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The littoral red alga Pyropia haitanensisuses rapid accumulation
of floridoside as the desiccation acclimation strategy
Feijian Qian &Qijun Luo &Rui Yang &Zhujun Zhu &
Haimin Chen &Xiaojun Yan
Received: 4 December 2013 /Revised and accepted: 8 May 2014# Springer Science+Business Media Dordrecht 2014
Abstract Intertidal marine algae experience various abiotic
stresses during low tide, such as desiccation. In this study,
a red alga, Pyropia haitanensis, which is extremely tolerantto desiccation, was selected to investigate the physiological,
chemical, and molecular responses of marine algae to des-
iccation. Osmoregulation and the synthesis of short-chain
volatile compounds were studied in detail. The results
showed that desiccation induced morphological and cellular
changes, as well as a loss of about 98 % of the cell water.
D e si c ca t i on m a r ke d ly i n cr e as e d t h e c o nt e nt o f
osmoregulator floridoside in the alga. Two genes,
PhNHO1, which encodes glycerokinase, and PhGPDH,
which encodes glycerol-3-phosphate dehydrogenase, are in-
volved in the biosynthesis of a floridoside precursor,
glycerol-3-phosphate. Both genes were upregulated during
desiccation. The species and content of short-chain volatiles
changed considerably after the exposure to desiccation-
inducing conditions. These changes included the production
of 5-octen-1-ol, E,E-2,4-octadien-1-ol, 1-octanol, (6Z)-
nonen-1-ol, and 2-nonenal, as well as the release of signif-
icant amounts of 3-octanone, dodecanoic acid, and 1-octen-
3-ol. PhLOX1 and PhLOX2, which facilitate the initiation
of production of downstream short-chain volatile
compounds via the oxylipin pathway, were also upregulat-
ed. In summary, when exposed to desiccation conditions
during low tide, stress-related responses were trigged in thealga. The concentration of floridoside, a solute involved in
the osmoregulation and the expression of genes responsible
for its synthesis, was increased to protect the cell from
dehydration damage. Short-chain volatiles may act as pher-
omones and antibacterial agents.
Keywords Pyropia haitanensis . Rhodophyta floridoside.
Desiccation . Volatile organic compounds. Gene expression.
Reactive oxygen species
Introduction
Intertidal marine macroalgae are subjected to repeated
immersion and emersion due to the periodic exposure to
tidal fluctuations. When the tide is high, they are sub-
merged in seawater. When the tide is low, intertidal
macroalgae are exposed to air and experience a number
of environmental stresses, such as intensive light, rapid
temperature changes, osmotic stress, salinity, radiation,
and desiccation (Burritt et al. 2002; Kumar et al. 2011).
Intertidal macroalgae must have developed effective strat-
e g ie s to o v erc o me th os e e n viro nme nta l s tres s es .
Desiccation is inevitable during low tide. The ability of
certain organisms to tolerate reversible desiccation and the
mechanisms underlying the tolerance has been extensively
studied. Anhydrobiotic organisms include nematodes,
plants, lichens, ferns, and seeds, yeasts, and bacteria.
These organisms can survive extended periods of desicca-
tion and recover completely upon rehydration (Crowe
2002). Reactive oxygen species (ROS) defense, repression
of membrane phase transition, and formation of a glassy
s ta te w it hi n t he c el l a re t he t hr ee m aj or k no wn
F. Qian:
Q. Luo:
R. Yang:
Z. Zhu:
H. ChenKey Laboratory of Applied Marine Biotechnology, Ministry of
Education, Ningbo, Zhejiang 315211, China
X. Yan (*)
School of Marine Science, Ningbo University, Ningbo, Zhejiang
Province 315211, China
e-mail: [email protected]
H. Chen (*)
Key Laboratory of Marine Biotechnology, Ningbo University, Post
Box 71, Ningbo, Zhejiang Province, China
e-mail: [email protected]
J Appl Phycol
DOI 10.1007/s10811-014-0336-0
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mechanisms of desiccation tolerance (Crowe et al. 1992;
Smirnoff 1993; Hoekstra et al. 2001). The latter two
mechanisms both involve the accumulation of high con-
centrations of disaccharide, such as trehalose (Mori et al.
