY_DN3255_c0_g1 UV-stimulated scaffold protein A homolog sp|Q9M358 2.0
TRINITY_DN5214_c0_g1 Protein-L-isoaspartate O-methyltransferase 2 sp|O27962 2.5
TRINITY_DN6179_c0_g4 Chromosomes flexible hinge domain-containing protein 1 sp|Q6P5D8 − 2.3
TRINITY_DN3517_c0_g1 Peroxide C1773.02c sp|O94561 2.2
TRINITY_DN5677_c0_g2 Protein anon-37Cs sp|O96570 2.4
TRINITY_DN5652_c0_g1 FAD dependent oxidoreductase IPR006076 2.0
TRINITY_DN3453_c0_g1 Photosystem II 22 kDa protein sp|Q9SMB4 2.5
TRINITY_DN530_c0_g1 Glutamate–glyoxylate aminotransferase 1 sp|Q9LR30 2.2
TRINITY_DN3658_c0_g1 Protein phosphatase 2C 11 sp|Q8VZN9 2.4
TRINITY_DN4973_c0_g3 Ariadne-like RING finger protein R811 sp|Q5UQ35 13.6
TRINITY_DN5054_c0_g1 Protein VMS1 sp|Q04311 14.2
TRINITY_DN6665_c2_g4 Malignant brain tumors 1 sp|Q9UGM3 2.2
Protein ­IDb Annotation Accession No Fold change
Maker03253 heat shock protein 90.1 IPR019805 1.9
Maker04122 Phospholipase D gamma 2 sp|Q9T051 2.0
Maker05396 Obg-like ATPase 1 sp|Q9SA73 2.5
Maker01999 Animal heme peroxidase homolog - 3.1
Maker02742 Acylamino-acid-releasing enzyme sp|Q84LM4 3.4
Maker05606 Peroxiredoxin Q sp|Q75SY5 2.1
Maker04445 Persulfide dioxygenase ETHE1 homolog sp|Q9C8L4 2.6
Maker05323 Glycine-rich RNA-binding protein 2 sp|Q9SVM8 1.8
Maker0494 Glutathione S-transferase DHAR2 sp|Q9FRL8 0.4
Maker00988 Mannose-1-phosphate guanylyltransferase 2 sp|Q941T9 0.6
Maker06519 ABC transporter C family member 2 sp|Q42093 0.5
Maker05807 Transmembrane 9 superfamily member 7 sp|Q9LIC2 0.3
Metabolites ­IDc Description Charge p value Fold change
M114T590 Proline - < 0.05 2.9
M118T553 Betaine + < 0.05 1.2
M147T712 Pyroglutamic acid + < 0.01
M179T504 D-tagatose - < 0.05 1.6
M227T79 Myristic acid - < 0.05 1.5
M105T606 Glyceric acid - < 0.05 1.9
a
1 | Log2 |Fold Change ||< 2 indicates a significant difference in gene expression (q-value [modified p-value after multiple hypothesis
tests] < 0.05), while |Log2 |Fold Change ||≥ 2 indicates an extremely significant difference in gene expression. n = 3 biologically independent
experiments
b
Fold Change ≥ 1.5 indicates that protein increased significantly (p value < 0.05), while Fold Change ≤ 0.6 indicates a significant decrease in pro-
tein. n = 3 biologically independent experiments
c
Fold Change > 1 indicates an increase in protein, while Fold Change (FC) < 1 indicates a decrease. p value < 0.05 means a significant difference,
p value < 0.01 means an extremely significant difference. n = 6 biologically independent experiments. “ + ” means cation of metabolite, while “-”
means anion of metabolite. In all selected metabolites above, the VIP score > 1.0
The description of “a, b, c” also applies to Tables 2–5

13
Journal of Applied Phycology

fatty acids for membrane repairing. Furthermore, glyceric
acid was used for detoxification by accelerating the oxidative
metabolism of ethanol and acetaldehyde.
Overall, according to the three omics above, it was indi-
cated that U. prolifera developed a resistance to acute copper
stress in many aspects.
Signal transduction of Ulva prolifera under copper
stress
Signal transduction-related genes were also upregu-
lated in response to resistance. Some signal receptors
were located on the cell surface, e.g., serine/threonine-
protein phosphatase 7 (cryptochrome that induces the
expression of heat shock proteins); and some were inside
the cell, including voltage-dependent N-type calcium
channel subunit alpha-1B (calcium ion transport), chlo-
ride channel protein CLC-e (chloride ion channel), and
sodium- and chloride-dependent gamma aminobutyric
acid transporter 2 (sodium channel). Others were related
to second messengers that convert extracellular signals
into intracellular signals, including calcium-dependent
protein kinase 1/3/30/34, mildew resistance locus pro-
tein (calcium signaling system), and RAF proto-oncogene
serine/threonine-protein kinase (initiating a mitogen-
activated protein kinase). Some were compounds that
acted as intracellular messengers, such as polyamine
transporter At1g31830 (polyamine) and isochorismate
synthase 1 (synthase of salicylic acid required for both
local and systemic acquired resistance). Meanwhile,

Table 2  Fluctuation of molecules in Ulva prolifera related to signal transduction after 12 h of exposure to 10 mg ­L−1 of copper sulfate
Transcript ­IDa Annotation Accession No Fold change

