ORIGINAL ARTICLE

Expression profile of desiccation tolerance factors in intertidalseaweed species during the tidal cycle

Camila Fierro1 • Camilo Lopez-Cristoffanini2 • Andres Meynard1 •

Carlos Lovazzano1 • Francisco Castaneda1 • Eduardo Guajardo1 •

Loretto Contreras-Porcia1,3

Received: 13 September 2016 / Accepted: 2 March 2017 / Published online: 13 March 2017

� Springer-Verlag Berlin Heidelberg 2017

Abstract

Main conclusion The transcriptional modulation of

desiccation tolerance factors in P. orbicularis explains

its successful recuperation after water deficit. Differ-

ential responses to air exposure clarify seaweed distri-

bution along intertidal rocky zones.

Desiccation-tolerant seaweed species, such as Pyropia

orbicularis, can tolerate near 96% water loss during air

exposure. To understand the phenotypic plasticity of P.

orbicularis to desiccation, several tolerance factors were

assessed by RT-qPCR, Western-blot analysis, and enzy-

matic assays during the natural desiccation–rehydration

cycle. Comparative enzymatic analyses were used to evi-

dence differential responses between P. orbicularis and

desiccation-sensitive species. The results showed that

during desiccation, the relative mRNA levels of genes

associated with basal metabolism [trehalose phosphate

synthase (tps) and pyruvate dehydrogenase (pdh)] were

overexpressed in P. orbicularis. Transcript levels related to

antioxidant metabolism [peroxiredoxin (prx); thioredoxin

(trx); catalase (cat); lipoxygenase (lox); ferredoxin (fnr);

glutathione S-transferase (gst)], cellular detoxification

[ABC transporter (abc) and ubiquitin (ubq)], and signal

transduction [calmodulin (cam)] increased approximately

15- to 20-fold, with the majority returning to basal levels

during the final hours of rehydration. In contrast, actin (act)

and transcription factor 1 (tf1) transcripts were down-reg-

ulated. ABC transporter protein levels increased in P.

orbicularis during desiccation, whereas PRX transcripts

decreased. The antioxidant enzymes showed higher speci-

fic activity in P. orbicularis under desiccation, and sensi-

tive species exhibited enzymatic inactivation and scarce

ABC and PRX protein detection following prolonged

desiccation. In conclusion, the reported findings contribute

towards understanding the ecological distribution of inter-

tidal seaweeds at the molecular and functional levels.

Keywords Desiccation stress � Intertidal distribution �Pyropia � Seaweeds

Introduction

The vertical distribution of macroalgae species along the

intertidal rocky zone is influenced by diverse abiotic fac-

tors, including desiccation due to air exposure (Helmuth

2002; Tomanek and Helmuth 2002; Lopez-Cristoffanini

et al. 2013). The previous studies have described that

extended periods of desiccation induce oxidative stress

through reactive oxygen species (ROS) overproduction

(Moore et al. 2009; Contreras-Porcia et al. 2011a, b; Kumar

et al. 2011), negative morphological alterations, life-cycle

disruption, and even cell death (Lopez-Cristoffanini et al.

2013; Flores-Molina et al. 2014). However, several

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00425-017-2673-0) contains supplementarymaterial, which is available to authorized users.

& Loretto Contreras-Porcia

lorettocontreras@unab.cl

1 Departamento de Ecologıa y Biodiversidad, Facultad de

Ecologıa y Recursos Naturales, Universidad Andres Bello,

Republica 440, Santiago, Chile

2 Departamento de Biologıa Evolutiva, Ecologıa y Ciencias

Ambientales, Facultad de Biologıa, Universidad de

Barcelona, Diagonal 643, 08028 Barcelona, Spain

3 Center of Applied Ecology and Sustainability (CAPES),

Pontificia Universidad Catolica de Chile, Avda. Bernardo

O’Higgins 340, Santiago, Chile

123

Planta (2017) 245:1149–1164

DOI 10.1007/s00425-017-2673-0

physiological mechanisms can be activated to attenuate

oxidative stress. For example, seaweeds inhabiting the

upper intertidal rocky zones display greater antioxidant

responses, an increased elimination of toxic compounds,

and better recovery after desiccation than lower intertidal

zone seaweeds (Lopez-Cristoffanini et al. 2013; Flores-

Molina et al. 2014). Moreover, the distribution of certain

species at lower latitudes, where a high environmental

stress pressure exist, is influenced by an efficient antioxi-

dant system (Lopez-Cristoffanini et al. 2013). However,

the temporality of activation and the participation of tol-

erance factors in desiccation stress tolerance strategies are

poorly known, despite the particular ecophysiological

characteristics of these organisms.

Red algae (Rhodophyta), the most ancient lineage of

photosynthetic eukaryotes, are distributed globally and

include several commercially important species. Worth

highlighting among these species is Pyropia orbicularis

Ramırez, Contreras-Porcia & Guillemin, which success-

fully grows along the upper intertidal rocky zone of the

Chilean coast (Ramırez et al. 2014; Guillemin et al. 2016).

P. orbicularis is a model species for understanding adap-

tations to desiccation in intertidal organisms. Recent EST

analyses determined that P. orbicularis undergoes signifi-

cant transcriptional-profile changes during the natural tidal

cycle (Contreras-Porcia et al. 2013). For example, the

enzymes trehalose phosphate synthase (TPS) and pyruvate

dehydrogenase (PDH) were identified. The expression of

these enzymes, which belong to the basal metabolism,

could be affected by the internal water content of tissue

(Contreras-Porcia et al. 2013). TPS is part of the biosyn-

thesis pathway for trehalose, a non-reductive sugar formed

by two glucose molecules, that has been related to envi-

ronmental stress tolerance in several vascular plants

(Goddijn and van Dun 1999; Whittaker et al. 2007; Gechev

et al. 2013). PDH is an essential enzymatic complex that

produces energy (ATP, GTP), NADH, and FADH2, thus

enabling the biosynthesis of complex molecules during

stressful environmental conditions and subsequent recu-

peration processes.

Other mRNAs identified include a 2-Cys peroxiredoxin

(PRX), thioredoxin (TRX), catalase (CAT), arachidonate

5-lipoxygenase (LOX), glutathione S-transferase (GST),

and membrane transporters such as ABC proteins. This

complex system for desiccation tolerance is activated

coordinately to reduce free radical molecules and com-

pounds overproduced under oxidative stress conditions. For

example, PRX activity increases during desiccation and

heavy metal exposure (Foyer and Noctor 2009; Lovazzano

et al. 2013) to reduce H2O2, alkylhydroperoxides, and

peroxynitrite. CAT catalyzes the reduction of H2O2 to

water, thus mitigating ROS excess during environmentally

stressful conditions (Contreras et al. 2005; Maharana et al.