2010; Sakamoto et al. 2009; Thorat et al. 2012) or sucrose
(Crowe 2002; Ghasempour et al. 1998; Sun et al. 1994).
These sugars are involved in the protection of biological
membranes and proteins from the destructive effects re-sulted from water removal. They act by replacing water
a nd thro ug h the fo rmation o f a morp ho us g la s se s
(vitrification) (Clegg 2001). However, there are still open
questions regarding the strategies that intertidal macroalgae
use to survive the harsh conditions. Studies on the mech-
anisms of desiccation tolerance in intertidal macroalgae are
mainly focused on the role of ROS in defense and pho-
tosynthesis. Increases in the concentrations of antioxidative
enzymes and antioxidants (Burritt et al. 2002; Kumar
et al. 2011), polyunsaturated fatty acids (PUFAs) (Kumar
et al. 2011) and decreases in the efficiency of photosyn-
thesis (Dring and Brown 1982; Bell 1993; Zou and Gao2002) and apparent carboxylating efficiency (Zou and Gao
2002) have been reported during desiccation. However, the
common features of protective sugars have not been ad-
dressed. The response of organisms to environmental stim-
uli is regulated by intricate networks of hormones. In
marine algae, a number of oxylipins, especially volatile
organic compounds (VOCs) have been identified. A great
deal of information supports the motion that VOCs are
involved in abiotic and biotic stress response and commu-
nication by chemical signals (pheromones) (Goulitquer
et al. 2009; Potin et al. 2002; Allison and Daniel Hare
2009; Heil and Karban 2010). During desiccation, UV
radiation and dehydration may kill the associated patho-
genic green algae and microbes, but it is not clear whether
altered VOCs act as pheromones or antibacterial agents.
For example, increased lipid peroxidation was observed
when the red alga Gracilaria corticata was subjected to
desiccation lasting 34 h (Kumar et al. 2011). When
Chlamydomonas reinhardtii cells were exposed to salt
stress, VOCs were released as pheromones and caused
C. reinhardtii cells to prepare for stress conditions (Zuo
et al. 2012).
High intertidal Pyropia is extremely tolerant to desicca-
tion. During the daytime at low tide, it experiences 6 h of
desiccation, resulting in loss of 8595 % water in the blades
and becoming dry sheets on the rocks (Blouin et al. 2011).
A better understanding of how these algae survive such
extreme conditions is of considerable scientific interest. It
seems that Pyropia does not contain trehalose or sucrose
(Holligan and Drew 1971; Kremer and Kirst 1982; Majak
et al. 1966). It is more likely that other molecules play the
same role as trehalose or sucrose do. In the red alga, there is
large amount of a photosynthetic carbohydrate, floridoside
(2-O-glycerol--D-galactopyranoside). It has been shown to
contribute to the osmotic acclimation in red algae (Reed
et al.1980). It has also be identified as the carbon precursor
for the synthesis of cell wall polysaccharides in the red
microalga Porphyridium sp. (Rhodophyta) (Li et al. 2002).
However, it is still unclear whether its physiological func-
tions are similar to those of trehalose during desiccation
stress.Because desiccation has far-reaching economic and eco-
logical consequence, in this study, Pyropia haitanensis
(Bangiales, Rhodophyta), an economically important red
alga, was studied to better understand the mechanism
underlying its desiccation tolerance. The desiccation toler-
ance of P. haitanensis was addressed from the point of
view of chemical strategies, gene expression and physio-
logical changes.