TRINITY_DN5765_c3_g1 Serine/threonine-protein phosphatase 7 sp|Q9FN02 2.4

TRINITY_DN4434_c0_g2 Voltage-dependent N-type calcium channel subunit alpha-1B sp|Q00975 6.9
TRINITY_DN4144_c0_g1 Chloride channel protein CLC-e sp|Q8GX93 2.8
TRINITY_DN6517_c0_g1 Sodium- and chloride-dependent GABA transporter 2 sp|A5PJX7 2.6
TRINITY_DN5948_c3_g9 Calcium/calmodulin-dependent protein kinase I sp|P27466 3.2
TRINITY_DN4928_c1_g1 Calcium-dependent protein kinase 3 sp|Q42479 2.1
TRINITY_DN6684_c0_g4 Calcium-dependent protein kinase 30 sp|Q9SSF8 5.0
TRINITY_DN5864_c0_g2 Calcium-dependent protein kinase 34 sp|Q3E9C0 3.8
TRINITY_DN3938_c0_g2 Protein MLO sp|P93766 3.3
TRINITY_DN6427_c3_g2 RAF proto-oncogene serine/threonine-protein kinase sp|P05625 2.2
TRINITY_DN6198_c2_g1 Polyamine transporter At1g31830 sp|Q9C6S5 8.4
TRINITY_DN5272_c0_g1 Isochorismate synthase 1 sp|Q9S7H8 2.1
TRINITY_DN5512_c0_g3 Protein-tyrosine-phosphatase MKP1 sp|Q9C5S1 − 6.2
TRINITY_DN5960_c0_g1 Serine/threonine-protein kinase ULK3 sp|D3ZHP7 7.7
TRINITY_DN6370_c0_g4 Serine/threonine-protein kinase PAK 7 sp|Q8C015 2.1
TRINITY_DN5897_c0_g1 Glucan 1,3-alpha-glucosidase sp|Q9FN05 3.2
TRINITY_DN4348_c0_g1 Riboflavin biosynthesis protein PYRR​ sp|Q9STY4 2.8
TRINITY_DN4380_c1_g1 Ethylene-responsive transcription factor ERF112 sp|P93007 2.0
TRINITY_DN6688_c1_g1 GTP-binding protein 1 sp|Q5R8Q7 2.4
TRINITY_DN5853_c1 Glutamate receptor 1 sp|P34299 3.0
TRINITY_DN6367_c2_g3 Serine/threonine-protein kinase SRK2D sp|Q39192 2.1
TRINITY_DN6245_c0_g1 8-amino-7-oxononanoate synthase sp|Q8GW43 − 8.4
TRINITY_DN5654_c0_g1 SAL1 phosphatase sp|Q42546 − 2.3
Protein ­IDb Annotation Accession No Fold change
Maker04327 Serine/threonine-protein kinase SAPK10 sp|Q75H77 2.5
Maker05358 Brefeldin A-inhibited guanine nucleotide-exchange protein 3 sp|Q9LPC5 5.2
Maker03329 Magnesium protoporphyrin IX methyltransferase sp|Q9SW18 0.6
Maker05329 Calcium-dependent protein kinase 22 sp|Q9ZSA3 0.4
Metabolites ­IDc Description Charge p value Fold change
M146T762 L-Glutamate - < 0.05 1.3
M296T111 S-methyl-5’-thioadenosine - < 0.05 2.2
M179T754 Myo-inositol - < 0.05 1.5
M243T296 Uridine - < 0.05 1.5