2015). Similarly, LOX, which catalyzes the formation of

fatty acid hydroperoxides, is involved in the tolerance

response to diverse stresses, such as pathogen attack,

wounding (Blee 2002), water deficit (Savchenko et al.

2014; Savchenko and Dehesh 2014), and anoxia (Pavelic

et al. 2000). The fatty acid hydroperoxides produced during

oxidative stress can be reduced to non-toxic compounds or

spontaneously decompose into toxic short-chain aldehydes

(Uchida 2003). These toxic compounds could be prevented

during desiccation through conjugation with glutathione

via involvement of the enzyme GST (Polidoros and

Scandalios 1999), followed by elimination from the cell by

means of membrane proteins.

In P. orbicularis, the cited factors maintain a stable state

of redox potential and detoxification to prevent the oxida-

tive stress triggered by air exposure. Consequently, a

transient and effective expressional modulation of these

factors is expected under the natural desiccation–rehydra-

tion cycle. Therefore, the aim of this study was to deter-

mine the expression of tolerance factors in P. orbicularis

involved in diverse metabolic pathways during the natural

tidal cycle. To understand the physiological responses that

support the ecological, vertical distribution of seaweed

species along the intertidal rocky zone, these responses

were additionally compared with those reported in sensi-

tive species [i.e., Mazzaella laminarioides (Bory de Saint-

Vincent) Fredericq, Scytosiphon lomentaria (Lyngbye)

Link, and Lessonia spicata (Suhr) Santelices] (Flores-

Molina et al. 2014). The expressional dynamics of these

factors were assessed by measuring mRNA levels using

real-time quantitative polymerase chain reaction (RT-

qPCR), western-blot analysis, and enzymatic assays.

Materials and methods

Sampling and desiccation experiments

The natural desiccation–rehydration cycle in P. orbicularis

Ramırez, Contreras-Porcia & Guillemin was studied by

monitoring and recording (1) the mRNA levels of selected

genes (Table 1), (2) the specific activity of enzymes from

the basal metabolism and antioxidant system (Table 1), and

(3) the induction of ABC membrane transport and PRX at

the proteomic level (Western-blot assay). Individuals

(100–150 plants) were collected along 200 m of coastline

at Maitencillo, Valparaıso, Chile (32�39.50S, 71�26.60W).

The tolerance factors were evaluated in fronds affected by

natural desiccation following 1, 2, and 4 h of air exposure

during low tide, as well as in fronds naturally rehydrated

during high tide for 2 and 4 h. Hydrated tissue ([8 h under

complete immersion) was used as the control condition.

After collection, fronds were promptly rinsed in 0.22 lm of

1150 Planta (2017) 245:1149–1164

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filtered seawater for 15–20 s, manually cleaned, and frozen

on-site in liquid nitrogen until further analyses.

Tolerance to the daily desiccation–rehydration cycle

was compared in vitro between individuals of P. orbicu-

laris and the sensitive species M. laminarioides (middle

intertidal distribution), S. lomentaria (middle distribution;

principally in pools), and L. spicata (lower distribution),

such as described in Contreras-Porcia et al. (2011b).

Briefly, hydrated plants were collected along 200 m of

coastline at Maitencillo, placed in plastic bags with sea-

water, and transported to the laboratory in a cooler kept at

5–7 �C. Once in the laboratory, plants were exhaustively

rinsed with seawater, manually cleaned, and acclimated in

0.22 lm filtered seawater for 12 h in a culture chamber

kept at 12–14 �C and under 30–50 lm of photon m-2 s-1

irradiance over a 12:12 light:dark cycle. Posteriorly, the

plants were blotted dry and exposed for 4 h to air in a

growth chamber kept at 14–16 �C and with 70–80 lm of

photon m-2 s-1 irradiance. Algae samples were collected

after 1, 2, and 4 h of desiccation. In addition, a subset of

fronds dehydrated for 4 h were immediately rehydrated

through 2 and 4 h of complete immersion in 0.22 lm of

filtered seawater (under the same culture conditions

described for the desiccation experiment).

Extent of desiccation

The extent of desiccation experienced by different algal

species during the in vitro trials was expressed through

relative water content [RWC (%)] following the formula

RWC% = [(Wd - Wdo) 9 (Wf - Wdo)-1] 9 100, accord-

ing to Flores-Molina et al. (2014).

RNA extraction

Total RNA was isolated from 1 g of fresh P. orbicularis

tissue from naturally hydrated, desiccated (1, 2, and 4 h),

and rehydrated (2 and 4 h) fronds according to Contreras-

Porcia et al. (2013). RNA yield and quality were assessed

with the NanoDropTM 1000 Spectrophotometer (Thermo

Fisher Scientific, Wilmington, DE, USA) and denaturing

1.2% formaldehyde denaturing agarose gel electrophoresis,

Table 1 Genes analyzed by RT-qPCR in P. orbicularis

Genea Functional categoryb Code Efficiency

(%)

Amplicon size

(bp)

Fragment Tm

(�C)

Senescence-associated

protein

Reference gene sen 109 114 77.6

Trehalose phosphate

synthasecBasal metabolism tps 100 146 72.6

Pyruvate dehydrogenasec Basal metabolism pdh 98 164 71.7

Peroxiredoxinc Antioxidant metabolism prx 108 148 70.6

Thioredoxinc Antioxidant metabolism trx 95 156 70.4

Catalasec Antioxidant metabolism cat 95 154 74.6

Lipoxygenasec Antioxidant metabolism lox 100 139 67.8

Glutathione S-transferasec Antioxidant metabolism gst 92 176 75.7

ABC transporter Cellular detoxification and redox potential abc 95 141 76.3

Ferredoxin

NADP? reductase

Cellular detoxification and redox potential fnr 102 105 79.5

Ubiquitin Cellular detoxification and redox potential ubq 107 122 81.5

Glycine hydrolase Transcription, signal transduction, and cellular

organization

glh 100 159 74.8

Actin 3 Transcription, signal transduction, and cellular

organization

act 102 152 83.7

Calmodulin Transcription, signal transduction, and cellular

organization

cam 93 118 79.2

Transcription factor 1 Transcription, signal transduction, and cellular

organization

tf1 90 145 79

The efficiency of amplification, amplicon size in base pairs (bp), and melting temperature (Tm) are showna The primer sequences used for each gene are shown in Table S1b According KEGG Orthology analysis (see Contreras-Porcia et al. 2013)c Enzymes selected for enzymatic assays

Planta (2017) 245:1149–1164 1151

123

respectively. Residual genomic DNA was removed using

DNase I Amplification Grade (Invitrogen, Carlsbad, CA,

USA) according to the manufacturer’s instructions. Sub-

sequently, 1 lg of RNA was reverse transcribed into cDNA

for 50 min at 42 �C using SuperScript II (Invitrogen)

according to the manufacturer’s protocol.