Materials and methods
Pyropia haitanensis thalli of about 510 cm long were col-
lected about 45 days after the germination of conchospores
released from free-living conchocelis. They were collected in
October 2012 from low intertidal zones in the coast of
Xiangshan, Ningbo, Zhejiang Province, China. Unwounded
and healthy thalli were selected and sealed in plastic bags with
seawater, and transported to the laboratory within 4 h in the
darkness in a cooler at 4 C. They were dried in the shade
and then stored at 20 C. The samples were rehydrated
with the filtered seawater at 20 C before use. The
rehydrated samples were rinsed manually with brush in
filtered seawater to remove visible epiphytic foreign mat-
ters, cleaned with 0.7 % KI (Wt/V) for 10 min, and main-
tained in autoclaved water in several glass aquaria at room
temperature (1820 C) under illumination of bout 50 mol
photons m2 s1 (light-dark cycle 12:12 h) for 24 h before use
in experiments.
Algae were desiccated in an incubator at 20 C, 75 %
relative humidity, and 100 mol photons m2 s1 as described
(Zou and Gao2002). Water loss was determined by weighing
the algae before and after desiccation. The water loss (WL, %)
was calculated as follows: WL=100(Wo Wt)/(Wo Wd),
where Wois the wet weight,Wt is the weight afterthours of
desiccation, andWdis the dry weight after drying at 90 C for
24 h.
Culture of thalli
The thalli were cultured in two ways, namely, rod culture and
floating. For rod culture, the thalli were hung on bamboo rods
suspended on the culture nets (Fig.1a) installed in intertidal
area. At low tide, the plants were surfaced and dried. For
floating culture, the alga was growing in nets installed in deep
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sea (Fig. 1b), where the alga was not dehydrated over theentire culture period.
Determination of ROS
ROS was measured as described by Bass et al. (1983) using
50 mol 2,7-dichlorodihydrofluorescein diacetate (DCFH-
DA) and 45 U mL1peroxidase for 45 min after the algae had
been desiccated for 4 h.
LC-MS analysis of floridoside
Of the alga samples, 100 mg were taken, extracted in 1 mL of
extraction solvent (methanol/ddH2O, 3:1, v/v), and centri-
fuged. The supernatant were used for floridoside analysis on
a TSQ Quantum Access analysis system (Thermo Fisher
Scientific, USA) using a Hypersil Gold C18analytical column
(1002.1 mm, 3 m, Thermo Fisher Scientific). Methanol
was used as mobile phase A, and 10 mmol L1 ammonium
acetate solution (1:9, v/v) was used as mobile phase B. The
elution lasted 25 min at a flow rate of 300 L min1.
Mass spectrometry was performed on a Thermo Q-TOF
Premier mass spectrometer using electron spraying ionization
(ESI) in negative modes. The capillary voltage was set to
2.65 kV. The pressure of sheath gas flow was set to
25 L min1 and the auxiliary gas flow was 5 arb. The temper-
ature of ion transport capillary temperature was set at 300 C.
Selected-reaction monitoring (SRM) scanning mode was used
at an acquisition time of 0.6 s. A quality scan was performedwithin the range of 50600m/z.
Analysis of volatile organic compounds by GC-MS
One thousand milligram of desiccated agla samples was ex-
tracted in 15-mL solid-phase microextraction (SPME,
Supelco) glass tubes as described (Croisier et al. 2010). The
tubes contained fibers that were coated with an absorbent
phase by exposing the headspace to polydimethylsiloxane/
carboxen/divinylbenzene at 201 C for 4 h.
The fiber was extracted and analyzed on a Shimadzu
QP2010 gas chromatography system equipped with a vocolcolumn (60 mm 0.32 mm1.8 m, Supelco) coupled with a
Shimadzu QP2010 mass spectrometer. Helium (99.995 %
purity) was used as the carrier gas at 1.99 mL min1. The
GC oven was programmed as follows: 35 C for 3 min, then to
40 C at 3 C min1, and finally to 210 C at 5 C min1, at
which temperature it was maintained for 35 min.
Mass spectra were obtained under electron impact ioniza-
tion at 70 eV, and data were acquired over an m/zrange of 45
1,000. The compounds were identified based on their reten-
tion times by comparing their mass spectra to those recorded
in Nist 147 and Wiley 7 Spectrometry Library and to those
obtained using commercially available references.
