(PDF) MAPK in Arctic kelps

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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy Environmental and Experimental Botany 91 (2013) 30–37 Contents lists available at SciVerse ScienceDirect Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot Rapid phosphorylation of MAP kinase-like proteins in two species of Arctic kelps in response to temperature and UV radiation stress María L. Parages a , Sandra Heinrich b , Christian Wiencke b , Carlos Jiménez a,∗ a Department of Ecology, University of Málaga, 29071 Málaga, Spain b Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany a r t i c l e i n f o a b s t r a c t Article history: Mitogen-activated protein kinases (MAPKs) are a group of cytoplasmic phosphoproteins that constitute Received 17 October 2012 the central core of the signalling network to respond to stress in most organisms. Their role in stress Received in revised form 31 January 2013 responses has been extensively studied in organisms from yeast to humans, and recently, their presence Accepted 5 February 2013 has also been described in higher plants as well as in micro- and macroalgae. In this study, we demon- strate via short experiments (1 h in duration), the rapid activation of two MAPKs similar to p38 and JNK Keywords: of mammalian cells, in the Arctic kelps Laminaria solidungula and Saccharina latissima exposed to tem- MAP kinases perature and UV stress. The molecular mass of p38 is 40 kDa in L. solidungula and 42 kDa in S. latissima, Arctic macroalgae Laminaria solidungula while two JNKs were detected in both species, of 36 and 42 kDa in L. solidungula, and 36 and 40 kDa in S. Saccharina latissima latissima. These MAPKs are highly phosphorylated in response to temperature and UV light. In S. latissima, UV stress both p38 and the JNK showed higher phosphorylation at 2 ◦ C than at 7 ◦ C, while the reverse response was Temperature stress shown for L. solidungula. In addition, a significant increase in phosphorylation of both kinases was found following exposure to UV radiation (UVR). Exposure to PAR + UVA + UVB induced higher phosphorylation than PAR + UVA in L. solidungula, especially at 7 ◦ C. In S. latissima, this response occurred only with JNK, and no differences in p38 phosphorylation between PAR + UVA and PAR + UVA + UVB at any temperature were observed. These results indicate the possible participation of MAPK-like proteins in response to stress in Arctic kelps, and that their activation is species-specific. © 2013 Elsevier B.V. All rights reserved. 1. Introduction have been proposed to exist in mammalian cells, for only three of which (p38, c-Jun and ERK) all the components have been com- All living organisms face changes in their environment, and pletely identified. These three cascades are involved in response must successfully respond to them in order to survive. To achieve, to a variety of stress conditions (i.e. UV radiation, heat, osmo- they have developed a complex signalling network that senses and larity, growth factors, hormones, etc.) and in the control of cell protects them in an ever-changing environment. Considerable evi- proliferation and differentiation. However, little information is dence has accumulated in recent years that has demonstrated that available for plants in general and for algae in particular, but an the central core of this signalling network is a group of cytoplasmic increasing number of reports indicate that different plant species phosphoproteins called mitogen activated protein kinases (MAPK). contain genes encoding MAPKs (around 20 MAPKs in Arabidop- Phosphorylation of the MAPKs leads to the activation of specific sis thaliana), MAPKKs, and MAPKKKs. All known plant MAPKs are groups of genes (Kyriakis and Avruch, 2001), allowing the cells similar to extracellular signal-regulated kinases (ERKs) (Yu and to survive under altered environmental conditions (Jiménez et al., Tang, 2004; Pitzschke et al., 2009) and are involved in various 2004, 2007, 2009). forms of biotic and abiotic stress responses, and not only in the According to Jonak et al. (1999), all eukaryotic cells possess mul- control of cell proliferation and division as in animal cells. Thus, tiple MAPK pathways. These pathways comprise a core module both p38-like and JNK-like cascades appear to be absent in higher of three protein kinases that are organised into functional units plants. or canonic tri-modules. These MAPKs, together with their acti- Even though the study of signal transduction in algae is vators (the so-called MAPK kinases – MAPKKs and MAPK kinase very recent, several studies with both macro- and microalgae kinases – MAPKKKs), form a cascade. To date, six MAPK cascades (Jiménez et al., 2004, 2007; García-Gómez et al., 2012; Parages et al., 2012) have clearly shown the presence of p38-like, JNK- like and ERK-like components, as well as their crucial role in ∗ Corresponding author. Tel.: +34 952134134. response to stress and in cell division (Jiménez et al., 2004, 2007; E-mail address:

(C. Jiménez). Parages, 2012). Parages (2012) demonstrated that blocking either 0098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.envexpbot.2013.02.005 Author's personal copy M.L. Parages et al. / Environmental and Experimental Botany 91 (2013) 30–37 31 the JNK-like or the p38-like pathways in stressed (high irradi- 2. Materials and methods ance, UV or desiccation) intertidal macrophytes leads to algal death. 2.1. Algal material, location and pretreatment The Arctic coastal ecosystem is one of the areas most affected by global warming (Zacher et al., 2011). It acts as a carbon sink, Young sporophytes of approximately 7–9 cm in length of S. contributing to 5–14% of the global CO2 balance (Bates and Mathis, latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W. Saun- 2009), with macroalgae being responsible for 62% of the total pri- ders and L. solidungula J. Agardh were collected by scuba divers mary productivity on these coasts (Rysgaard and Nielsen, 2006). at Kongsfjorden at Hansneset, Blomstrand Island (Blomstrand- The largest communities of benthic marine organisms in the Arc- halvøya), Svalbard, Norwegian Arctic (78◦ 39′ N, 11◦ 57′ E), between tic consist of kelp beds, mainly composed of macroalgae of the 4–6 m deep for S. latissima and 12–15 m deep for L. solidungula. order Laminariales. Among the most common species, Saccha- Thalli were transported to the laboratory in cold black plastic con- rina latissima is a perennial kelp that forms huge beds from polar tainers in the dark. After cleaning of the epiphytes, whole thalli to temperate latitudes (Bolton et al., 1983; Borum et al., 2002); were kept in 100 L containers under continuous air bubbling, and another conspicuous species on Arctic coasts is the endemic species continuous filtered seawater circulation at around 2 ◦ C under low Laminaria solidungula. There is great concern about the speed of light (10 ␮mol photons m−2 s−1 ). Thalli were preincubated under changes occurring in the Arctic, and how these will affect the these conditions for 3 d before performing experiments. aquatic environment, and especially endemic species or those with a small distribution range. Current increase in the ocean temper- ature caused by global warming as well as the increase in UV 2.2. Experimental design radiation (UVR) due to the development of an ozone hole over the Arctic might represent a real threat for sessile marine Arc- Experiments consisted of exposure of thalli of S. latissima and tic organisms. Research on the ecophysiology of polar macroalgae L. solidungula to increased temperature (7 ◦ C) and UVR for 1 h. has shown some of the effects of excess UVR, such as photoinhi- In order to avoid the induction of MAPK phosphorylation due to bition and eventual photodamage of the photosynthetic apparatus touching or wounding, thalli were carefully removed from the (Hanelt et al., 1997), impaired spore germination (Wiencke et al., preincubation containers, held by the stipe with plastic twee- 2004, 2006; Steinhoff et al., 2011), inhibition of DNA replication zers and placed in shallow (13 cm deep) rectangular containers (Wiencke et al., 2000; Pakker et al., 2000; van de Poll et al., 2002), (50 cm long × 15 cm wide) at the start of each experiment. Thalli and the production of reactive oxygen species, responsible for were provided with continuous air bubbling, and irradiated with a oxidative damage of cell components (Aguilera et al., 2002). It has combination of Osram-Biolux L36W/965 (Osram, Germany) and Q- been previously shown that a dramatic decrease in growth rate and Panel UVA-340 (Q-Panel Lab Products, Cleveland, OH, USA) lamps photosynthetic performance occurs in Arctic kelps exposed to UVR at the doses detailed in Table 1. To generate three different irradi- (Karsten et al., 2001; Roleda et al., 2006), and this reduction was ation conditions, three types of filter foils were used to selectively more pronounced in L. solidungula than in S. latissima (formerly L. exclude different wavelength ranges from the spectrum emitted saccharina; Roleda et al., 2006). L. solidungula occurs at depths of by the fluorescent tubes: two types of Ultraphan filters (Dige- 12–15 m in the Kongsfjord, where no biologically-significant UV- fra GmbH, Munich, Germany) with transmission at > 395 nm B irradiance reaches (Hanelt et al., 2001). In contrast, S. latissima (Ultraphan 395) and > 295 nm (Ultraphan 295) for only PAR (P) occupies shallower waters (2–12 m deep). Exposure to natural solar and PAR + UVA + UVB (PAB), respectively, and a Folex PR filter (Dr. radiation (7 d at 1 m depth) without UV-A/B was accompanied by Schleussner, Germany) with transmission at > 320 nm for the a small decrease in photosynthetic performance in L. solidungula, PAR + UVA (PA) treatment. Spectral irradiance under the filters was reaching 86% of the control value; treatment with UV-A led to a measured with a cosine sensor connected to a spectoradiometer value of 64%, while in the presence of UV-B, only 17% of maxi- 320D (Instrument Systems, Munich, Germany) (Fig. 1). To com- mum photosynthesis was reached (Karsten et