13

protein-tyrosine-phosphatase MKP1, a negative regula-
tor that modulates defense response by repressing sali-
cylic acid production, was extremely downregulated. In
addition, genes involved in a reversible phosphorylation
system of proteins downstream of second messengers,
such as serine/threonine-protein kinase ULK3 (a regula-
tor of autophagy), serine/threonine-protein kinase PAK
7 (playing a role in cytoskeleton regulation, cell migra-
tion, and proliferation or cell survival), were signifi-
cantly upregulated. Moreover, genes involved in defense
signaling were also overexpressed, including glucan
1,3-alpha-glucosidase (involved in defense response to
pathogen-associated molecular patterns), riboflavin bio-
synthesis protein PYRR (involved in riboflavin biosyn-
thesis pathway), ethylene-responsive transcription factor
ERF112 (involved in the regulation of gene expression),
GTP-binding protein 1 (regulation of circadian mRNA
stability), glutamate receptor 1 (controlling movement in
response to environmental factors), and serine/threonine-
protein kinase SRK2D (key component and activator of
the abscisic acid signaling pathway). However, 8-amino-
7-oxononanoate synthase (enzyme that synthesizes biotin
to avoid activation of defense signaling) and SAL1 phos-
phatase (negative regulator of stress response genes) were
downregulated (Table 2).
Signal transduction-related proteins actively responded
to copper damage. For example, serine/threonine-protein
kinase SAPK10 (accelerating the abscisic acid-activated
signaling pathway) and brefeldin A-inhibited guanine nucle-
otide-exchange protein 3 (regulation of ADP-ribosylation
factor signal transduction involved both in nuclear division
and fusion phase) both increased. Simultaneously, magne-
sium protoporphyrin IX methyltransferase (inhibitor of pho-
tosynthetic gene expression) was down-regulated. However,
calcium-dependent protein kinase 22 (involves calcium as
a second messenger in signal transduction pathways) also
decreased, which was the opposite of that observed in the
transcriptome.
Signal transduction-related metabolites increased to
respond actively to the damage. e.g., L-glutamate (activation
of gamma aminobutyric acid branch, carbon and nitrogen
metabolism, and salicylic acid pathway, while inhibiting the
ethylene/jasmonic acid pathway), S-methyl-5’-thioadenosine
(bypass product that reduces ethylene synthesis), myo-
inositol (second messenger in releasing calcium ion from
the endoplasmic reticulum), uridine (regulation of glucose
metabolism by the leptin signaling pathway), and guanosine
(second messenger in biological function regulation, e.g.,
bio-membrane formation).
Signal transduction in all three omics described above
appeared to be actively responsive to the damage caused
by copper.
13
Journal of Applied Phycology
Metabolism of Ulva prolifera under copper stress
According to the transcriptome, the metabolism of U. prolifera
also changed. Metabolism includes substance metabolism
and energy metabolism. In terms of substance metabolism,
anabolism in the algae was found to be enhanced in terms
of nitrogen, lipid, and carbon. For example, genes related
to high-affinity nitrate transporter 2.2 and delta(7)-sterol-
C5(6)-desaturase 1 (involved in the biosynthesis of sitosterol
and campesterol) were significantly upregulated, while the
cholesterol 25-hydroxylase gene, involved in cholesterol
biosynthesis repression, was remarkably downregulated.
In addition, many genes related to carbohydrate produc-
tion were significantly overexpressed, e.g., UDP-glucuro-
nate 4-epimerase 5 (involved in the synthesis of negatively
charged monosaccharides), peptidyl-prolyl cis–trans isomer-
ase FKBP20-2 (accumulation of PSII complex), chloro-
phyll a-b binding protein type 2 member 1A (light trapping
and delivering complex), soluble starch synthase 3 (starch
synthesis), and UPF0187 protein At3g61320 and protein
TOC75 (both involved in voltage-dependent translocation
channels in chloroplasts). Regarding energy metabolism,
many genes related to energy metabolism were downregu-
lated, e.g., dynein heavy chain 7 (ATPase activity in ciliary