Primer design

Primers for the qPCR analysis of selected genes (Table S1)

were designed from the EST sequences previously obtained

for P. orbicularis (Contreras-Porcia et al. 2013). To obtain

high-quality primers and avoid secondary structures, the

programs Oligoanalyzer 3.1 (http://www.idtdna.com/analy

zer/applications/oligoanalyzer/) and Primer 3 Plus (http://

primer3plus.com/cgi-bin/dev/primer3plus.cgi) were used.

To ensure gene specificity, primer pairs were compared

using the GenBank database tool BLAST (http://www.

ncbi.nlm.nih.gov/tools/primer-blast/).

qPCR

qPCR analysis was performed using the StepOneTM Real-

Time PCR System (Thermo Fisher Scientific). Each qPCR

reaction mixture contained 5 lL of the Fast SYBR Green

Master Mix, 125 ng of cDNA, 400 nM of each primer

(Table S1), and RNase-free water to a final volume of 10 lL.

Amplifications were performed in triplicate with the follow-

ing thermal cycling conditions: the initial activation at 95 �Cfor 3 min, followed by 40 cycles of 3 s at 95 �C and 30 s at

60 �C. In addition, a no template control (NTC) reaction was

included. SYBR Green fluorescence was consistently recor-

ded during the linear phase of cycling. To confirm the pres-

ence of a single PCR product, a dissociation curve analysis of

PCR products was performed (Fig. S1). With the purpose of

estimating assay efficiency, fivefold dilution series were cre-

ated from a cDNA pool for each set of primers.

Efficiency values were estimated from the slope of the

curve following the efficiency equation Eff = 10-1/slope

(see Table 1 for efficiency results). qPCR data were ana-

lyzed using Ct values and the reference gene senescence-

associated protein (sen) (Contreras-Porcia et al. 2013;

HE859069). For analyses of mRNA levels, graphs were

expressed as fold changes over the basal levels found in the

tissue after[8 h hydration (control condition).

Extraction and quantification of proteins

for enzymatic analysis

Algal tissue (5 g) from the in vitro experiments was frozen

in liquid nitrogen and homogenized with a mortar and

pestle in the presence of a 0.1 M phosphate buffer pH 7.0

containing 5 mM of 2-mercaptoethanol. Afterwards, the

homogenate was filtered through Miracloth paper (Cal-

biochem, Billerica, MA, USA) and centrifuged at

7400g for 15 min at 4 �C. Proteins were precipitated with

0.7 g of ammonium sulfate per mL of extract over 2.5 h at

4 �C. The proteins were dissolved in 2 mL of 0.1 M

phosphate buffer pH 7.0, and quantified using the Pierce

BCA Protein Assay Kit (Thermo Fisher Scientific) (Smith

et al. 1985) according to manufacturer’s instructions.

Enzymatic activity assays

TPS and PDH activity assays

Trehalose phosphate synthase (TPS) activity was deter-

mined by coupling the reaction to the pyruvate kinase

enzyme (Cabib and Leloir 1958) and by quantifying the

pyruvate formed through the appearance of the colored

pyruvate 2,4-dinitrophenyl hydrazine compound (Sch-

wimmer and Weston 1961). The reaction mixture con-

tained 15–50 lg of protein extract, 5 mM of UDP-glucose,

25 mM of magnesium sulfate, 10 mM of EDTA, and

10 mM of glucose 6-phosphate, which was added to con-

trol reactions after 30 min at 37 �C. To stop the reaction,

the samples were incubated for 3 min at 100 �C. After

subsequent cooling, 60 mM of phosphoenolpyruvate and

1 U of pyruvate kinase were added to the samples, fol-

lowed by incubation for 15 min at 37 �C. The reaction was

stopped by adding 10% trichloroacetic acid and cen-

trifuging at 7700g for 10 min to precipitate the proteins. An

aliquot of supernatant was mixed with 0.2 mM 2,4 dini-

trophenyl hydrazine and 0.4 M of NaOH to form a reddish

complex, the concentration of which was measured at

450 nm, using a standard curve of 1–20 nM pyruvic acid.

Pyruvate dehydrogenase (PDH) activity was measured by

coupling the assay to the citrate kinase enzyme (Szutowicz

et al. 1981). Using a final volume of 1 mL, 15 lg of protein

extract was incubated for 15 min at 37 �C in the presence of

50 mM of Tris–HCl pH 8, 10 mM of sodium pyruvate, 2 mM

of NAD?, 2 mM of thiamine pyrophosphate, 1 mM of mag-

nesium chloride, 10 mM of dithiothreitol, and 0.2 mM of

coenzyme A (CoA), which was omitted from control reac-

tions. Subsequently, samples where incubated for 15 min at

37 �C, and 2.5 mM of oxalacetic acid and 0.013% 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB) were added. Finally,

4 U of citrate synthase was incorporated, and the reduction

from DTNB to 2-nitro-5-thiobenzoate (TNB) anion was

determined by spectrophotometry at 412 nm using the molar

extinction coefficient of TNB (e = 13.6 mM-1 cm-1).

Antioxidant enzymatic assays

PRX activity was determined using the method previously

described by Lovazzano et al. (2013). For TRX activity,

1152 Planta (2017) 245:1149–1164

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the reaction mixture was incubated for 20 min at 37 �C.

This mixture contained 10 lg of protein extract, 85 mM of

the Hepes buffer pH 7.6, 3 mM of EDTA, 660 lM of

NADPH, 0.3 mM of insulin, and 50 nM of the TRX

reductase enzyme, which was omitted from control reac-

tions. To stop the reaction, 1 mM of DTNB prepared in

6 M of guanidine-HCl was added. The quantitation of thiol

reduction was determined through spectrophotometry at

412 nm and using the TNB molar extinction coefficient.

CAT and LOX activities were determined as described

by Contreras et al. (2009) and Flores-Molina et al. (2014).

GST enzyme activity was determined by monitoring the

thio-ether formation at 340 nm and by using 1-chloro-2,4-

dinitrobenzene as a substrate, according to the method

described by Sparnins et al. (1982).