al., 2001). Compared pare experimental light conditions to solar conditions in nature, to other brown algae, oxygen production by L. solidungula is also wavelength-dependent effectiveness of the UVR applied in each strongly inhibited by UV stress (Aguilera et al., 1999), indicating a treatment (biologically effective dose) was weighted using three high sensitivity to solar radiation. action spectra for some well-known biological responses, i.e. DNA The hypothesis to be tested in this study is whether cell systems damage for Escherichia coli (Setlow, 1974), generalised plant dam- for the control and response to environmental stress (tempera- age (Cadwell, 1971), and the inhibition of O2 evolution of isolated ture and UVR) via activation of MAPK-like components are present spinach chloroplasts (Jones and Kok, 1966) (Table 1). in these algae. Organisms living in polar regions have developed Two temperatures were tested, namely 2 ◦ C and 7 ◦ C, to analyse several unique strategies of adaptation and acclimation, differ- the effect of increasing temperature on MAPK activation in these ent to those already described for algae from temperate latitudes macrophytes. The former temperature is within the range found at (i.e. cold-adapted species have differential genetic expression as the surface in the sampling area at the harvesting time (May 2010). well as enzymatic activities to maintain high rates of enzyme- catalyzed reactions at low temperature, and modifications in the thylakoid membrane system as well as in the carbon acquisi- 2.3. PAM fluorometry tion mechanism; Ensminger et al., 2006; Gordillo et al., 2006; Gómez et al., 2011). Thus, the main objective of this study was The physiological fitness of both macrophytes was followed by to determine the presence of MAPK-like components in selected measuring the photosynthetic efficiency using PAM-fluorometry Arctic macroalgae, and to study their response to environmen- (Imaging PAM M-Series MAXI version; Heinz Walz GmbH, Effel- tal stress caused by increased temperature and UV radiation, trich, Germany). For this, optimal quantum yield (Fv /Fm ) was focusing on four partial objectives: (1) detection of the pres- measured at the beginning and at the end of the experiments in five ence of MAPKs (p38-like and JNK-like) in the Arctic kelps S. independent thalli, with three replicate measurements in different latissima and L. solidungula; (2) MAPK activation in response to parts of the blades to avoid the effect of blade heterogeneity. Results temperature increase; (3) MAPK activation under UV radiation of the photosynthetic measurements were analysed by two-way stress and (4) detection of interactive effects between both stress- ANOVA (P < 0.05). Significant differences as well as interaction of ors. means were compared with the post hoc Tukey test (HSD, P < 0.05). Author's personal copy 32 M.L. Parages et al. / Environmental and Experimental Botany 91 (2013) 30–37 Table 1 Experimental irradiance for each light treatment indicating the weighted irradiance applied according to three biologically effective weighting functions for UV effects. Treatments Experimental irradiance (W m−2 ) Weighted irradiance (W m−2 ) UVB UVA PAR (␮mol m −2 s −1 ) Chloroplast inhibition Generalised plant DNA damage (Jones and Kok, 1966) damage (Cadwell, 1971) (Setlow, 1974) Ultraphan 295 0.48 5.01 23.41 1.723 0.011 0.03 Folex 320 0.04 4.13 22.57 1.138 0.0496 × 10−4 3.4 × 10−4 Ultraphan 395 0.00 0.13 23.18 0.018 0.4271 × 10−4 1.2 × 10−4 2.4. Sample processing confirms the presence of proteins with epitopes similar to those used to raise the specific antibodies. Whole thalli were frozen in liquid N2 , ground by hand and Equal amounts of protein extracts (100 ␮g) were loaded in stored at −80 ◦ C until further processing. MAPK extraction, gel each lane. Western blots were analysed using a Kodak Gel Logic electrophoresis, immunoblot and phosphorylated MAPK detection 1500 Image System, using Amersham ECL Advance (GE Health- were performed according to Parages et al. (2012). This method is care, Buckinghamshire, UK) as a chemiluminescence agent. Band based on the use of commercial RNA purification kits (RNeasy Plant intensity analysis was performed using the ImageJ 1.440 free soft- Mini Kit, Qiagen; Hilden, Germany), which produce clean protein ware (National Institute of Health, USA). Band intensities of the extracts where interference with polyphenols, acidic polysaccha- blots were compared by means of Student’s t-tests and two-way rides and nucleic acids is avoided and the phosphorylation state of ANOVA, with three to five independent replicates for each data the protein kinases is kept intact. Protein content in the extracts point (P < 0.05). was estimated by means of the bicinchoninic acid method, after which extracts were maintained at −80 ◦ C until further analysis. 