Quantitative reverse transcriptase PCR
For quantitation of gene expression by quantitative reverse
transcriptase PCR (qRT-PCR), total RNA was isolated with
RNAiso Plus Reagent (TaKaRa, China) according to the
manufacturers protocol. Reverse transcription (RT) was per-
formed using 2 g RNA at 37 C for 15 min in a volume of
40L reaction containing oligo dT primer, random 6 mers, 5
PrimeScript Buffer, and PrimeScript RT Enzyme Mix
(TakaRa).
Samples were collected after the alga had been subjected to
desiccation for 0 (control), 0.5, 1, 2, 3, and 4 h under con-
trolled conditions, flash frozen in liquid nitrogen, and stored at
70 C.
The primers for PCR were designed according to the
sequences available in the transcriptome ofP. haitanensis
and GenBank. qRT-PCR was performed with the SYBR
Premix Ex Taq (TakaRa, China) on a Mastercycler EP
realplex real-time PCR system (Eppendorf, Germany) to in-
vestigate relative levels of expression ofPhNHO1,PhGPDH,
Fig. 1 Culture ofP. haitanensis. a Rod culture, the thalli are hung on
bamboo rods suspended on the culture nets installed in intertidal area. At
low tide, the plants are on the surface and dry. b Floating culture, the alga
grown on nets installed in the deep sea, where the alga is not dehydrated
over the entire culture period
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PhLOX1, and PhLOX2 messenger RNA (mRNA). Primers
qPhNHO1-F (5-CAACCTGCACCTCATCCACACG-3)
and qPhNHO1-R (5-CATCACCTGAAACGCAATCGCC-
3) were used to amplify a PhNHO1 fragment of 214 bp;
primers qPhGPDH-F (5-AACCTCACGGACATCATCAA
C-3) and qPhGPDH-R (5-CGGCAGCACAAACACCAG-
3) were used to amplify aPhgdhfragment of 138 bp; primers
qPhLOX1-F (5-TGCCCCACTTCGCCGACACC-3) andqPhLOX1-R (5-GCCGCCGAGAAGACGTCCATCC-3)
were used to amplify a PhLOX1 fragment of 130 bp; and
primers qPhLOX2-F (5-TCCTTCGTGCTCTTGTTGGTT-
3) and qPhLOX2-R (5-GCTGCTGTTGTTGGGTTCCT-3)
were used to amplify a PhLOX2 fragment of 108 bp. Two
Ph18Sribosomal RNA (rRNA) primers, qPh18S-F(5-AGTT
AGGGGATCGAAGACGA-3) andqPh18S-R(5-CAGCCT
TGCGACCATACTC-3), were used to amplify a 18S rRNA
gene fragment of 153 bp as the internal control for qRT-PCR
(Yang et al. 2013). The PCR amplification procedure
consisted of initial denaturation at 94 C for 3 min, followed
by 40 cycles of denaturation at 94 C for 10 s of annealing at61 C for 18 s for PhNHO1; 55 C forPhGPDH,PhLOX1,
andPh18S-rRNA;63CforPhLOX2; and elongation at 72 C
for 15 s. The disassociation curve was analyzed to determine
target specificity. Negative controls and a reference gene were
included on each plate. Three PCR reaction replicates were
setup. The concentration of cDNA in each sample was deter-
mined by the Ct (threshold cycle) value (Livak and
Schmittgen 2001). The relative mRNA expression of each
gene of interest was normalized to that of the housekeeping
gene rRNA 18S.
Statistical analysis
Data from floridoside determinations and qRT-PCR were
analyzed with one-way ANOVA. All the experiments were
performed at least three times.
Results
Morphological and ultrastructural changes under desiccation
conditions
Lost from the alga during the first 1 h of desiccation was 60
70 % of water. The water loss slowed down after that. Water
loss reached 92 and 98 % after desiccation for 2 and 4 h,
respectively. Water loss also caused morphological changes.