dynamin), adenosine monophosphate kinase (maintenance
of cellular energy balance), RNI-like protein and copropor-
phyrinogen-III oxidase 1 (catalyzes oxidative decarboxy-
lation), 6-phosphogluconate dehydrogenase (reduction of
NADP to NADPH by oxidative decarboxylation), serine/
threonine-protein kinase SRK2G and developmentally-reg-
ulated protein kinase 1 (both for ATP binding), and SNF1-
related protein kinase catalytic subunit alpha KIN11 (central
regulator of cellular energy homeostasis) (Table 3).
Consistent with the transcriptome, the expression of many
metabolism proteins increased in relation to glycometabolism
(e.g., glutamine-fructose-6-phosphate aminotransferase, for
converting fructose-6P into glucosamine-6P using glutamine
as a nitrogen source), lipids (e.g., ABC transporter A family
member 7, for lipid transporter activity), protein thylakoid for-
mation 1 (for promoting vesicle-mediated thylakoid membrane
biogenesis), ions (e.g., sodium/sulfate cotransporter 3, a cation
transmembrane transporter), and other proteins (e.g., vesicle
transport v-SNARE 12, for accelerating endoplasmic reticulum
to Golgi vesicle-mediated transport). However, the content of
photosystem I reaction center subunit II (ferredoxin-docking
protein in chloroplasts) and VI (docking of the LHC I antenna
complex to the core complex) protein decreased, which was
related to carbon metabolism (photosynthesis). Furthermore,
the expression of alpha-glucan water dikinase 2 (promote
carbohydrate metabolic process) increased to supply energy,
which was in accordance with the major facilitator superfamily
general substrate transporter in the transcriptome.
Journal of Applied Phycology

Table 3  Fluctuation of molecules in Ulva prolifera related to metabolism after 12 h of exposure to 10 mg ­L−1 of copper sulfate
Transcript ­IDa Annotation Accession No Fold change

TRINITY_DN6185_c1_g1 High-affinity nitrate transporter 2.2 sp|P0DKH0 2.4

TRINITY_DN6180_c0_g6 Delta(7)-sterol-C5(6)-desaturase 1 sp|Q39208 2.6
TRINITY_DN6214_c2_g3 Cholesterol 25-hydroxylase sp|Q4G1G8 − 5.7
TRINITY_DN5862_c0_g1 UDP-glucuronate 4-epimerase 5 sp|Q9STI6 5.6
TRINITY_DN92_c0_g1 peptidyl-prolyl cis–trans isomerase FKBP20-2 sp|Q0WRJ7 2.4
TRINITY_DN5697_c4_g1 Chlorophyll a-b binding protein type 2 member 1A sp|P15193 2.5
TRINITY_DN6037_c4_g10 Soluble starch synthase 3 sp|Q43846 2.2
TRINITY_DN5826_c1_g1 UPF0187 protein At3g61320 sp|Q9M2D2 2.1
TRINITY_DN5572_c3_g2 Protein TOC75 sp|Q43715 2.0
TRINITY_DN6580_c0_g1 Dynein heavy chain 7 sp|Q8WXX0 − 2.4
TRINITY_DN4831_c0_g2 Adenosine monophosphate kinase sp|Q9D2H2 − 2.7
TRINITY_DN6318_c1_g2 RNI-like protein gi|545,358,254 − 2.7
TRINITY_DN5830_c0_g4 6-phosphogluconate dehydrogenase sp|Q9FFR3 − 2.1
TRINITY_DN4472_c0_g1 Serine/threonine-protein kinase SRK2G sp|P43292 − 2.3
TRINITY_DN6233_c3_g1 developmentally-regulated protein kinase 1 sp|P34100 − 3.1
TRINITY_DN4450_c0_g1 SNF1-related protein kinase catalytic subunit alpha KIN11 sp|P92958 − 2.5
TRINITY_DN4847_c0_g1 MFS general substrate transporter gi|545,365,626 5.2
Protein ­IDb Annotation Accession No Fold change
Maker00961 Glutamine-fructose-6-phosphate aminotransferase sp|O19908 1.9
Maker0448 ABC transporter A family member 7 sp|Q9STT5 6.8
Maker03909 Protein thylakoid formation 1 sp|Q7XAB8 1.9
Maker02160 Sodium/sulfate cotransporter 3 sp|A8IHV3 6.2
Maker02164 Vesicle transport v-SNARE 12 sp|Q9SEL5 4.9
Maker06423 Photosystem I reaction center subunit II sp|Q39615 0.6
Maker02433 Photosystem I reaction center subunit VI sp|P13352 0.4
Maker02568 alpha-glucan water dikinase 2 sp|Q9STV0 3.8
Metabolites ­IDc Description Charge p value Fold change
M132T772 L-aspartate - < 0.01 1.6
M160T733 Cyclohexylamine + < 0.01 1.5
M141T654_2 2-oxoadipic acid - < 0.01 1.1
M164T477 L-phenylalanine - < 0.05 1.3
M279T709 γ-glutamyl-L-methionine + < 0.05 1.8
M113T298 uracil + < 0.05 1.5
M503T869 Maltotriose - < 0.01 2.2
M565T858 Uridine diphosphate glucose - < 0.05 0.8
M162T671 L-carnitine + < 0.01 2.4
M133T780 L-malic acid - < 0.05 0.6
M133T118 Ethylmalonic acid + < 0.05 0.6
M117T750 Succinate - < 0.05 0.8

