Responses of cyclic phosphorylation of MAPK-like proteins in intertidal macroalgae after...

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Journal of Plant Physiology 171 (2014) 276– 284

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Journal of Plant Physiology

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Responses of cyclic phosphorylation of MAPK-like proteins inintertidal macroalgae after environmental stress

María L. Parages ∗, Juan M. Capasso, F. Xavier Niell, Carlos JiménezDepartment of Ecology, Faculty of Sciences, University of Málaga, 29071 Málaga, Spain

a r t i c l e i n f o

Article history:Received 5 July 2013Received in revised form 26 August 2013Accepted 27 August 2013Available online 11 October 2013

Keywords:High irradianceIntertidal macroalgaeLow tideMAP kinasesStress

a b s t r a c t

The presence and activation of MAPK-like proteins in intertidal macroalgae is described in the currentstudy. Two MAPK-like proteins of 40 and 42 kDa in size similar to p38 and JNK, of mammalian cells havebeen identified in six representative species of intertidal macroalgae from the Strait of Gibraltar (South-ern Spain), namely in the chlorophytes Ulva rigida and Chaetomorpha aerea, the rhodophytes Corallinaelongata and Jania rubens, and the phaeophytes Dictyota dichotoma and Dilophus spiralis. Phosphorylationof MAPK-like proteins was studied during semi-tidal cycles. Analysis of p38-like and JNK-like MAPKsin macroalgae protein extracts was carried out by using specific antibodies against the phosphorylatedforms of both MAPKs. Protein blot analysis of samples collected from 2009 to 2011 in natural growingsites on days when either low or high tide occurred at midday, indicated that MAPK-like proteins in allspecies were highly phosphorylated in response to desiccation imposed by low tide or high irradiance.Phosphorylation of p38-like MAPK always preceded that of JNK-like MAPK. In addition, phosphorylationof MAPKs was fastest in rhodophytes, followed by chlorophytes and then finally phaeophytes. In the firstgroup, phosphorylation was mostly dependent on desiccation, whereas both high irradiance and desic-cation were responsible for p38-like and JNK-like phosphorylation in chlorophytes. In phaeophytes, highirradiance was mostly responsible for MAPK-like activation.

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Introduction

Algal growth, development, reproduction and productivity aredrastically affected by changes in temperature, humidity, salinity,irradiance (both PAR and UV radiation) and nutrient availability.Among algal communities, intertidal macroalgae are permanentlyexposed to a constantly fluctuating and potentially stressful envi-ronment. In addition, intertidal systems are highly variable and canbe divided according to the degree of air exposure. As an extremelyharsh environment, organisms inhabiting intertidal systems mustbe very well-adapted to the severe conditions, and natural selectionhas ensured survival of intertidal macroalgae that have developedacclimation strategies in response to dehydration, high irradiation,high UV, etc.

In general, plants can survive in constantly changing environ-ments due to complex metabolic networks capable of detectingstress signals that allow them to respond appropriately and adapt tonew conditions. This sensing of stress signals and their transductioninto appropriate responses is crucial to the acclimation and survivalof intertidal macroalgae. These stress response mechanisms must

∗ Corresponding author. Tel.: +34 952 13 1844; fax: +34 952 73 86.E-mail address: [email protected] (M.L. Parages).

be rapid as well as perfectly regulated. If not, intertidal macroalgaewould be seriously damaged or even die during periods of low tide.

In animal cells and yeast, the described stress mechanismshave been extensively characterised to involve the activationof a well-regulated signalling network, where a central role isplayed by a specific group of cytoplasmic phosphoproteins calledMAPKs (mitogen-activated protein kinases). Transduction of exter-nal signals to the cell nucleus occurs through a series of MAPKphosphorylation events, leading to expression of specific groupsof genes (Kyriakis and Avruch, 2001). Specifically, extracellularstimuli initiate cascades of events that can entail activation of oneor more subsets of MAPK families (Widmann et al., 1999). Theseproteins participate in response to a wide range of environmentaland extracellular stimuli, such as growth factors, drought, UV radi-ation, osmotic changes, temperature stress and so on. Ultimately,they are involved in the activation of several cellular programmes,such as proliferation, motility and programmed cell death. Jonaket al. (1999) determined that MAPKs are ubiquitously expressed,and their sequences are highly conserved with serine/threonineresidues in the active core (Kyriakis and Avruch, 2001). All eukary-otic cells possess multiple MAPK pathways. To date, five distinctgroups of MAPKs have been characterised in vertebrates: extracel-lular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases(JNKs), p38 isoforms, ERKs 3/4; and ERK5 (Cobb, 1999; Davis, 2000;Nebreda and Porras, 2000; Widmann et al., 1999). Conversely, the