Fully hydrated fronds were expanded and translucent, and
dehydrated fronds became tightly folded, stiff, and brittle
(Fig.2a). Cells were observed and photographed under mi-
croscopes (Fig. 2b). Many cells were visibly shrunken after
dehydration. The volume was reduced to 45.910.32 % of
control (n=18, P
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to 1.02 mg g1, which was 1.40-fold higher than that of the
floating-cultured algae (P
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However, the maximum relative mRNA expression ofPhLOX2was about twice as high as that ofPhLOX1.
Discussion
Many marine algae can tolerate even more severe and
more rapid desiccation than resurrection plants do.
However, the mechanism underlying the tolerance is not
clearly understood. These algae are marine macrophytes
that inhabit in the intertidal zone of rocky shores (Liu
2009). P. haitanensis is mainly distributed in southern
coastal areas in China, and has a dried-out phase of 2 to
3 h every day during low tide and of up to 4 h during the
largest low tide. The data obtained in the present study
clearly demonstrated that desiccation-induced morphologi-
cal changes in the cells were associated with chemical and
gene expression responses to the stressful condition.
When the plant was subjected to continuous desiccation, up
to 98 % of the water in the thalli might be lost, resulting in
considerable cellular changes such as significantly reduced
cell volume and increased the cell wall. The reduction in cell
size may be due to the folding of the cell walls as a result of
decreased water content. This folding may prevent the plas-
malemma from being teased away from the cell wall during
desiccation. In addition, in the desiccated alga, cells exhibited
elevated levels of ROS as indicated by increased DCF flores-
cence. ROS may affect the cellular morphology through its
impact on ROS-dependent changes in the cell membrane
integrity. TEM showed that the inner membrane system had
become blurred after desiccation. This might be resulted from
burst of ROS. In anhydrobiotic organisms, high concentra-
tions of low-molecular-weight nonreducing sugars build up
during desiccation. These can help organisms tolerate
desiccation. Contreras-Porcia et al. (2011) found that
Porphyra columbina has a similar strategy. In the present
work, an increase in the floridoside content ofP. haitanensis
was discovered when the thalli was subjected to desiccationconditions. These findings were closely consistent with those
reported by Goulard et al. (2001) in Solieria chordalis and
Karsten et al. (1995) in Catenella nipae. Moreover, to verify
the early results,P. haitanensisharvested from the rod culture
and floating culture were assayed for floridoside content. For
the rod culture, the thalli were hung on bamboo rods
suspended on the culture nets installed in intertidal area. At
low tide, the plants were surfaced and dried. For the floating
culture, the alga was growing in nets installed in deep sea,
where the alga was not dehydrated over the entire culture
period. The results showed that the contents of floridoside in
algae harvested from the rod culture were higher than those
from the floating culture, indicating that the rod culture in-
duces the formation of more floridoside. Floridoside is a major
photos ynthetic pro duc t in mem bers of mos t ord ers of
Rhodophyta. The occurrence of floridoside has received sig-
nificant attention. The physiological roles of this compound in
carbon storage and transport and as a regulator of osmotic
balance have also been partially elucidated (Li et al.2002). In
the present study, water content in the alga decreased during
dry-out phase, which may result in increased cellular osmotic
pressure. Since floridoside is an effective osmoregulator, it
can protect organelle, especially chloroplasts and mitochon-
dria, from high ion concentrations. According to the present
hypothesis, if floridoside plays a role similar to that of treha-
lose or sucrose in other anhydrobiotic organisms under desic-
cation conditions, it may replace the structural water of mem-
branes and macromolecules as water is lost during dehydra-
tion. This process may involve the formation of biological
glasses in the cells at low water levels, forming a largely inert
protective matrix. TEM results showed that the cell wall were
thickened in the desiccated algae. Recently, it has been pro-
posed that floridoside is the carbon precursor for the synthesis
0
0.2
0.4
0.6
0.8
1
1.2
Full-floating culture Insert rod culture
Floridside
content
mg.g
-1
Fig. 5 The contents of floridside under different aquaculture culture
models. P. haitanensis was harvested from rod culture and floating
culture, respectively. The data were analyzed and bars represent the mean
standard deviation (SD) from three separate experiments (n=3).*P
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o f c e ll wa ll p o ly s a c c h a rid e s in th e re d mic ro a lg a
Porphyridium sp. (Rhodophyta) (Li et al. 2002). Therefore,
floridoside in P. haitanensis may also be involved in the
synthesis of cell wall polysaccharides as a protective
strategy to secure cell integrity and to prevent further
damage to cells due to temperature fluctuations or UV
radiation during desiccation. It has been demonstrated
that floridoside possesses significant antioxidant activity
(Li et al. 2009). The observed increase of floridoside
content during desiccation may also be a cellular re-
sponse to increase compatible solute to reduce ROS-
generated cytoplasmic and membrane.