Consistent with the transcriptome and proteome, many
metabolites increased in relation to nitrogen (e.g., L-aspar-
tate, in assimilation of nitrogen), lipid (e.g., cyclohexylamine,
in lipid metabolism), amino acid (e.g., 2-oxoadipic acid,
L-phenylalanine, L-leucine, and γ-glutamyl-L-methionine),
and nucleic acid (e.g., uracil, the precursor of uridine)
metabolism. In addition, the content of maltotriose (promot-
ing starch hydrolysis) increased according to supply energy,
which was consistent with the transcriptome and proteome.
Correspondingly, uridine diphosphate glucose (precursor
for sugars synthesis) decreased. Furthermore, the content of
L-carnitine (promoting the oxidative decomposition of fat for
energy) also increased. However, L-malic acid, ethylmalonic
acid (derivative of L-malic acid), and succinate decreased,
which indicated a block of the tricarboxylic acid cycle.

13

In conclusion, the metabolism of U. prolifera was
enhanced after 12 h of exposure to copper sulfate, while the
energy metabolism rate decreased.
Gene expression of Ulva prolifera under copper
stress
Many genes related to gene expression were significantly
down-regulated in U. prolifera. Some acted in transcription,
e.g., transcriptional activator Myb, transcription elongation
factor S-II, and Poly(A) RNA polymerase GLD2-A, while
WD repeat-containing protein 35 was involved in ciliogen-
esis and ciliary protein trafficking (Table 4).
In the proteome, some proteins related to development
significantly decreased in U. prolifera. Among them, some
were related to transcription and translation, including DNA-
directed RNA polymerases II and V subunit 6B (component
of RNA polymerase II), phenylalanine-tRNA ligase (charg-
ing of tRNA with phenylalanine), and elongation factor
G-2 (involved in protein synthesis), while protein BTR1
increased (inhibiting the efficiency of translation).
These results indicate that the gene expression of U. prolifera
was inhibited under copper stress.
Cell division of Ulva prolifera under copper stress
According to the transcriptome, many genes promoting cell
division were downregulated. Some were involved across the
whole process, including citron rho-interacting kinase, cyc-
lin-a3-1, leishmanolysin-like peptidase, catalytic domain of
cyclin-dependent protein kinase like serine/threonine kinases,
centromere-associated protein E, and cell division control
protein 7. Others played a role in particular period, e.g., S
phase (e.g., Myb-related protein B), G2 phase (e.g., Fanconi
anemia group D2 protein, kinesin-1 heavy chain), G2/M tran-
sition (e.g., aurora kinase A, helicase and polymerase-con-
taining protein TEBICHI, condensin complex subunit 1, and
Table 4  Fluctuation of Transcript ­IDa
molecules in Ulva prolifera
related to growth after 12 h TRINITY_DN5881_c1_g1
of exposure to 10 mg ­L−1 of TRINITY_DN6749_c0_g2
copper sulfate
TRINITY_DN6546_c1_g1
TRINITY_DN6831_c2_g3
Protein ­IDb
Maker03615
Maker01471
Maker06147
Maker00667
13
Journal of Applied Phycology
cryptochrome-2), and M phase (e.g., DNA mismatch repair
protein MSH4, NPK1-related protein kinase 3, NPK1-related
protein kinase 1S, and cell division cycle protein 20). Some
were spindle-associated proteins (e.g., MAP65/Ase1/PRC1,
and Mad2), and others were repair proteins in DNA replica-
tion, e.g., DNA mismatch repair protein MLH3, minichro-
mosome maintenance 9 homologous recombination repair
factor, and Werner syndrome ATP-dependent helicase. Fur-
thermore, histone-lysine N-methyltransferase ATX1, which
modulates transition from cell proliferation to cell elonga-
tion, was upregulated. Some antioxidants that have a peak in
expression during cell division were also downregulated, e.g.,
glutathione-S-transferase (S and G2 phase) and ascorbate per-
oxidase (M phase). Moreover, those involved in gametogen-
esis (e.g., serine/threonine-protein kinase TIO), initiation of
meiosis during spermatogenesis (e.g., membrane-associated
tyrosine, threonine-specific cdc2-inhibitory kinase wee-1.3),
recombination and segregation of homologous chromosomes
at meiosis (e.g., DNA mismatch repair protein MSH5), sporu-
lation (e.g., SP5S_BACSU stage V sporulation protein S),
and gamete flagellum axoneme organization and function
(e.g., cilia- and flagella-associated protein 44) were all down-
regulated. However, several genes involved in the initiation
of mitosis (e.g., protein kinase gsk3) or G1/S transition (e.g.,
tyrosine-protein kinase, lipid phosphate phosphatase 2) were
upregulated (Table 5).
In the proteome, serine/threonine-protein kinase aurora-3
(necessary for cytokinesis and with the microtubule spin-
dle) and glutathione reductase (retards the glutathione meta-
bolic process) decreased, while 14–3-3 protein (inhibiting
cell cycle regulation during the early stages of mitosis) and
glutathione S-transferase Z2 (promoting the conjugation
of reduced glutathione to a wide number of exogenous)
increased. However, Rab GTPase A2A (involved in cell divi-
sion in interphase) increased, while histidine biosynthesis
bifunctional protein hisIE (retards the histidine biosynthetic
process) decreased.
Annotation Accession No Fold change
Transcriptional activator Myb sp|Q08759 − 3.3
Transcription elongation factor S-II smart00510 − 2.0
Poly(A) RNA polymerase GLD2-A sp|Q641A1 − 2.1
WD repeat-containing protein 35 sp|A6N6J5 − 2.6
Annotation Accession No Fold change
DNA-directed RNA polymerases II and sp|Q9SJ96 0.5
V subunit 6B
Phenylalanine-tRNA ligase sp|Q94K73 0.6
Elongation factor G-2 sp|I1K0K6 0.6
Protein BTR1 sp|Q9LZ82 1.5
 Table 5  Fluctuation of molecules in Ulva prolifera related to cell division after 12 h of exposure to 10 mg ­L−1 of copper sulfate
Transcript ­IDa Annotation Accession No Fold change