Western-blot assay for ABC transporter and PRX detection

Protein extracts from in vitro hydrated, desiccated (4 h), and

rehydrated (2 h) fronds were analyzed by Western-blot to

verify the presence and expression levels of ABC transporter

and 2-cys PRX. Proteins were extracted using the phenol

method as previously described by Contreras et al. (2008) and

Lopez-Cristoffanini et al. (2015). Posteriorly, the protein

pellet was re-suspended in PBS at pH 7.2, and separated

according to molecular weight by one-dimensional elec-

trophoresis for 75 min at 150 V using 40 lg of proteins in a

15% acrylamide gel. The proteins were then transferred to a

0.45 lm nitrocellulose membrane (BioRad, Hercules, CA,

USA) for 1.5 h at 70 mA and 4 �C. The non-specific-binding

sites were blocked for 4 h at 4 �C in PBS pH 7.2 containing

3% BSA and 0.05% Tween-20. The membrane was incubated

for 12 h with the primary anti-II PRX and the anti-ABC

transporter antibody (Prestige Antibodies, Sigma-Aldrich, St.

Louis, MO, USA) diluted to 1:2000 or 1:300, respectively.

The antibody excess was removed by rinsing twice with PBS

pH 7.2 and 0.05% Tween-20 for 10 min. The membrane was

then incubated for 2 h with the secondary antibody diluted to

1:1000 (goat anti-rabbit conjugated horseradish Px; BioRad).

Finally, blots were visualized using the Opti-4CN ECL Sub-

strate (BioRad) according to the manufacturer’s instructions.

Statistical analyses

For RWC, mRNA levels, and enzymatic activities, the

significance of differences was determined by two-way

analysis of variance (ANOVA) followed by Tukey’s mul-

tiple comparisons tests (T), considering species and treat-

ment as fixed-effect factors, with three levels in the latter

(control or natural hydration, desiccation, and rehydration).

Prior to the statistical analyses, data were checked for

homogeneity of variance and normal distribution (Zar

2010).

To establish relationships among the relative transcrip-

tion profiles of the genes analyzed in P. orbicularis, a heat

map summary and hierarchical clustering analysis of global

gene expression were carry out. Analysis was performed by

calculating the Euclidean distance for each functional

category of genes (i.e., antioxidant, transcriptional factors,

and basal metabolism), and McQuitty clustering was then

performed (see Clifford et al. 2011). In the resulting

heatmap, color intensity is a proportional representation of

the absolute value of log2 for the fold differences in

expression for all analyzed genes in each treatment. Three

biological repetitions were used with three technical

replicates in each repetition. For gene information, see

Table 1, Table S1, and Fig. S1. All statistical analyses were

performed using the R software version 3.0.1 (The R

Foundation for Statistical Computing).

Results

Level of desiccation

During desiccation, water content decreased in all species.

At 4 h of desiccation, P. orbicularis showed an 88%

decrease in RWC, which was lower than registered in M.

laminarioides (90%), S. lomentaria (91%), and L. spicata

(94%) (Table 2). During rehydration, 100% RWC was

recovered in P. orbicularis and M. laminarioides, while S.

lomentaria and L. spicata only recovered 67 and 90%,

respectively.

Relative transcript levels of tolerance factors

Standard and melting curve analyses revealed an amplifi-

cation efficiency of 100 ± 10% (Table 1) and a single

sharp melting peak between 67.8 and 81.5 �C for each

primer set design (Fig. S1). During desiccation, mRNA

levels for basal metabolism genes (tps and pdh) showed

differential transcription patterns (Fig. 1). After 1 h of

desiccation, tps and pdh transcripts increased 45-fold

(T = 13.13, P\ 0.001) and 5-fold (T = 5.59, P\ 0.005)

higher than during hydration, respectively. After prolonged

desiccation stress, pdh transcripts continued to increase,

whereas tps transcript levels decreased. Both transcripts

reached maximum levels during the early phase of rehy-

dration (2 h), achieving 70- and 40-fold higher levels than

during hydration, respectively.

Regarding antioxidant metabolism, prx transcripts

increased five-fold during the first hour of desiccation as

compared to hydration (Fig. 1). Levels subsequently

decreased under 2 and 4 h of desiccation. Moreover, the

relative transcript levels of trx increased nearly 30-fold

under both 1 and 4 h of natural desiccation (T = 49.29 and

Planta (2017) 245:1149–1164 1153

123

T = 51.85, respectively; in both cases, P\ 0.001). Simi-

larly, cat transcript levels were six-fold (T = 3.54,

P\ 0.05) and 14-fold (T = 9.02, P\ 0.001) higher under

1 and 4 h of desiccation, respectively, than during hydra-

tion (Fig. 1). Maximum transcript levels for prx, trx, and

cat were observed after 2 h of natural rehydration, reaching

levels 14-, 60-, and 17-fold greater than hydration values,

respectively.

Furthermore, lox transcripts increased three to five-fold

during desiccation exposure as compared to hydrated

conditions, and basal levels were reestablished during the

4 h rehydration period (Fig. 1). Transcript levels related to

gst slightly, but significantly, increased after 4 h of desic-

cation (1.3-fold; T = 7.74, P\ 0.001), and a significant

decrease in transcripts occurred during rehydration

(*90%; T = 16.91, P\ 0.001) (Fig. 1).

mRNA levels of the detoxification transporter gene

(abc) increased[6-fold during the first hour of desiccation

as compared to hydration (T = 9.41, P\ 0.001), with high

levels maintained during all desiccation periods (Fig. 1). In

contrast, abc transcripts decreased during rehydration,

reaching basal values by 4 h of rehydration. Chloroplast

gene fnr transcripts decreased ten-fold during the first hour

of desiccation (Fig. 1). However, transcripts increased[8-

fold during prolonged stress, evidencing maximum tran-

script levels, during the second hour of rehydration. The

relative transcript levels of ubq continually increased dur-

ing desiccation, reaching a maximum of four-fold greater

than hydration values (T = 11.28, P\ 0.001) (Fig. 1).

However, ubq transcripts decreased during rehydration

until reaching basal levels. Similarly, while the signal

transduction gene cam showed elevated transcript levels

during the entire desiccation period (Fig. 1), cam levels

returned to basal values during rehydration.

Actin 3 (act) transcript levels decreased 100-fold com-

pared to hydration (T = 61.78, P\ 0.001) (Fig. 1) during

the first hour of desiccation. However, during extended

desiccation exposure, act transcripts increased seven-fold.