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M.L. Parages et al. / Journal of Plant Physiology 171 (2014) 276– 284 277

complete genome sequence of Saccharomyces cerevisiae indicatesthat six different MAPKs exist in these organisms. The most exten-sively studied groups of vertebrate MAPKs are the ERK1/2, JNKs andp38 kinases. In yeast, the HOG1 cascade (similar to the p38 cascadein vertebrates) is the most characterised (Widmann et al., 1999).JNKs were identified based on their ability to specifically phos-phorylate the transcription factor c-Jun, mediating transcription ofspecific genes in response to exposure to ultraviolet radiation, pro-inflammatory cytokines and environmental stress. The p38 MAPKsare activated in response to endotoxin from Gram-negative bacte-ria, interleukin-1 and hyperosmotic stress, while the ERK cascade issolely involved in mitogenic stimuli and differentiation (Widmannet al., 1999).

Recent studies have shown that MAPK signalling cascades arealso present in vascular plants, such as Arabidopsis, tobacco and rice(Nakagami et al., 2005). However, while the ERK, JNK and p38 MAPKsubfamilies are present in animal cells, plant kinase genes appearto belong solely to the ERK subfamily (Zhang et al., 2006). PlantERK-like MAPKs have been further subdivided into at least fourdistinct subgroups (Machida et al., 1997; Ligterink, 2000; Nakagamiet al., 2005; Zhang et al., 2006). Subgroups I and II are believedto be involved in environmental and hormonal response, while,according to Bögre et al. (1999, 2000), a few MAPKs of subgroup IIIappear to be involved in cell cycle regulation.

The presence of MAPK-like pathway components in algae hasbeen previously described by our group (Jiménez et al., 2004,2007). In these previous studies, it was revealed that the microalgaDunaliella viridis possesses a 57 kDa protein (detected by cross-reactivity with mammalian p38 MAPK antibody and even moreso with the antibody against the yeast homologue, HOG1), whichwas induced in response to increasing tonicity. Furthermore, expo-sure of D. viridis to hypertonicity was quickly followed by rapidphosphorylation of this 57 kDa protein, which was detected usinga highly specific antibody raised against the phospho-p38 MAPKmotif (T*GY*). The possibility that JNKs were activated in D. viridisby environmental stresses was also investigated. Using a highlyspecific antibody raised against the phospho-JNK motif (T*PY*), apattern of three protein bands was detected and resembled thatfound in mouse inner medullary collecting duct cells (Berl et al.,1997). Lastly, expression of ERK1/2 in D. viridis was also detected,and their role in control of cell division was demonstrated. In addi-tion, partial cloning of ERK1/2 MAPKs showed a high degree ofhomology with other previously described ERK1/2 MAPKs from awide variety of different animal cells (Jiménez et al., 2007). Specificinhibitors of MAPK activity impaired cell division and had delete-rious effects on the ability of the algae to overcome hyperosmoticstress, revealing the critical role of MAPKs in acclimation and sur-vival of D. viridis under such environmental conditions.

Macroalgae, as any other organism, must respond to changesin the environment in order to survive. The intertidal zone ischaracterised by large environmental variations, primarily due toalternation of emersion and immersion periods (Connan et al.,2007). In such a hostile environment, organisms are subjected toimmense stress. Although well-adapted to the harsh conditionsimposed by emersion/immersion cycles, the severity and frequencyof environmental fluctuations ultimately dictate the survival strate-gies undertaken by inhabitants of the intertidal zone.