Although floridoside has been the subject of extensive
research since the 1930s, biosynthesis of the compound has
not been completely reported. However, it is clear that in the
cell, two precursors glycerol-3-phosphate (G3P) and UDP-
galactose, are responsible for the synthesis of floridoside
phosphate synthase. Analysis of all genomics information of
the red alga has not found any floridoside-phosphate synthase
gene. This may be because the annotated gene information
currently available for this species is limited. The two genes,
PhNHO1 andPhGPDH, have been identified and they encode
enzymes responsible for synthesis of G3P. Recent studies
show that G3P serves as the inducer of an important form of
Table 1 Effect of desiccation treatment on the production of VOCs inP. haitanensis
NFnot found*P
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systemic acquired resistance (SAR) in plants. Genetic mutants
that lack G3P cannot induce SAR (Chanda et al. 2011; Xia
et al. 2009). Correspondingly,NHO1 has been reported to be adefense-related gene in Arabidopsis, whose expression could
be induced by flagellin, which can activate plant defense
responses through signal transduction pathway (Li et al.
2005). In this study, we found that relative mRNA expression
ofPhGPDHand PhNHO1were upregulated during desicca-
tion treatment and the timing of the gene expression reached
maximum earlier than floridoside content did. These findings
suggest that floridoside synthesis may become activated in
response to matric water stress (desiccation). However, these
two genes showeddifferent tendencies over time in term of the
magnitude of upregulation. PhNHO1 had much stronger re-
sponse. The maximum mRNA expression ofPhNHO1 was
twice as high as that ofPhGPDH. In addition to the difference
in increases in expression during dry-out phase, the two genes
also differed in time of transcription. Although PhNHO1was
upregulated to a higher level, it was upregulated later, as
compared with PhGPDH. These differences may be because
of their different roles in the production of down-stream
products. G3P transcripted from the two genes may function
to resist hemibiotrophic pathogen, Colletotrichum
gloeosporioides as it is in Arabidopsis. Plants deficient in
the GPDH gene is more susceptible to Colletotrichum
higginsianum than those deficient the gli 1 (a glycerol kinase)
gene, indicating that G3P encoded by GPDHis more impor-
tant in resistance to C. higginsianum(Venugopal et al.2009).
When responding to desiccation, G3P derived fromPhGPDH
may be synthesized first in the red alga to play certain role,
although there is no direct evidence about this in algae. The
other role is to produce downstream product floridoside to
regulate the cellular osmotic pressure during desiccation or to
provide precursors for cell wall synthesis, as observed in the
study. Therefore, increased G3P might lead to increased syn-
thesis of floridoside downstream to some extent. Although it
is currently not clear about the downstream products and their
roles, our study showed thatPhNHO1 was one of the genes
that encode for the same product that responded dramaticallyduring desiccation; as such, its product would play major role
during the stress response that requires large amount of the
product but not urgently. Products from PhGPDHmay also
participate in the stress response, but in a fast reaction way.
More studies on expression, cloning, and in vitro and in vivo
analysis are needed to elucidate the functions of the two genes
in the stress response. It may be that onlyPhNHO1 is involved
in the stress response inP. haitanensis. Its roleis similar tothat
of glycerol-insensitive 1 (NHO1) in Arabidopsis (Eastmond
2004). As the concentration of G3P increases, it can be pre-
sumed that the concentration of floridoside, which is the final
effector of the response to desiccation stress, will increase as
well.