TRINITY_DN6428_c1_g3 Citron Rho-interacting kinase cd05601 − 5.1

TRINITY_DN2018_c0_g1 Cyclin-A3-1 sp|Q9FMH5 − 3.7
TRINITY_DN6620_c0_g1 Leishmanolysin-like peptidase sp|Q8BMN4 − 3.4
TRINITY_DN5839_c1_g2 Catalytic domain of cyclin-dependent Protein kinase-like Serine/threonine Kinases (STKc_CDKL) cd07833 − 2.1
TRINITY_DN6372_c2_g3 Centromere-associated protein E gi|760,444,529 − 2.1
Journal of Applied Phycology

TRINITY_DN6194_c0_g7 Cell division control protein 7 sp|P41892 − 2.4

TRINITY_DN6651_c0_g2 Myb-related protein B sp|P10244 − 2.2
TRINITY_DN6728_c0_g4 Fanconi anemia group D2 protein sp|Q80V62 − 2.1
TRINITY_DN5981_c1_g2 Kinesin-1 heavy chain sp|P33176 − 2.7
TRINITY_DN3716_c0_g1 Aurora kinase A sp|Q2TA06 − 5.4
TRINITY_DN6523_c0_g9 Helicase and polymerase-containing protein TEBICHI sp|Q588V7 −3
TRINITY_DN5153_c0_g1 Condensin complex subunit 1 sp|Q15021 − 3.9
TRINITY_DN4899_c0_g1 Cryptochrome-2 sp|Q96524 −2
TRINITY_DN5584_c0_g1 DNA mismatch repair protein MSH4 sp|F4JP48 − 2.5
TRINITY_DN4612_c0_g1 NPK1-related protein kinase 3 pfam00069 − 3.3
TRINITY_DN4693_c0_g1 NPK1-related protein kinase 1S sp|Q54R82 −2
TRINITY_DN6653_c0_g2 Cell division cycle protein 20 sp|Q62623 − 1.1
TRINITY_DN5628_c0_g1 Microtubule-associated protein, MAP65/Ase1/PRC1 gi|302,828,180 − 5.1
TRINITY_DN5812_c0_g2 Mitotic spindle checkpoint protein Mad2 sp|Q9LU93 − 4.2
TRINITY_DN6619_c2_g4 DNA mismatch repair protein MLH3 sp|F4JN26 − 4.8
TRINITY_DN4880_c0_g1 Minichromosome Maintenance 9 Homologous Recombination Repair Factor sp|Q69QA6 − 3.9
TRINITY_DN6800_c0_g3 Werner syndrome ATP-dependent helicase sp|Q14191 −2
TRINITY_DN6235_c0_g2 Histone-lysine N-methyltransferase ATX1 sp|Q9C5X4 2.2
TRINITY_DN5704_c0_g2 glutathione-S-transferase sp|Q9Y7Q2 − 4.1
TRINITY_DN6585_c1_g5 Ascorbate Peroxidase sp|Q539E5 − 2.6
TRINITY_DN5722_c0_g1 Serine/threonine-protein kinase TIO sp|Q2QAV0 − 2.4
TRINITY_DN6550_c2_g1 Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase wee-1.3 sp|O18209 −2
TRINITY_DN6206_c3_g1 DNA mismatch repair protein MSH5 sp|Q6L4V0 − 2.3
TRINITY_DN5925_c0_g3 SP5S_BACSU Stage V sporulation protein S sp|P45693 − 2.1
TRINITY_DN5015_c0_g1 Cilia- and flagella-associated protein 44 sp|A8J1V4 − 2.1
TRINITY_DN5948_c2_g2 Protein kinase gsk3 sp|Q10452 2
TRINITY_DN5759_c1_g5 Tyrosine-protein kinase IPR008266 4.7
TRINITY_DN3745_c0_g1 Lipid phosphate phosphatase 2 sp|Q9XI60 2.2
Protein ­IDb Annotation Accession No Fold change
Maker06972 Serine/threonine-protein kinase Aurora-3 sp|O64629 0.4
Maker02897 Glutathione reductase sp|P48642 0.5
Maker00702 14–3-3 protein IPR000308 1.7
Maker02971 Glutathione S-transferase Z2 sp|Q9ZVQ4 1.8
Maker01273 Rab GTPase A2A cd01868 4.6
Maker01370 Histidine biosynthesis bifunctional protein hisIE sp|O82768 0.6
Metabolites ­IDc Description Charge p value Fold change
M136T314 Adenine + < 0.05 1.3

13
 Journal of Applied Phycology

Finally, the growth of U. prolifera was inhibited accord-

Fold change
ing to the metabolome. In this case, we inferred that adenine
(increased) may decompose into allantoic acid (increased)
which inhibits growth and development through feedback
0.7
1.2
1.4
1.5
1.5
inhibition of allantoin (decreased) synthesis. Additionally,
reproduction was also inhibited by L-glutamine (increased),
which is a NO synthase inhibitor, since NO promotes the
Accession No

maturation of gametangium in U. prolifera.