This behavior was also registered during the maximum

rehydration period. tf1 transcripts continually decreased

during desiccation, and reached maximum levels during the

second hour of rehydration (Fig. 1). Glycine hydrolase glh

transcripts were only slightly detected during rehydration.

Hierarchical clustering of tolerance factors in P. orbic-

ularis during the natural desiccation–rehydration cycle

showed transcriptional variations among the different

pathway components (Fig. 2). Four clades were catego-

rized based on gene functions related to (1) basal meta-

bolism (tps, pdh), (2) antioxidant function (prx, trx, cat,

lox, gst), (3) cellular detoxification and redox potential

(abc, fnr, ubq, glh), and (4) transcription, signal transduc-

tion, and cellular organization (cam, act, tf1). The distances

between clades and the formation of sub-clades evidence

the dissimilar transcription patterns of the assessed genes,

consequently indicating a potential modulation of these

tolerance factors during natural daily desiccation–rehy-

dration cycles.

Functional comparison between P. orbicularis

and sensitive species to desiccation

Regarding basal metabolism, TPS activity was higher at all

desiccation times in P. orbicularis (Fig. 3a). The TPS

activity in P. orbicularis compared to M. laminarioides

was higher, although not significantly so, after 1 and 2 h of

desiccation stress (Fig. 3a).

Moreover, no significant differences in PDH activity

between hydration and desiccation were observed in P.

orbicularis (Fig. 3b), although a slight, 1.4-fold increase

was observed during the second hour of rehydration

(Fig. 3b). During desiccation and rehydration, PDH activ-

ity decreased in the sensitive species in relation to natural

Table 2 Relative water content (RWC, %) of P. orbicularis, M. laminarioides, S. lomentaria, and L. spicata during in vitro desiccation and

rehydration experiments

Time of exposure (h) Pyropia orbicularis Mazzaella laminarioides Scytosiphon lomentaria Lessonia spicata

Hydration [8 100 100 100 100

Desiccation 1 50 ± 13* 75 ± 4* 40 ± 2* 46 ± 5*

2 35 ± 12* 42 ± 4* 16 ± 8* 12 ± 3*

4 12 ± 4* 10 ± 3* 9 ± 3* 6 ± 1*

Rehydration 1 85 ± 3* 93 ± 2ns 26 ± 4* 86 ± 3*

2 94 ± 2ns 97 ± 2ns 53 ± 3* 88 ± 4ns

4 99 ± 1ns 100 ± 0.2ns 67 ± 10* 90 ± 2*

Values are mean ± SD of five replicates

ns not significant

* Significant (P\ 0.05) differences with values measured in hydrated plants

1154 Planta (2017) 245:1149–1164

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hydration, with activity levels three and four-fold less than

basal values (Fig. 3b).

Concerning the antioxidant enzymes, maximum activi-

ties were registered in P. orbicularis (Fig. 3c–g). In

particular, PRX activity increased during all desiccation

periods (Fig. 3c) and returned to basal values during

rehydration. In M. laminarioides, PRX activity increased in

the first hour of desiccation and was maintained at this

Fig. 1 Relative transcription levels expressed as fold change of

hydration (control values) during the natural desiccation–rehydration

cycle in P. orbicularis. Values represent the mean ± SD of three

replicates. Letters above histograms indicate the results of Tukey

tests; means with the same letter are not significantly different at

P = 0.05

Planta (2017) 245:1149–1164 1155

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level over a 4 h stress period. S. lomentaria and L. spicata

did not show PRX activation during the entire desiccation–

rehydration cycle (Fig. 3c). Enzymatic activity of TRX

increased in P. orbicularis during all desiccation–rehy-

dration cycles, and in S. lomentaria, TRX reached maxi-

mum activity during full desiccation (Fig. 3d). In contrast,

TRX activity in L. spicata decreased below basal values

during rehydration (Fig. 3d).

CAT activity increased in all species, although the highest

activity levels were recorded in P. orbicularis (Fig. 3e). In

this species, maximum activity was registered following 4 h

of desiccation, while in sensitive species, CAT activity sig-

nificantly increased during rehydration (Fig. 3e). For LOX,

all species showed increased activity after 4 h of desiccation

as compared to the hydrated condition (Fig. 3f). However,

maximum activity was once again found in P. orbicularis.

Finally, an increase of GST activity was only found in P.

orbicularis after 4 h of desiccation (Fig. 3g).

Western-blot analysis showed that the ABC transporter

was only overexpressed in P. orbicularis during desicca-

tion in relation to the hydrated control condition (Fig. 4a).

In turn, PRX expression significantly decreased during

desiccation (Fig. 4b), as was also detected at the tran-

scriptional level (Fig. 1). In M. laminarioides, the ABC

transporter was slightly detected, and a continuous

decrease for PRX expression was recorded. Finally, in S.

lomentaria and L. spicata, both proteins were scarcely

detected under all experimental conditions (Fig. 4a, b).

Fig. 2 Heat map summary and hierarchical clustering of the genes

involved in air exposure desiccation tolerance in P. orbicularis. Red

and green indicate higher and lower expression values, respectively.

Analysis was performed by calculating Euclidean distance for each

functional category of genes (i.e., antioxidant, transcriptional factors,

and basal metabolism), and McQuitty clustering was then performed.

Color intensity is a proportional representation of the absolute value

of log2 of the fold difference in expression for all genes analyzed in

each treatment. Three biological repetitions were performed with

three technical replicates in each repetition. For gene information, see

Table 1, Table S1, and Fig. S1

cFig. 3 Specific activity for basal metabolism [TPS (a) and PDH (b)]

and antioxidant enzymes [PRX (c), TRX (d), CAT (e), LOX (f), and

GST (g)] during in vitro desiccation and rehydration in P. orbicularis,

M. laminarioides, S. lomentaria, and L. spicata. Values represent the

mean ± SD of three replicates. Letters above histograms indicate the

results of Tukey tests; means with the same letter are not significantly

different at P = 0.05

1156 Planta (2017) 245:1149–1164

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Planta (2017) 245:1149–1164 1157

123

Discussion

Pyropia orbicularis evidenced distinguished expressions of

tolerance genes during the distinct stages of the natural

desiccation–rehydration cycle. Moreover, functional com-

parisons between P. orbicularis and the sensitive species

revealed significant differences in tolerance responses in

enzymatic activity and protein expression during in vitro

desiccation. Consequently, the present data help to explain

the intertidal distribution of these species, as well as the

tolerance mechanisms existing in P. orbicularis when

under desiccation stress.