Recently, a procedure has been developed to detect the pres-ence of MAPK homologues in several species of Chlorophyta,Rhodophyta and Phaeophyta (Parages et al., 2012) revealing thatphospholyration of p38- and JNK-like MAPKs also occurs. Fur-thermore, phosphorylation of p38- and JNK-like MAPKs occursin Arctic kelps in response to increasing UV exposure and tem-perature (Parages et al., 2013). Taken together with our previousresults, we hypothesise that intertidal macroalgae cyclically phos-phorylate and dephosphorylate p38-like and JNK-like MAPKs in

response to tidal movements. The current study investigates thepresence of homologues of the p38 and JNK MAPK cascades inthe intertidal macroalgae Ulva rigida, Chaetomorpha aerea, Dicty-ota dichotoma, Dilophus spiralis, Jania rubens and Corallina elongata.Moreover, we sought to determine the phosphorylation status ofthese kinase homologues in response to environmental stressestypically imposed at low tide and by high irradiance.

Materials and methods

Working site and sampling

Ulva rigida C. Agardh, Chaetomorpha aerea (Dillwyn) Kütz-ing, Dictyota dichotoma (Hudson) J.V. Lamouroux, Dilophus spiralis(Montagne) G. Hamel, Jania rubens (Linnaeus) J.V. Lamouroux andCorallina elongata J. Ellis and Sollander were collected from theintertidal system of Punta Carnero, Algeciras (36◦04′ N, 05◦25′ W)in the Strait of Gibraltar in Southern Spain. In these coasts, tides aresemidiurnal, and a tidal semiperiod lasts from 6 to 7 h with a tidalamplitude of approximately 1 m. In addition to the long emersionperiods in this region of Southern Spain, air temperatures averagemore than 30 ◦C during the daytime in the summer while the win-ter mean air temperature is around 15 ◦C. Air temperatures above30 ◦C are too high to permit adequate metabolism in aquatic plantsexposed to the air at low tide in temperate zones (Niell et al., 1989).

The structure of the intertidal system in the transit regionbetween the Atlantic Ocean and the Mediterranean Sea comprisesseveral levels, ranging from supralittoral to infralittoral zones(Fernández, 1982; Niell et al., 1989). All species used in this workwere harvested from the mid and low horizons of the eulittoralzone. Due to severe conditions in the intertidal systems of SouthernSpain, macroalgae are nearly entirely absent from the upper eulit-toral horizon, where mostly dwarf Fucus spiralis form conspicuouspopulations. In the mid eulittoral zone, there is a horizon between0 and 0.6 m above mean sea level where C. elongata is the mostabundant species. All the species used in the current work werecollected from the described horizon in the mid eulittoral zone. U.rigida was found forming patches in the mid-upper part of the hori-zon. C. aerea was distributed between 10 and 35 cm above mean sealevel, whereas J. rubens was located slightly higher between 15 and45 cm above mean sea level. Although occasionally found in pondsformed at low tide, D. dichotoma and D. spiralis were predominantlyrestricted to a very narrow belt at 50–55 cm. C. elongata was equallydistributed throughout the horizon.

Radiation data were continuously collected using a NILU-UV6multifilter radiometer (NILU Products AS, Norway).

Whole thalli of the six described species were harvested fromgrowing sites at different times during high and low tides. Due topopulation dynamics all species were not present throughout theentire year, then different but similar species of the same groupwere used at the different seasons of the year. U. rigida, C. elongataand D. dichotoma were used in spring and summer experiments,while C. aerea, J. rubens and D. spiralis were used in autumn andwinter.

Epiphytes were removed on site, and thalli were immediatelyplaced into liquid N2 for transport. Specimens were kept at −80 ◦Cuntil further analysis.

Protein extraction, electrophoresis and immunoblot analysis

Frozen thalli were ground using a Retsch MM400 mixer mill(Retsch, Haan, Germany). Proteins were extracted according themethod recently described by Parages et al. (2012), using a com-mercial RNA purification kit (RNeasy Plant Mini Kit, Qiagen, Hilden,Germany) to yield clean protein extracts, avoiding interference


278 M.L. Parages et al. / Journal of Plant Physiology 171 (2014) 276– 284

Fig. 1. Dot blot analysis of the phosphorylated forms of MAPK-like proteins detected by specific antibodies against phospho-p38 and phospho-JNK in (A) the absence (−BP)and (B) presence (+BP) of specific blocking peptides. Dun: Dunaliella, Cor: Corallina, Dict: Dictyota, Dil: Dilophus, Ulv c: Ulva compressa, Jan: Jania, Ulv r: Ulva rigida.

from polyphenols, acidic polysaccharides and nucleic acids. Fur-thermore, the phosphorylation state of protein kinases is preserved.Protein extracts were stored at −80 ◦C until further analysis.