These results suggest that cell division and reproduction
< 0.01
< 0.01
< 0.01
< 0.05
< 0.01

were gradually inhibited after 12 h of exposure to copper

sulfate in U. prolifera.
+

-
-
-
-

Discussion

Antioxidants in U. prolifera under copper stress

Plants growing in environments contaminated by heavy met-

als over a long period gradually develop resistance through
chemical detoxification and/or physical isolation (Foster
1977). The development of this resistance can be verified
in short-term experiments. Ulva prolifera inhibits partial
oxidative damage through its antioxidant components. For
example, in response to 10 μM of copper sulfate exposure,
U. compressa produces a large amount of active oxygen after
7 days, which consumes intracellular proline, ascorbic acid,
phenol, polyunsaturated fatty acids, nitric oxide, and other
antioxidant components (Kováčik et al. 2018). Glutathione
is an important antioxidant, and the ratio of glutathione in
the reduced and oxidized state of the Ulva cell can be used
as a non-specific indicator of copper-induced oxidative stress
(Rijstenbil et al. 1998). Although stimulated by 0.92 mg ­L−1
of copper sulfate, Ulva lactuca produced glutathione peroxi-
dase within a week which interfered with glutathione pro-
duction and distribution (Jervis et al. 1997). However, when
Ulva compressa was exposed to 2.5–5 μM C ­ u2+ for 1 day,
the intracellular glutathione content began to increase, peak-
ing on the ­5th day (Navarrete et al. 2019). After increasing
the exposure to 10 μM ­Cu2+, the activity and gene expres-
sion of glutathione, ascorbic acid, peroxidase, and thiore-
doxin were increased within 3 days (Contreras-Porcia et al.
Allantoic acid
allantoic acid

L-glutamine
Annotation

2011), among which the level of reduced glutathione peaked

Allantoin
Adenine

on the 5th day. The content of oxidized glutathione and glu-

tathione synthase increased continuously in 7 days, but the
reduced ascorbic acid, which also had antioxidant effects,
was consumed on the 1st day and did not recover (Mellado
et al. 2012). After further elevation to 50 µM C­ u2+, the gene
expression levels of Ulva fasciata intracellular ferritin, as
Table 5  (continued)

well as hydrogen peroxide-induced glutathione reductase

and cyclophilin peaked at 3 h. This was followed by type I
iron superoxide dismutase (6 h) and thioredoxin (9 h), while
Transcript ­IDa

M145T708_2
M134T313
M177T691
M175T689
M157T336

the gene expression and enzyme activity of antioxidant

enzyme, such as manganese superoxide dismutase, ascorbate

13
Journal of Applied Phycology
peroxidase, catalase, and methionine sulfoxide reductase,
significantly increased after 4 days, but this did not offset
the damage of hydrogen peroxide to cells under excessive
­Cu2+ stress (Wu and Lee 2008; Wu et al. 2009). However,
U. compressa, which was sampled in oceanic areas contami-
nated by low-concentration copper mines (10 μg ­L-1) for an
extended period of time, mainly relied on an extremely high
content of ascorbate peroxidase and recycled a large amount
of dehydroascorbic acid to maintain antioxidant activity, in
the presence of a large amount of lipid peroxides in vivo
(Ratkevicius et al. 2003). In addition, the content of soluble
phenolic compounds was very low, while gutathione and
glutathione peroxidase, dehydroascorbate reductase, and
catalase were not detected.
Glutathione S-transferase is a key enzyme in the glu-
tathione binding reaction and catalyzes the initial step of
the glutathione binding reaction. In this current study, the
gene expression of glutathione S-transferase and lactoylglu-
tathione lyase were downregulated, while the proteomic data
showed that glutathione S-transferase, glutathione reductase,
and glutathione S-conjugate were downregulated. Further-
more, the up-regulation of glutathione S-transferase Z2
activity inhibited the coupling of glutathione to a variety
of exogenous and endogenous hydrophobic electrophiles.
Meanwhile, in the transcriptome and proteome, ascorbate
peroxidase, methionine sulfoxide reductase, and mannose-
1-phosphate guanylate transferase 2 that are involved in plant
ascorbic acid biosynthesis were all downregulated, while
the amount of thioredoxin, peroxidase, and thiol-specific
peroxidase peroxiredoxin Q were all upregulated. There-
fore, it could be inferred that glutathione, which was down-
regulated, did not play a significant role in the antioxidative
stress in U. prolifera in response to 10 mg L ­ −1 of copper
sulfate after 12 h of exposure. This was similar to reduced
ascorbic acid, which had not recovered from consumption.
At this time point, peroxidase was the main factor.
Clearance and excretion of copper by U. prolifera
The resistance of U. prolifera to copper also manifests
through detoxification effects including excretion and chela-
tion. When U. compressa was exposed to 10 μM C ­ u2+ for
­ u2+
5 days, followed by the removal of the stress for 3 days, C
exudation occurred, which might be mediated by glutathione
(Navarrete et al. 2019). In the present study, transcripts of
protein phosphatase 2C 11, ariadne-like RING finger protein
R811, and protein VMS1 were upregulated. Furthermore,
the activity of ABC transporter C family member 2, trans-
membrane 9 superfamily member 7 in the proteome also
increased. It appeared that U. prolifera accelerated the efflux
reaction to excrete excessive copper and damaged proteins
out of the cell. Approximately 60% of the unexhausted cop-
per is enriched in the cell wall (Lee et al. 2005), resulting
in a thickening of the cell wall (U. flexuosa (Andrade et al.
2004), U. compressa (Kováčik et al. 2018), Ulva rigida, and
Ulva intestinalis (Zeroual et al. 2020)). Metallothionein and
phytochelin are capable of direct copper chelation, and the
threshold of ­Cu2+ stimulation in cell synthesis is higher than
that of the antioxidant glutathione (Navarrete et al. 2019).
The three types of metallothionein peaked, respectively, at
3 days (MT7), 9 days (MT3), and 12 days (MT6), under
exposure to 10 μM C ­ u2+ (Laporte et al. 2016; Navarrete
et al. 2019), while PC2, PC3, and PC4 took more than 5 days
to peak (Mellado et al. 2012).
Repairing and assimilation of Ulva prolifera
under copper
Ulva prolifera secretes antioxidants by speeding up cell
structure repair. Within 24 h of exposing U. compressa to
10 μM of copper sulfate, the expression of some enzyme
genes involved in carotenoid synthesis and the Calvin
cycle peaked at 3 h; the expression of some involved in the
assembly, repair, and protection of PSII and PSI peaked
at 6 h, while the expression levels of carotenoids and
chlorophyll a peaked at 12 h (Rodriguez et al. 2018). The
12-h data of the current study also showed similar trends.
For example, transcript in the light-harvesting complex
chlorophyll a-b binding protein type 2 member 1A and
1D, and protochlorophyllide reductase during chlorophyll
biosynthesis, were all upregulated. Furthermore, the peptidyl-
prolyl cis–trans isomerase FKBP20-2, which is related to PSII
complex accumulation, and protein TOC75, the constitute
protein of the outer membrane of chloroplasts, were also
upregulated. In the proteome, the glutamate-1-semialdehyde
2,1-aminomutase was reduced, which is involved in the
chlorophyll biosynthesis pathway, as well as geranylgeranyl
diphosphate reductase, which provides plant alcohol
for chlorophyll synthesis. Although the activity of PSII
component protein thylakoid formation 1 was upregulated, the
photosystem I reaction center subunit II was downregulated.
Therefore, it was speculated that copper might destroy the
protein structure of the photosynthetic system, resulting in a
large number of related genes becoming overexpressed in the
transcriptome, which indicates short-term antagonism of U.
prolifera to copper stimulation. According to a previous study,
the expressions of many components in the photosynthetic
system of U. compressa peaked from the 3rd to the 5th day
after exposure to copper (Laporte et al. 2020). Genes related
to carbon and nitrogen assimilation also peaked during the
same period (Laporte et al. 2020).
In the present study, there was a similar trend regard-
ing the upregulation of carbon and nitrogen assimilation in
U. prolifera. In carbon assimilation, the transcript of solu-
ble starch synthase 3 was upregulated, while the amount
of glucose-1-phosphate adenylyltransferase large subunit 1
13