Pyropia orbicularis gene behaviors and functions

Basal metabolism: tps and pdh

TPS is coded by the tps gene and is part of the synthesis

pathway for trehalose, a non-reducing disaccharide formed

from two glucose molecules (El-Bashiti et al. 2005). This

sugar can be produced by different organisms and can

function as an energy reserve and/or potent membrane and

macromolecule stabilizer (Franca et al. 2007; Grennan

2007). In P. orbicularis, tps expression varied during the

desiccation–rehydration trial. The rapid gene expression

and activity increase during the first hour of desiccation

concur with desiccation responses in desert organisms,

such as the plant Selaginella lepidophylla (Hook. and

Grev.) Spring; some yeasts; and in the common wheat

Triticum aestivum L. These organisms show increased

trehalose levels, as well as increased levels of a respective

synthesizing enzyme (TPS) (Goddijn and van Dun 1999;

Franca et al. 2007; Pandey et al. 2010). Furthermore, yeast-

based observations indicate a relationship between

increased trehalose levels and greater environmental tol-

erance, as well as a modulation of glycolysis through the

inhibition of the enzyme hexokinase (Wingler 2002;

Grennan 2007). During rehydration, trehalose synthesis

could return to basal levels in P. orbicularis, as evidenced

by decreased gene expression and enzyme activity after 4 h

of rehydration. Nevertheless, trehalose is not the only non-

reducing sugar involved in desiccation tolerance. A number

of vascular plants, including the resurrection grass

Sporobolus stapfianus Gand and the plant Haberlea

rhodopensis Friv., redirect glycolytic metabolism towards

sucrose synthesis instead of trehalose when under desic-

cation (Whittaker et al. 2007; Gechev et al. 2013). There-

fore, P. orbicularis tps appears to change its expressional

and functional levels to stabilize membranes and macro-

molecules according to cell needs during the tidal cycle.

The PDH complex is essential to basal metabolism, and

the oxidative decarboxylation of pyruvate to acetyl-CoA

determines the amount of carbon in the citric acid cycle. In

P. orbicularis, the expression of pdh changed under all of

the evaluated conditions. Positive pdh regulation was

observed after 1 h of desiccation, which would be con-

gruent with cellular demand during desiccation acclimati-

zation. Further supporting this observation, a desiccation-

tolerant wheat species evidenced a stress-induced increase

in gene transcriptions for PDH and the malic enzyme

NADP. These enzymes allow organisms to generate car-

bonated skeletons for molecule synthesis that additionally

facilitate metabolic changes, detoxification, and cellular

stability during stress (Guo et al. 2009).

Although P. orbicularis evidenced positive pdh

expression during the entire desiccation period, PDH

activity slightly, but significantly, decreased after 4 h of

desiccation as compared to the first hour of stress. Func-

tionally, vascular plants decrease acetyl-CoA mitochon-

drial production under abiotic stress due to inactivating

components derived from lipoic acid that are essential for

the proper functioning of the PDH complex (Rapala-Kozik

et al. 2008). Desiccation increases the activity of the

enzymes phosphoenolpyruvate carboxylase and pyruvate-

phosphate dikinase, which belong to cellular pathways that

produce amino-acid precursors from pyruvate and that

Fig. 4 Western-blot analysis

for ABC transporter (a) and

peroxiredoxin (b) detection in

protein extracts (50 lg) from P.

orbicularis, M. laminarioides, S.

lomentaria, and L. spicata under

exposure to hydration (H,

[8 h), desiccation (D 4h, 4 h),

and rehydration (R 2h, 2 h). ?,

high detection; -, low detection

using the UN-SCAN-IT gel 6.1

software

1158 Planta (2017) 245:1149–1164

123

provide the NADPH needed for biosynthetic and antioxi-

dant processes (Doubnerova and Ryslava 2011). Therefore,

these prior reports support the presently unchanged PDH

activity registered in P. orbicularis during desiccation, a

condition in which certain enzymatic factors would be

inactivated and where substrate availability would be

limited.

During rehydration, pdh transcript expression was high,

which is in line with the high energy requirements needed

during this period to biosynthesize proteins and complex

molecules (Lopez-Cristoffanini et al. 2015). Likewise,

maximum PDH activity was observed after 2 h of rehy-

dration. In this context, high pdh expression and func-

tionality appear needed to activate the tolerance

mechanisms that allow P. orbicularis to attenuate the

detrimental effects of prolonged desiccation. As such, pdh

expression would need to vary during the natural tidal

cycle on a basis of cellular demand for energy, reductive

power, and biosynthetic precursors.

Antioxidant metabolism

prx transcripts mostly increased during the first hours of

desiccation and rehydration. The expressions of trx and cat

were highest at the end of the desiccation period and during

the second hour of rehydration. Since ROS production

increases during desiccation, efficient elimination through

antioxidant enzymes, such as PRX, TRX, and CAT, is

needed to maintain cellular functions during the tidal cycle

(Pearson et al. 2010; Contreras-Porcia et al. 2011b).

P. orbicularis PRX, a protein possibly located in the

chloroplast (Contreras-Porcia et al. 2013), possesses two

active-site cysteine residues that are functional when in a

reduced form (–SH). Differential prx expression during

tidal cycle can be explained by the high levels of ABA, as

was recently recorded in P. orbicularis under in vitro

desiccation experiments (Guajardo et al. 2016). ABA

negatively regulates the gene associated with type 2-Cys

PRX in Tamarix hispida Willd, a bush native to dry areas

(Gao et al. 2012), and positively regulates genes related to

other PRX types. Therefore, the distinct types of PRXs

have varied functions under stress, and the observed dif-

ferential induction of 2-Cys PRX could be involved in the

transduction of signals needed to tolerate desiccation.

Both trx expression and TRX activity increased in P.

orbicularis during desiccation, reaching maximum levels

during the second hour of rehydration. The enzymatic

activity of PRX detected after 4 h of desiccation could be

explained by the high trx transcript levels and TRX

activity. The functionality of P. orbicularis PRX is a result

of the enzymatic thioredoxin/thioredoxin reductase system

(Contreras-Porcia et al. 2011b). Therefore, the varied trx

expression likely corresponds to cellular needs to maintain

the reduced form of enzymes associated with antioxidant

metabolism. Besides this, high ferredoxin (fnr) transcript

levels were detected during high desiccation. This protein

plays a key role in chloroplast electron distribution by

regulating the chloroplast metabolic network through the

TRX system, thus contributing to PRX regeneration

(Ceccoli et al. 2011; Heinrich et al. 2012). Consequently,

diverse attenuation systems might also be operating under

stress to normalize redox state imbalances.