Dot blots were carried out using Schleider and Schnell dot blotequipment (Bio-Rad Laboratories). PVDF membranes were pre-activated with 100% methanol for 15 s and washed with ddH20for 5 min. Membranes were then kept for 3–5 min in PBS 1×, afterwhich 200 �l of 1 mg/ml protein extract (200 �g of protein) wasloaded into each well. Vacuum pressure (400 mbar) was applied,then membranes were processed according to standard protein blotprocedures as described in detail below.

Gel electrophoresis, immunoblotting and detection of phos-phorylated MAPKs were also performed according to methodsdescribed by Parages et al. (2012). Protein blots were analysedusing Amersham ECL Advance (GE Healthcare, Buckinghamshire,UK) as chemoluminiscence agent and visualised on a Kodak GelLogic 1500 Imaging System (Eastman Kodak Company, Rochester,NY, USA). All antibodies and blocking peptides were purchasedfrom Cell Signaling Technology (Beverly, MA, USA) and used accord-ing to manufacturer’s recommendations. Band intensity analyseswere carried out using ImageJ 1.440 software (National Institute ofHealth, USA).

Results

Detection of MAPK-like proteins in intertidal macrophytes

Dot blots from protein extracts of C. elongata, D. dichotoma,D. spiralis, U. compressa, J. rubens and U. rigida were performedto determine the presence of MAPK-like proteins using antibod-ies raised against the phosphorylated forms of p38 and JNK frommammalian cells. D. viridis extracts were used as a positive control(Fig. 1). In all species from the three groups of macroalgae, specificcross-reaction of p38 and JNK antibodies was observed (Fig. 1A).Significant variation between species was noted. The highest dotintensity was observed in U. compressa protein extracts, while C.elongata extracts had the lowest observed intensity.

Nevertheless, this cross-reactivity may not be sufficient evi-dence to support the presence of MAPK-like proteins in themacroalgae. Thus, in order to confirm initial observations, bindingbetween proteins immobilised on the PVDF membrane and primaryphospho-antibody was blocked using specific blocking peptidesthat bound to the specific primary antibodies. Inhibition of bindingto phosphorylated p38-like or JNK-like proteins immobilised onthe membrane from protein extracts permitted evaluation of thespecificity of antibody reactivity. As shown in Fig. 1B, a significantreduction in signal intensity of the bands occurred in all species,indicating the presence of both p38-like and JNK-like MAPKs inthese intertidal macroalgae. In U. compressa, a partial signal was stilldetected, but may simply be attributed to the extremely high signalinitially observed in the non-blocked dot blots. Intensity reductionof the dot was determined to be nearly 90% in U. compressa. Forall other species, dot blot signal was reduced nearly 100% in thepresence of blocking peptides.

Induction of phosphorylation of MAPK-like proteins during lowtide

The behaviour of p38- and JNK-like phosphorylation in the sixdescribed macroalgae was analysed separately based on the occur-rence of low tide and high tide during midday. Since some specieswere not present throughout the entire year, different species wereused during the different seasons of the year.

Fig. 2 shows the response of p38- and JNK-like proteins in thechlorophyte C. aerea during a semi-tidal cycle. In the top panel,temporal changes in protein band intensity as well as PAR, UV-A and UV-B radiation are displayed. The middle inset illustratesprotein bands detected in the presence (+BP) and absence (−BP)of specific blocking peptides, while the bottom inset summarisesthe height of the tides throughout the daily sampling period. Lowtide occurred at approximately 11:00 h, and maximum radiationexposure occurred between 12:00 and 14:00 h. Very low levels ofphosphorylation of both p38-like (Fig. 2A) and JNK-like (Fig. 2B)proteins were detected during the early morning. However, 1.5 hafter initial exposure of C. aerea to the air at low tide (exposure