VOCs are defense compounds associated with oxidative
responses to various biotic and abiotic causes. When plants
are subjected to pathogen and herbivore attacks or to mechan-
ical wounding, defensive genes are activated to express relat-
ed proteins/enzymes and VOCs are released. Some secondary
signal molecules are produced for strengthening the informa-
tion exchange between their own cells and different individ-
uals (Farmer and Ryan1990). The regularity of the immersed
state plays an important role in healthy aquaculture of
P. haitanensis. The immersed state may involve high temper-
atures, ultraviolet radiation, high salinity, and other severe
conditions. These can kill epiphytic algae and pathogens on
the thalli surface. However, it is generally believed that algae
do not have a cell-based acquired immunity as their defense
mechanisms. They have evolved several innate immune traits
that provide them with an efficient means of coping with
pathogens. VOCs have been recognized as an effective de-
fense strategy for marine algae. For example, fatty acid-
derived C8 and C11 hydrocarbons and sulfated C11 com-
pounds in marine heterokont algae can act as both sexual
0
2
4
6
8
10
0 0.5 1 2 3 4
Time h
noisserpxeANRmevitaleR
PhLOX1
PhLOX2
*
**
****
*
*
Fig. 7 Relative mRNA
expression level ofPhLOX1and
PhLOX2 under desiccation
treatment conditions.
P. haitanensiskept in seawater
were used as the control and
compared to individuals treated
with desiccation for 0.5, 1, 2, 3,
and 4 h. The data were analyzed
andbarsrepresent the triplicatemean standard deviation (SD)
from three separate individuals
(n=3).*P
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pheromones and herbivore deterrents (Potin et al. 2002).
Sulfated C11 compounds and their possible by-products, such
as 9-oxo-nonadienoic acid, also act as chemical defenses
against amphipod grazers in the brown algae Dictyopteris
spp. (Schnitzler et al. 2001). In diatoms, polyunsaturated
aldehydes, mostly octenals and decenals, have been reported
to inhibit the reproduction of the planktonic predators, the
copepods (dIppolito et al. 2003). In the present study, thered macroalga P. haitanensis was exposed to desiccation
conditions for 4 h. The concentration of dodecanoic acid, 3-
octanone, and 1-octen-3-ol increased markedly. New VOCs,
such as 1,9-nonanediol, 5-octen-1-ol, 1-octanol, E,E-2,4-
octadien-1-ol, 2-nonenal, decanal, and 1,4-dimethoxy-ben-
zene are also generated. The majority of these news VOCs
are C8 compounds or C8 methylated product. These are
mainly derived from the C20 PUFAs through LOX oxidation
to generate 12-hydrogen peroxide lipids, and then the lipid
hydroperoxide lyase cleavage formation. The results are con-
sistent with those reported by Wang et al. (Wang et al. 2013).
The lipid metabolic defense mechanism takes place mainlythrough the C20 metabolic pathway in P. haitanensis.
Previous research has shown that 1-octen-3-ol can be consid-
ered as an antimicrobial compound and that it can influence
different developmental processes during the life cycle of
Penicillium paneum, including inhibition of conidia germina-
tion (Chitarra et al.2004). (E)-2-nonenal was reported to have
a strong inhibitory effect on the plant pathogenic fungus
Botrytis cinerea (Abanda-Nkpwatt et al. 2006). The use of
(E)-2-nonenal to nondormant tubers could terminate sprout
growth and prevent regrowth for 23 months (Knowles and
Knowles2012). In this way, short chain volatiles may have a
positive effect on innate immune defense in P. haitanensis.
However, only the largest marine algae have primitive vascu-
lar systems. Red algae have no internal convey signals to elicit
systemic-induced defenses. Hence, the question is whether
marine algae have any fully developed mechanism for the
signal transconduction between different thalli or signal ag-
gression to the whole plant (systemy). Intertidal algae may use
airborne signals. During desiccation, there is a perfect oppor-
tunity for algae to communicate each other using volatiles.