Also recorded was an increased expression of cat, a

gene homologous to that recently described in the red

macroalga Pyropia yezoensis (Ueda) Hwang and Choi (Li

et al. 2012). The highest mRNA levels in P. orbicularis

were recorded after 4 h of desiccation and 2 h of rehy-

dration, with transcriptional regulation of the enzyme. The

functional increase of this enzyme during desiccation has

previously been documented in P. orbicularis (Contreras-

Porcia et al. 2011b) and vascular plants (Vicre et al. 2004).

The increased enzymatic activity of CAT during desicca-

tion was associated with increased mRNA expression, but

after 2 h of rehydration, differences were detected between

activity and expression levels. Specifically, while the gene

coding for the mRNA levels of cat increased at a tran-

scriptional level, the activity was less than expected. This

indicates that there might not be a direct relationship

between mRNA expression and the protein product.

Alternatively, slight enzymatic inhibitions could main-

tain transient ROS levels, principally of H2O2, thereby

inducing certain intracellular signaling mechanisms during

stress (Blokhina and Fagerstedt 2010). Supporting this, P.

orbicularis evidenced high lox expression and high LOX

activity, an enzyme activated by ROS. LOX is involved in

the production of fatty acid derivatives termed oxylipins

(Blee 2002; Howe and Schilmiller 2002), which, in vas-

cular plants, are involved in water deficiency tolerance

mechanisms (Savchenko and Dehesh 2014). Therefore, the

regulation of ROS levels under desiccation stress would

require an effective maintenance of diverse tolerance

mechanisms.

Cellular detoxification, transcription, signal transduction,

and cellular organization

During desiccation, an overexpression of ABC transporters

and a slight increase of gst transcripts were evidenced in P.

orbicularis. These proteins are involved in translocating a

wide variety of compounds across cell membranes (Poli-

doros and Scandalios 1999; Ritter et al. 2014). Further-

more, the overexpression of ABC transporters and GST in

seaweed species modulates copper homeostasis and growth

(Contreras et al. 2010; Contreras-Porcia et al. 2011a; Wang

et al. 2015). Considering this information, ABC trans-

porters and GST could be involved during desiccation in

Planta (2017) 245:1149–1164 1159

123

recovery processes, specifically in the elimination of toxic

compounds. Methylglyoxal, overproduced during stressful

conditions, is a mutagenic and genotoxic compound that

can degrade proteins, inactivate the antioxidant defense

system, and provoke cell death (Martins et al. 2001). A

recent study by Fierro et al. (2016) assessed methylglyoxal

production during desiccation in seaweeds, finding that

only P. orbicularis maintains reduced methylglyoxal levels

during this condition. Therefore, ABC transporters and

GST are likely implicated in the elimination of toxic

compounds generated during air exposure, such as

methylglyoxal.

In diverse organisms, the proteolysis of oxidized and

inactivated proteins can occur through the ubiquitin-26S

proteasome system. Related to this, it is reasonable to

suggest that the increased expression of ubq in P. orbicu-

laris would be involved in the degradation of damaged

proteins, thereby impeding a harmful stress condition

during air exposure. Supporting this supposition, the pre-

vious studies in P. orbicularis have reported high oxidized

protein levels during desiccation that are efficiently

reduced during rehydration (Contreras-Porcia et al. 2011b),

a case not found in sensitive species (Flores-Molina et al.

2014).

Rearrangements of the microtubule actin (act) and of

actin-associated proteins have been observed under dif-

ferent stressful conditions, including low temperatures

(Khokhlova et al. 2003) and desiccation (Faria et al. 2005).

Similarly, the present study detected reduced act expres-

sion during desiccation in P. orbicularis. Furthermore, a

recent proteomic analysis found that reduced actin quantity

is due to an upregulation of a cofilin/actin depolymerizing

factor (Lopez-Cristoffanini et al. 2015). This factor disas-

sembles actin filaments and prevents denaturation (Maciver

and Hussey 2002). Ultrastructural analyses of the desic-

cation-tolerant algae P. orbicularis and Klebsormidium

crenulatum (Kutzing) Lokhorst (Holzinger et al. 2011)

evidenced tissues with irregular contours, plasma mem-

brane folding, and a notable accumulation of electron-

dense bodies inside the chloroplast. These observations

support that cytoplasmic components agglomerate, such as

with compatible solutes, and that cytoplasm viscosity

increases under low water content. Therefore, low act fil-

ament expression, and consequent cell-wall flexibility,

decreases the probability of molecular interactions that

may cause protein denaturation and membrane fusion

during desiccation.

Calmodulin (cam), an ubiquitous Ca2?-binding protein

related to many biochemical reactions, is activated by Ca2?

released in response to multiple environmental stimuli

(Yang and Poovaiah 2003). In P. orbicularis, cam tran-

script levels increased during the entire desiccation period,

such as reported in other seaweed species under copper and

temperature stress (Contreras-Porcia et al. 2011a; Liu et al.

2016), as well as in vascular plants during desiccation

(Zeng et al. 2015). Interestingly, cam/Ca2? is required for

the induction of ABA-mediated antioxidant responses in

plants. Therefore, the high expression of cam during the

desiccation–rehydration cycle in P. orbicularis suggests an

active role for cam/Ca2? in efficient antioxidant response

activation in tolerant seaweeds during air exposure.

A transcription factor gene (tf1) was also studied, with

results showing a significant decrease in transcript levels

during desiccation. Transcriptional factors (TF) regulate

the spatial and temporal biosynthesis of salicylic acid in

vascular plant (Zheng et al. 2015), as well as the tolerance

to dryness in some resurrection plants (Gechev et al. 2012).

Literature implicating tf1 in desiccation stress is scarce, but

more information is abundant for other genes involved in

transcription. However, the present results are in accor-

dance with those of Contreras-Porcia et al. (2013), research

in which other TFs (BTF3 homolog 4, TFIIH basal TF

complex P44 subunit, and RNA polymerase beta subunit,

among others) were identified under natural hydration but

not during desiccation. Nevertheless, other authors have

shown that several TFs regulate desiccation tolerance. For

example, WRKY may regulate the expression of a gene

involved in galactinol (compatible solute) synthesis in the

resurrection plant Boea hygrometrica R. Br. (Wang et al.

2009). The TF CpR18 in the resurrection plant Crater-

ostigma plantagineum Hochst, is needed for the expression

of the lea-like CDeT27-45 gene (Hilbricht et al. 2002).

Moreover, there is evidence for cross-talk between differ-

ent stress-response TFs (Liu et al. 2014), suggesting

complex interactions for these elements.