Fig. 2. Temporal changes in phosphorylation of (A) p38 and (B) JNK MAPK-like proteins in the absence (−BP) and presence (+BP) of specific blocking peptides in C. aerea duringlow tide. Top panel: Irradiance curves of PAR, UV-A and UV-B radiation, as well as protein gel blot band intensity. Middle panel: Protein gel blot analysis using phospho-MAPKantibodies. Bottom panel: Height of the tides and a semi-quantitative estimate of the degree of phosphorylation of the MAPK-like proteins.

began between 09:00 and 09:30 h), very high levels of a phospho-rylated 40 kDa protein were detected by the antibody raised againstphospho-p38 MAPK (Fig. 2A). After the initial peak that coincidedwith the lowest tide, residual phosphorylation was detectable forseveral hours following the initial peak, indicating that increasingirradiance (either PAR or UVR) may induce phosphorylation of this40 kDa p38-like protein. Signal reduction with the use of a specificblocking peptide provided further evidence that the 40 kDa proteinwas, in fact, similar to mammalian phospho-p38. In addition, non-specific cross-reactivity was ruled out by incubating the blots withsecondary antibody only, after which no signal was detected (datanot shown).

C. aerea protein extracts were also analysed for the presence ofJNK-like MAPKs. As shown in Fig. 2B, a conspicuous band 42 kDa insize was detected by the phospho-JNK antibody. The 42 kDa phos-phoprotein was present at very low levels during high tide, butincreased significantly for 1 h after the lowest tide. Interestingly,phosphorylation of this JNK-like protein peaked 1 h after (12:00 h)the peak expression of the 40 kDa p38-like protein (Fig. 1). When C.aerea was first covered by water as the tide rose, phosphorylation ofthe JNK-like protein decreased to levels similar to those in the earlymorning hours. The use of a specific blocking peptide reduced bandintensity to nearly undetectable levels, corroborating the nature ofthe phosphorylated protein. Phosphorylation of the 42 kDa JNK-likeMAPK was extremely transient, peaking 1 h after the lowest tide.Conversely, the p38-like MAPK exhibited maximum phosphoryla-tion during lowest tide and persisted during hours of maximumirradiance.

Similar results were obtained from the phaeophyte D. spiralis(Fig. 3) and the rhodophyte J. rubens (Fig. 4). Specifically, in D. spiralisnearly undetectable levels of phospho-p38 occurred during the firsthours of the morning (Fig. 3A). However, a very strong 40 kDa bandappeared between 12:00 and 13:00 h then decreased significantlyas the afternoon progressed. For the JNK-like protein (Fig. 3B),

elevated levels of a 42 kDa phosphoprotein were detectable for alonger period of time and began to decline at 14:00 h. Similar toC. aerea, the emersion period lasted from 09:00–09:30 to 13:00 h,and maximum intensity of the JNK-like phosphoprotein occurred1 h after maximum expression of the 40 kDa p38-like phosphopro-tein. Lastly, negligible levels of both the 40 kDa and 42 kDa bandswere detected in the presence of specific blocking peptides, pro-viding further support of the homology between these proteins inD. spiralis and their corresponding MAPKs in mammalian cells.

J. rubens behaved more similar to C. aerea than D. spiralis (Fig. 4).Specifically, levels of the 40 kDa p38-like phosphoprotein showed adramatic increase shortly before the lowest tide, peaking at approx-imately 10:00 h (Fig. 4A). Expression then progressively decreasedto undetectable levels with no further activation observed duringmidday exposure to maximum irradiance. In contrast to C. aereaand D. spiralis, which remained air-exposed for nearly 4 h due totheir low position in the eulittoral zone, the emersion period for J.rubens could last for more than 6 h from 08:00 to 14:30 h. As forthe 42 kDa JNK-like phosphoprotein in J. rubens, expression peakedduring lowest tide between 11:00 and 12:00 h, which was 1–2 hafter peak of the 40 kDa p38-like phosphoprotein, regardless of theirradiance level (Fig. 4B). Similar to results from all other reportedmacroalgae, blocking peptides resulted in nearly complete inhibi-tion of the signal detected by protein blot.