One recent report demonstrated that exposure to methyl
jasmonate (MeJA) can cause the common rockweed Fucus
vesiculosus to accumulate phlorotannins at low tide (Potin
et al.2002). MeJA and jasmonic acid may play a role in the
development of antiherbivore responses in Fucus tissues,
including those that involve inter-plant communication.
Richard M. Seifert found that (Z)-5-octen-1-ol was an attrac-
tant for the fruit fly (Seifert1981). The attraction of female
Microplitis demolitor to 3-octanone was demonstrated in ol-
factometer tests by Ramachandran et al. (1991). The attraction
of gravid female Megaselia halterata to 1-octen-3-ol was
reported by Grove and Blight (1983). Then VOCs, whose
concentrations are increased or whose production is generated
during desiccation may act as pheromones for the communi-
cation between thalli or transmit of information to other part of
thallus.
The lipoxygenase/hydroperoxide lyase is responsible for
biosynthesis of VOCs from oxylipin pathway in living organ-
isms. In P. haitanensis, two genes (PhLOX1 and PhLOX2)
encoding LOX have been identified in the transcriptome. No
candidates for allene oxide synthase, allene oxide cyclase, orhydroperoxide lyase were found. These results are consistent
with those reported by Collen et al. (2013) in their study on
genome of Chondrus crispus. The two enzymes used here
have different catalytic activities. PhLOX2 only shows the
function of LOX, which catalyzes the conversion of -
linolenic acid, C20, and C22 PUFAs to hydroperoxide prod-
ucts, but PhLOX1 is a multifunctional enzyme. It exhibits
LOX, allene oxide synthase, and hydroperoxide lyase (data
not shown) activities. In the present study, the relative mRNA
expressions of PhLOX1 and PhLOX2 were all upregulated.
PhLOX1 was activated earlier than PhLOX2. The level of
PhLOX1 expression increased obviously at 1 h. PhLOX2was activated at 2 h, and then two genes reached the
ma x imu m e x p re s s io n le v e l a t 4 h . S imila r k in d o f
information has been demonstrated by Kumar et al. (2011),
wherein two LOX isoforms were found with maximum activ-
ity in 4 h of desiccation. After exposure to desiccation for 3
4 h, the enhanced production of ROS and increased lipid
peroxidation generated hydrogen peroxide products which
lead to VOCs synthesized in the oxylipins pathway.
However, in our results, the level of PhLOX2 expression
was twice that of PhLOX1 at the maximum. This might be
because LOXs are from a multigene family and some isoen-
zymes may be present. Studies showed that different adverse
stresses or different developmental stages could induce the
expression of the different LOX genes (Li and Ma 2006).
Under desiccation stress conditions, PhLOX1 and PhLOX2
in P. haitanensis may be induced to express and function to
generate a series of volatiles against the stress.
In summary, the present study have illustrated that desic-
cation can induce the production of ROS. Meanwhile, ROS
acts as a signal molecule to modify the expression of the
PhLOXgene, modulate the protein responses, and produce
oxylipins as stress responses. However, long-term des-
iccation could lead to loss of 98 % of the organisms
water. This could increase osmotic pressure and cellular
dehydration, and alter the membrane-bound structures
within the cells. Floridoside may perform physiological
functions similar to trehalose or sucrose in desiccation
stress conditions. These physiological responses, includ-
ing floridoside accumulation, ROS production, and the
generation of volatile compounds, might play a role in
the extreme desiccation tolerance thatP. haitanensis has.
However, the molecular mechanisms underlying these
processes remain to be further elucidated.
J Appl Phycol
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8/10/2019 Qian Et Al 2014_floridoside
11/12
Acknowledgments This project was funded by NSFC project
(81370532), National Spark Major Project (No. 2013GA701001), Ning-
b o P r o g r a m s f o r S ci e n ce a n d Te ch n o lo g y D ev e l op m e nt
(201201C1011016), Zhejiang Major Technology Project for Breeding
of New Variety (2012C12907-6), K.C. Wong Magna Fund in Ningbo
University; 151 Talents Project.
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