Functional analysis in desiccation-sensitive species

Basal metabolism

The studied desiccation-sensitive species maintained rela-

tively constant levels of enzymatic activity for TPS and

PDH over the course of the desiccation–rehydration cycle.

In Scytosiphon gracilis Kogame and L. spicata, desiccation

and metal stress result in increased levels of lipoperoxides

such as 4-hydroxy-2-nonenal (Lovazzano et al. 2013;

Guajardo et al. 2016). Data obtained from mitochondria

derived from rat hearts incubated with 4-hydroxy-2-none-

nal evidence that this toxic product can reduce PDH

activity by 46% as it oxidizes lipoic acid, the enzyme

cofactor of PDH (Humphries et al. 1998). Considering this,

if sensitive species cannot attenuate the effects of ROS

generated during desiccation, then an inhibitory effect on

cellular metabolism would be expected. Moreover, low

TPS activity could explain the morpho-functional damage

observed in sensitive macroalgae during desiccation

1160 Planta (2017) 245:1149–1164

123

(Contreras-Porcia et al. 2012; Flores-Molina et al. 2014).

This was compared to P. orbicularis, which does not evi-

dence enzymatic inhibition or ultrastructural changes under

stress. Alterations in the breakdown of cell membranes

could also explain the low enzymatic levels of PDH reg-

istered in sensitive species, considering that this enzyme is

associated with the mitochondrial and chloroplast mem-

branes. Therefore, the inactivation of basal metabolism

enzymes suggests that sensitive species (1) are unable to

overproduce the non-reducing sugar trehalose and (2) have

a limited ability to decrease ROS generation, an effect

evidenced by the alteration of essential biomolecules.

Antioxidant metabolism

The assessed sensitive species demonstrated low activity

levels for all enzymes, with only very slight variations

during the desiccation–rehydration cycle. PRX activity

slightly increased during desiccation in only M. laminari-

oides. In addition, low PRX protein levels were detected in

the sensitive species. These observations could be the result

of the following: (1) cells do not have enough energy to

reactivate tolerance factors or (2) the negative effects of

desiccation affect cellular mechanisms at a transcriptional

and/or translational level. In fact, L. spicata tolerance

genes are inactivated under metal stress, which is in con-

trast to tolerant species that activate diverse tolerance

factors (Contreras et al. 2005, 2007, 2009).

TRX activity was constant in M. laminarioides, but

TRX was inactivated at the end of the rehydration period in

L. spicata. This inactivation could be the result of direct

alterations to the enzymatic system by ROS or as a result of

inefficient activation of the TRX enzyme. The functional

loss of TRX would explain the low variation in PRX

activity observed under desiccation in these species. In

fact, low PRX activity was related to null TRX activity in

L. spicata under metal stress (Lovazzano et al. 2013). In S.

lomentaria, TRX activity increased during desiccation and

decreased during the final hours of rehydration, but no

increase in PRX activity was observed. Considering this, it

is probable that the increased TRX activity was insufficient

to recover the activity of certain enzymes, such as PRX,

essential to attenuating the overproduction of ROS during

desiccation.

The most significant differences between species were

observed in CAT activity. While the sensitive species

presented higher CAT activity during desiccation, levels

were significantly less than the activity registered in P.

orbicularis. The observed CAT activity increase in sensi-

tive species is similar to that found in the leaves and seeds

of other organisms exposed to desiccation, such as in

sunflowers, corn, tomatoes, and beans. In these organisms,

there is a negative correlation between intracellular water

content and CAT activity (Hsieh et al. 2002; Jiang and

Zhang 2002; Bailly 2004). Gene transcription associated

with CAT is likely related to interactions of H2O2 and ABA

with antioxidants and ABA response elements, respectively

(Polidoros and Scandalios 1999; Guan et al. 2000). Under

desiccation, sensitive species overproduce H2O2 (Flores-

Molina et al. 2014), suggesting that the increased CAT

activity would be related to a transcriptional regulation

mediated by H2O2. However, CAT activity in P. orbicu-

laris is regulated by H2O2 and ABA, which would explain

the higher CAT activity.

In all, lesser antioxidant enzyme responses would

explain the lower tolerance of sensitive algae species to

desiccation stress. In the case of M. laminarioides, proxi-

mal intertidal localization to P. orbicularis likely allows

for the acquisition of soluble products liberated by P.

orbicularis, which would confer a certain degree of toler-

ance. In fact, in vitro life-cycle studies using P. orbicularis

compounds have found these to protect sensitive species

from the negative effects of desiccation (Contreras-Porcia

et al. 2012; Guajardo et al. 2016). Therefore, the present

study provides evidence that differences in the ability to

attenuate natural desiccation during daily tidal changes

regulate the ecological distribution of algae species along

the intertidal zone. Adaptations at the transcriptional and

transductional levels would confer some organisms, such

as P. orbicularis, with ecological strategies for surviving

under diverse environmental conditions (e.g., temperature,

relative humidity, and photosynthetically active radiation,

among others). In addition, understanding the differential

physiological responses of seaweeds to environmental

stress will help to predict future alterations to seaweed

community structures under a model of global climate

change.

Conclusions

The selected tolerance genes in P. orbicularis presented

differential expressions during the natural desiccation–re-

hydration cycle. During desiccation, this species activated

enzymes involved in the non-reducing sugar production of

membranes and macromolecules, in addition to inhibiting

and attenuating ROS production (Fig. 5). During rehydra-

tion, P. orbicularis would generate the energy needed to

recover functions that had been depressed during desicca-

tion and to control the ROS derived from the reactivation

of metabolic and photosynthetic processes (Fig. 5). The

transcriptional regulations and pathway interactions that

confer desiccation tolerance to P. orbicularis were found

deficient in desiccation-sensitive seaweed species. Due to

this, these species are unable to mitigate the structural and

oxidative damage generated during desiccation stress.

Planta (2017) 245:1149–1164 1161

123

Author contribution statement LC-P and CF conceived

and designed the research. CF, CL, and EG conducted the

experiments. AM, CL-C, EG, and FC contributed to the

analytical procedures. LC-P and CF analyzed the data. LC-P,

CL-C, and FC wrote the manuscript. All authors read and

approved the manuscript. The comments and suggestions of

anonymous reviewers aided in improving the final version of

the manuscript, and their assistance is deeply appreciated.

Acknowledgements This work was supported by FONDECYT

1120117, and DI-501-14/R and DI-1245-16/R (Universidad Andres

Bello) awarded to LC-P. The authors acknowledge language support

provided by Ashley VanCott (BioPub, Chile).

Compliance with ethical standards

Conflict the interest The authors have declared no conflict of

interest.

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