Induction of phosphorylation of MAPK-like proteins during hightide

As previously mentioned, the behaviour of p38- and JNK-likephosphorylation was analysed separately based on the occurrenceof low tide and high tide during midday. As explained previously,different species were used throughout different periods of the yeargiven that most species are not perennial. To represent chloro-phytes, U. rigida was selected for the described experiments. Fig. 5



Fig. 3. Phosphorylation in D. spiralis of (A) p38 and (B) JNK MAPK-like proteins in the absence (−BP) and presence (+BP) of specific blocking peptides during low tide. Contentof the panels is identical to Fig. 2.

Fig. 4. Phosphorylation in J. rubens of (A) p38 and (B) JNK MAPK-like proteins in the absence (−BP) and presence (+BP) of specific blocking peptides during low tide. Contentof the panels is identical to Fig. 2.



Fig. 5. Phosphorylation in U. rigida of (A) p38 and (B) JNK MAPK-like proteins in the absence (−BP) and presence (+BP) of specific blocking peptides during high tide. Contentof the panels is identical to Fig. 2.

Fig. 6. Phosphorylation in D. dichotoma of (A) p38 and (B) JNK MAPK-like proteins in the absence (−BP) and presence (+BP) of specific blocking peptides during high tide.Content of the panels is identical to Fig. 2.



Fig. 7. Phosphorylation in C. elongata of (A) p38 and (B) JNK MAPK-like proteins in the absence (−BP) and presence (+BP) of specific blocking peptides during high tide.Content of the panels is identical to Fig. 2.

shows the temporal expression of p38-like and JNK-like phospho-proteins in U. rigida during a semidiurnal tide cycle. For the 40 kDap38-like phosphoprotein (Fig. 5A), three transient phosphorylationperiods were detected. The first, at 10:00 h, coincided with the finalhour of emersion of the thalli. The second phosphorylation periodbetween 13:00 and 17:00 h (peaking at 14:00 h) corresponded tothe time of maximum irradiance. Lastly, the third period, whichpeaked at 20:00 h, was caused by a second emersion period. Simi-lar results were obtained for the JNK-like phosphoprotein (Fig. 5B).As before, the peak of phosphorylation of the JNK was delayed incomparison to the corresponding peak of the p38-like phospho-protein. In all cases, very faint bands were detected when blockingpeptides were used.

The second species used for the “high tide” experiments wasthe phaeophyte Dictyota dichotoma. A clear peak of phosphor-ylation of a 40 kDa protein was detected by the phospho-p38antibody at the beginning of the emersion period (Fig. 6A).Minor levels of phosphorylation were detected throughout therest of the day until a second peak appeared during the secondemersion period at 21:00 h during late sunset. The phosphoryla-tion/dephosphorylation cycle of the MAPKs was most evident whenusing the phospho-JNK antibody (Fig. 6B). An initial peak of phos-phorylation of a 42 kDa protein was detected at the end of themorning emersion period. A second intense peak was detected2 h after maximum irradiance. Lastly, a third and final peak wasdetected in the late afternoon at the start of a new emersionperiod. As with all previous species, band intensity was significantlyreduced in the presence of specific blocking peptides.

High tide studies were performed using Corallina elongata asa representative rhodophyte. No protein phosphorylation wasdetected by the phospho-p38 antibody during the first few hours

of sampling, but two distinct transient peaks of phosphorylation ofa 40 kDa protein were detected later on during the day at the timeof maximum irradiance and, finally, at the onset of the eveningemersion period (Fig. 7A). As for a JNK-like phosphoprotein, asimilar temporal expression pattern was observed. While just asmall increase was detected at the time of maximum irradiance, asharp and transient increase in phosphorylation of a 42 kDa proteinoccurred at the onset of the evening emersion period.

In all species studied a 40 kDa p38-like MAPK was transientlyphosphorylated as a result of either increased radiation or air expo-sure during low tide. Phosphorylation of a 42 kDa JNK-like MAPKwas also transiently induced by the same environmental condi-tions. However, clear differences were observed between low andhigh tide, as well as between algal groups. As a general rule, phos-phorylation of the p38-like MAPK preceded that of the JNK-likeMAPK, which typically occurred 1–2 h after peak detection of thep38-like phosphoprotein.

