Qian Et Al 2014_floridoside

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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

J Appl Phycol


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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

J Appl Phycol


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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


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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