protein in the corresponding proteome was downregulated.
In nitrogen assimilation, the transcript of high-affinity nitrate
transporter 2.2 was upregulated. In the metabolome, gluta-
mate and aspartic acid involved in nitrogen assimilation were
both upregulated, but uridine diphosphate glucose, which is
involved in carbon assimilation, was downregulated, indi-
cating glycogen consumption. In summary, the expression
and activity of carbon and nitrogen assimilation-related sub-
stances were upregulated, which might promote the replace-
ment of proteins damaged by oxidative stress.
Copper inhibition of photosynthesis in Ulva prolifera
In a previous study investigating photosynthetic physiology,
the quantum output of energy dissipation increased, and the
maximum relative electron transfer rate decreased within
24 h of exposure to 10–100 µg L ­ −1 of CuO in U. lactuca,
indicating destruction of the electron transfer chain (Sid-
diqui and Bielmyer-Fraser, 2019). However, after exposure
to 250–500 μg L ­ −1 of ­Cu2+ for 5 days, the total photosynthe-
sis rate of U. flexuosa was completely suppressed (Andrade
et al. 2004). The transcriptome data of the present study
showed that the expression of 6-phosphogluconate dehy-
drogenase, which participates in the photosynthetic electron
transport chain and generates NADPH, was downregulated.
This phosphate dehydrogenase participates in the first stage
of oxidation of the pentose phosphate pathway, which
inhibits subsequent reactions and reduces the production of
NADPH required for anabolism and pentose required for
phosphate synthesis. In addition, the expression of succinate
dehydrogenase [ubiquinone] iron-sulfur subunit 2 in prot-
eomics in the tricarboxylic acid cycle was downregulated, as
well as the content of succinate and malic acid in the metab-
olome. This indicates that the regeneration of oxaloacetate
in the tricarboxylic acid cycle was inhibited, and the produc-
tion of FADH2 and NADPH was also reduced. Meanwhile,
as a component of the respiratory electron transport chain
complex II involved in transferring electrons from succinate
to coenzyme Q, the decrease in succinate dehydrogenase
[ubiquinone] iron-sulfur subunit 2 resulted in a reduction in
the number of electrons that were transferred from succinate
to ubiquinone, which reduced ATP synthesis (Andrade et al.
2004; Lee et al. 2005; Mellado et al. 2012; Laporte et al.
2016; Kováčik et al. 2018; Navarrete et al. 2019; Zeroual
et al. 2020).
Copper inhibition of U. prolifera reproduction
A high concentration of copper significantly affected the
growth and reproduction of U. prolifera. Within 5 days of
10-μM copper sulfate exposure, the expression of a large
number of genes related to protein synthesis and degrada-
tion, signal transduction, and DNA replication and repair
13
Journal of Applied Phycology
in cells decreased in U. compressa (Laporte et al. 2020).
After 8 days of exposure to 9.8 mM of C ­ u2+, the growth
rate of U. fasciata decreased by 50% (Lee et al. 2005). In
the present study, the expression of transcriptional activa-
tor Myb, transcription elongation factor S-II, and the DNA
helicase Werner syndrome ATP-dependent helicase, which
are involved in the transcription process, were all downregu-
lated. Furthermore, the proteomic data showed that the func-
tional proteins involved in the transcription and translation
process (DNA-directed RNA polymerases II and V subunit
6B, phenylalanine-tRNA ligase, and elongation factor G-2)
were also downregulated, and the negative regulatory pro-
tein glycine-rich RNA-binding protein 2 and protein BTR1
were upregulated. These data were consistent with those pre-
viously reported. Regarding asexual reproduction, even a
low concentration of copper solution could reduce the spore
formation rate of U. pertusa within 4 days (Lee et al. 2019),
thereby affecting spore germination and gametophyte growth
(Han et al. 2009). In the transcriptome data of the current
study, the expression level of the transcript SP5S_BACSU
Stage V sporulation protein S that induces sporulation was
downregulated, and the expression levels of transcripts dur-
ing meiosis, such as DNA mismatch repair protein MSH4
and Fanconi anemia group D2 protein homolog, were also
downregulated. Allantoic acid participates in the purine deg-
radation pathway and has a feedback inhibitory effect on the
synthesis of allantoin. In the present study, allantoic acid was
significantly upregulated in the metabolome, which slowed
the growth and development of algae. The joint analysis of
these omics showed that U. prolifera slowed down its growth
and reproduction after being exposed to 10 mg ­L−1 copper
sulfate for 12 h.
Copper enrichment in Ulva as an environmental
indicator
Fresh Ulva has a significant effect of copper enrichment. At
a concentration of 10 μM C ­ u2+, most of the copper from a
solution was found to be enriched in two strains of U. lactuca
after 24 h (Valdés et al. 2018). Ulva flexuosa grown in 500 μg
­Cu2+ ­L−1 showed a copper accumulation of 5284 mg g­ -1 (dry
weight) after 5 days, which was 556 times that of the control
group (Andrade et al. 2004). The complexation ability of U.
rigida to copper is as high as 13 μM ­Cu2+, which may be
derived from the Triton-X-100 ligand (Scoullos et al. 2004)
secreted on the surface; however, metallothionein plays
an auxiliary role in the enrichment of copper ions (Zúñiga
et al. 2020). The selective adsorption capacity of copper by
Ulva indicates that it could become an indicator organism.
Laboratory experiments have indicated that the accumulation
of copper in U. lactuca and U. compressa cells was linearly
correlated with the increase of copper concentration in the
medium within 48 h (Siddiqui and Bielmyer-Fraser, 2019)
Journal of Applied Phycology
and 10 days (Navarrete et al. 2019), respectively. Furthermore,
the enrichment rate of U. rigida is also proportional to the
concentration, which indicates that the algae can be used to
monitor copper ions in coastal areas (Barraza and Carballeira
1999). Under highly variable estuarine conditions, Ulva could
also be used as a biological indicator for the quantitative
monitoring of copper (Seeliger and Cordazzo 1982), but it is
worth noting that, as salinity decreased, copper accumulation
in Ulva reticulata increased, resulting in a decrease of effect
concentration 50% and no observed effect concentration,
indicating increased copper toxicity at low salinity (Mamboya
et al. 2009). The copper accumulation effects of U. prolifera
and U. linza also indicated similar trends at salinities of 6–9
and 25–35, respectively (Rijstenbil et al. 1998). Additionally,
in an alkaline solution rich in sodium bicarbonate, the effect
of U. fasciata on copper enrichment was also significantly
reduced (Geddie and Hall 2019). Therefore, when using
Ulva as an indicator for copper detection in areas of heavy
rainfall or underground freshwater invasion, acidic waters, and
estuaries, the negative effects of excessive metal accumulation
caused by the decrease in salinity should be considered. In
addition, glyphosate combined with copper was incidentally
adsorbed by U. lactuca, which could become an auxiliary
indicator of copper concentration (Trinelli et al. 2013).
Conclusion
Ulva prolifera exposed to 10 mg ­L −1 of copper sulfate
for 12 h responded to the stress as follows: (1) developed
resistance to acute copper stress regarding innate immu-
nity, repairing, antioxidant, osmotic regulation, detoxifica-
tion, elimination reaction, and exocytosis regulation; (2)
upregulated the genes, proteins, and metabolites associ-
ated with second messengers, e.g., defense signaling; (3)
enhanced the metabolism of nitrogen, lipid, and carbon,
while reducing energy metabolism; and (4) inhibited algal
cell division and gene expression. These results elucidate
the mechanisms of the responses of U. prolifera to copper
exposure for 12 h.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 10811-0​ 21-0​ 2527-8.
Acknowledgements We are very grateful to Thomas Wichard for
helpful discussions. We thank all of the reviewers, who contributed to
improving the manuscript. We would also like to thank Sangon Biotech
Co. (Shanghai) and Applied Protein Technology Co. (Shanghai) for
their technical support.
Authors’ Contribution Chuner Cai, Hui Zhao, Rui Jia, and Peimin He
conceived the study. Chuner Cai designed the experiments. Ting Jiang
collected, cultured, and prepared the algae. Xuanhong Liu, Chuner Cai,
and Hui Zhao analyzed the data. Chuner Cai and Xuanhong Liu wrote
and finalized the manuscript. All authors have read and approved the
manuscript.
Funding 1. Key Laboratory of Integrated Marine Monitoring
and Applied Technologies for Harmful Algal Blooms, S.O.A.
(MATHAB201817).
2. Shanghai Port and Offshore Ecological Environment Technology
Service Platform Project (19DZ2292500).
3. Natural Science Foundation of Shanghai (18ZR1417400).
Declarations
Conflict of interest The authors have declared that no competing in-
terests exist.
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