Discussion

In the current study, macroalgae from an intertidal systemof Southern Spain showed clear protein cross-reactivity withvery specific antibodies against the phosphorylated forms of p38and JNK, which are established players in the mammalian stressresponse. The observed response of both MAPK-like proteins in thenatural environment varied depending of the species of macroal-gae. The rhodophytes (Jania rubens and Corallina elongata) showedthe fastest response after onset of the emersion period, followedby the chlorophytes (Chaetomorpha aerea and Ulva rigida) and,finally, the phaeophytes (Dilophus spiralis and Dictyota dichotoma)(Fig. 8). In rhodophytes, temporal analysis of the expression of



Fig. 8. Phosphorylation levels of (A) p38 and (B) JNK MAPK-like proteins inRhodophyceae, Chlorophyceae and Heterokontophyceae from onset of the emer-sion period. The vertical radius of each oval is a semi-quantitative estimate of thedegree of phosphorylation.

the p38-like phosphoprotein (Fig. 8A) showed initial detection just30–60 min after onset of the emersion period. However, in phaeo-phytes, phosphorylation was first detected 3 h after initial exposureto air, peaking 1.5 h later. Chlorophytes showed a somewhatintermediate response, with maximum phosphorylation of p38-like MAPK occurring 3 h after emersion of the thalli. For JNK-likeMAPK phosphorylation, a similar pattern was observed (Fig. 8B). Inaddition, phosphorylation of the p38-like protein always precededphosphorylation of the JNK-like protein.

The selected rhodophytes, C. elongata and J. rubens, possesscalcified thalli. Based on Figs. 4 and 7, it can be concluded that des-iccation is a primary inducer of stress in these species at low tide.When both types of irradiance stress (UV + PAR) and desiccationwere concurrent (e.g. low tide at midday), a distinct peak in phos-phorylation of both p38-like and JNK-like proteins was detected.This peak activation was not observed when high tide occurredduring midday. Taken together, calcification appeared to play aprotective role against PAR and UVR, providing the layer of cal-cium carbonate acts as a shield to solar radiation (Beach et al.,2006). Sharp peaks in MAPK-like protein phosphorylation duringthe late evening (i.e. rhodophytes are air-exposed with very lowirradiance levels) provides further evidence of the crucial role des-iccation plays in stress induction (Fig. 7). High light-induced stresscan be ruled out in the late evening. Thus, desiccation (togetherwith other environmental factors not controlled in this work) plays

the predominant role in MAPK induction. This peak of phosphor-ylation was much more pronounced than the one detected duringmidday at high tide, where high irradiance was the main envi-ronmental stress factor. Calcified macroalgae are highly resistantto biotic and abiotic disturbances. As such, they are some of thefew primary producers able to occupy higher levels of the inter-tidal system, where strong wave forces and intense herbivoryoccur (Beach et al., 2006), presenting advantageous strategies thatallow them to perfectly tolerate high irradiance. However, dur-ing long emersion periods they are very susceptible to waterloss.

The representative chlorophytes, C. aerea and U. rigida, usedin this work showed a different response than the rhodophytes.These particular species showed increased phosphorylation of p38-like and JNK-like MAPKs at both low tide and high irradiance, yetdesiccation did not appear to have a significant effect. High irra-diance at midday induced a drastic increase in phosphorylationof both MAPKs, either at high or low tide, indicating the abilityof these species to cope well with large water losses. However,in all cases, the greatest level of phosphorylation was detectedwhen emersion and maximum irradiance coincided. These speciesappeared to be vulnerable to both desiccation and high irradiance,but showed a high capacity to quickly recover from desiccation orexposure to PAR and UV. It is well known that Ulva species (i.e.U. lactuca) are highly susceptible to desiccation during emersiondue to their thin sheet-like thallus morphology (Zou et al., 2007).Desiccation may affect reactions catalysed by enzymes located atthe water/membrane interface, such as ATPase or soluble enzymes(Kaiser et al., 1987), and excessive loss of water may lead toincreased surface pH, resulting in reduction or cessation of pho-tosynthesis (Bidwell and McLachlan, 1985). According to Häderet al. (1999), algae with flat thalli suffer more pronounced pho-toinhibition than algae with massive thalli, even if the former aresun-adapted plants. However, the former ones are able to startrecovering from high irradiance as soon as the source of stress issuppressed, while massive thalli from shaded sites do not recoverfrom the effects, even after prolonged periods of time.

The phaeophytes D. spiralis and D. dichotoma behaved mostlysimilar to chlorophytes than to rhodophytes. Specifically, they wereshown to be sensitive to high UV and PAR, but not to desiccation.This made that phosphorylation of MAPKs was less pronouncedthan in chlorophytes. Resistance to desiccation in brown algaemay be related to the presence of polyphenols in specialisedvesicles called physodes (Baardseth, 1958). No anatomical fea-tures or impermeable cuticles similar to terrestrial vascular plantsthat might effectively protect them against water loss have beendescribed in phaeophytes that could explain their high resistanceto dessication (Oates, 1985, 1986; except in the case of saccatealgae such as Colpomenia and Halosaccion; Lüning, 1990). Thesepolyphenols consists of a single structural class, the phlorotan-nins, which are formed by the polymerisation of phloroglucinol.In addition, physodes may contain polysaccharides (i.e. fucoidan).Numerous reports since the beginning of the 20th century showdiurnal variations in the number of physodes and fucoidan content(Pontillon, 1926), as well as seasonal variations in physode num-ber (Hunger, 1902; Evans and Holligan, 1972a,b). At low tide, thesestudies have also shown that air exposure can lead to accumula-tion of significant amounts of fucosan in physodes. Conversely, athigh tide, most of fucosan was found outside of the physodes, sug-gesting that these variations may be caused by desiccation ratherthan by changes in intensity of incident light. The high content ofpolyphenols and polysaccharides in brown algae favours formationof a mucilaginous layer with high water retention capabilities. Thenwater loss during emersion is retarded. In addition, these organicosmolytes participate in the maintenance of intracellular osmoticpressure during desiccation and other forms of water stress. A very



well-known compatible solute in phaeophytes is d-mannitol,which is found in the Laminariales, Dictyotales and Fucales species,aiding retardation of water loss. Moreover, accumulation of smallorganic compounds is part of the physiological and biochemicalmechanisms used by macroalgae to adapt or tolerate water lossduring emersion (Sáiz, 1984; Hand and Menze, 2007). Water reten-tion capacity and the ability to maintain osmotic pressure due tothe presence of certain osmolytes at low tide may delay the onsetof desiccation stress and MAPK phosphorylation.

In summary, the current study demonstrate that MAPK-likeproteins are involved in the response of intertidal macroalgae toenvironmental stresses imposed by low tide and/or high irradiance,and that phosphorylation/dephosphorylation of several kinases(p38-like and JNK-like MAPKs) occurs under these conditions.

The phosphorylation response of MAPKs (40 kDa p38-like pro-tein and 42 kDa JNK-like protein) in the natural environmentdiffered between MAPKs and varied between macroalgae species.Rhodophytes (J. rubens and C. elongata) showed the fastest responseafter onset of the emersion period. This phosphorylation occurredat the moment of lowest tide regardless of irradiance levels. Chloro-phytes (C. aerea and U. rigida) had peak MAPK-like phosphorylationwhen low tide coincided with maximum irradiance. Lastly, thetemporal phosphorylation pattern determined in Phaeophytes (D.spiralis and D. dichotoma) was likely reduced at low tide com-pared to other algal groups due to their higher water retentioncapacity.

Further studies are been carried out to demonstrate direct impli-cation of MAPKs cascades phosphorylation and survival of themacroalgae under high irradiance and UV radiation, by means ofspecifically blocking the phosphorylation pathways.

Acknowledgments

This work has been supported by the Ministry of Science andInnovation of Spain, Grants Nos. CGL05-01071/BOS and CTM2011-24007 to C.J., and CTM-08-04453 to F.X.N. Radiation data wereprovided by the Photobiology Unit of the University of Málaga.The authors gratefully acknowledge the skilful technical assistanceduring fieldwork of V. Meco, T. Camarena, G. Amaya and C. Íniguez.Authors are indebted to Proof-Reading-Service.com for manuscriptcorrection.

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