3. Results Antibodies (anti-phospho-p38 and anti-phospho-JNK of mam- malian origin) as well as their specific blocking peptides (BP) were 3.1. PAM fluorometry purchased from Cell Signaling Technology (Beverly, MA, USA), and used according to Parages et al. (2012) and the manufacturer’s rec- Fig. 2 summarises the results of the optimal quantum yield ommendations. The blocking peptides consist of small peptides (Fv /Fm ) for both S. latissima (Fig. 2A) and L. solidungula (Fig. 2B) after that bind to the specific primary antibodies (Ab), thus blocking the 1 h of exposure to P, PA and PAB, at 2 ◦ C and 7 ◦ C. Initial Fv /Fm values signals from the indicated phospho-p38 or phospho-JNK MAPKs were slightly, but significantly (P < 0.05), higher for L. solidungula and permitting the evaluation of the specificity of antibody reactiv- (0.68) than for S. latissima (0.56). Temperature increase following P ity. For their use, specific antibodies were incubated in the presence treatment for 1 h did not cause significant differences between 2 ◦ C of the blocking peptides for 30 min, after which, Western blot mem- and 7 ◦ C in either species, however, exposure to UVR induced a clear branes were exposed to the mixture Ab + BP as for a standard and significant decrease of the efficiency of PSII reaction centres at western blot process. Thus, a significant reduction in the band both temperatures and in both species. The drop of Fv /Fm in UVR intensity when the Ab was preincubated in the presence of the was similar at 2 ◦ C and 7 ◦ C, and no significant differences (P < 0.05) respective blocking peptides, as compared with the non-blocked were observed between these two temperatures under any light antibodies, eliminates unspecific cross-reactivity or artefacts, and treatment in S. latissima (Fig. 2A). UVR was the only variable that induced a significant loss of photosynthetic efficiency, however, no significant differences were found between PA and PAB treat- ments. The interactive effect of temperature and UVR stress was 0.25 not significant (two-way ANOVA, P > 0.05). PAB PA Similar results were obtained with L. solidungula (Fig. 2B); UVR P induced a significant drop of Fv /Fm at both temperatures com- 0.2 pared with PAR treatment, however, no significant differences were Spectral Irradiance (relative units) observed between PA and PAB treatments. Furthermore, as for S. latissima, Fv /Fm was similar at 2 ◦ C and 7 ◦ C, and the interactive effect of temperature and UVR stress was not significant (two-way 0.15 ANOVA, P > 0.05). 3.2. Detection of p38-like and JNK-like MAPKs in L. solidungula and S. latissima 0.1 As no information was available regarding the presence or phos- phorylation of MAPK-like proteins in Arctic macrophytes, random protein extracts from both species were initially submitted to gel 0.05 electrophoresis, western blotting and immunodetection by means of specific antibodies against the phosphorylated forms of stress- related MAPKs (e.g. phopho-p38 and phospho-JNK) (Fig. 3). In the case of the phospho-p38 Ab, a band of 42 kDa was detected in S. 0 300 400 500 600 700 latissima (Fig. 3A), while a band of 40 kDa was found in L. solidun- gula (Fig. 3B), both under UV stress. The phospho-JNK Ab also Wavelength (nm) cross-reacted with two main bands of 40 and 36 kDa in S. latis- sima (Fig. 3A) and bands of 42 and 36 kDa in L. solidungula (Fig. 3B), Fig. 1. Three types of filter foils allow the selective exclusion of different wavelength ranges. Spectral irradiances used in PAR, PAR + UVA and PAR + UVA + UVB treatments exposed to UV. Antibodies against the phosphorylated forms of the used in this study. ERK MAPK have not been tested in this study, since the treatments Author's personal copy M.L. Parages et al. / Environmental and Experimental Botany 91 (2013) 30–37 33 Fig. 3. The use of blocking peptides confirms the presence of phospho-p38- and phospho-JNK-like proteins. Western blot analysis of phosphorylated forms of the MAPKs as detected by specific phospho-p38 and phospho-JNK antibodies in Saccha- rina latissima (A) and Laminaria solidungula (B) under UV stress (PA and PAB), both in the presence (+BP) and absence (−BP) of the specific blocking peptides of each antibody. Each lane contains 100 ␮g protein extract. blocking peptides (Fig. 3) clearly reduced the intensity of the bands detected by both phospho-antibodies; band intensity was reduced by between 60% and 98% when the specific blocking peptides were added, confirming the presence of phospho-p38- and phospho- JNK-like proteins. 3.3. Phosphorylation of the two MAPKs in response to temperature and UV stress Once the presence of p38-like and JNK-like MAPKs in these Arc- tic kelps had been established, the effect of stress was analysed Fig. 2. Physiological fitness analysed by measuring photosynthetic efficiency (Figs. 4–7). In L. solidungula, two slightly phosphorylated bands through PAM-fluorometry. The optimal quantum yield (Fv /Fm ) of young sporophytes of 36 and 42 kDa were detected in PAR treatment when the anti- of Saccharina latissima (A) and Laminaria solidungula (B) measured through imaging phospho-JNK Ab was used (Fig. 4). The results indicate that at both PAM after 1 h of exposure to PAR (P, open columns), PAR + UV-A (PA, grey columns) and PAR + UV-A + UV-B (PAB, black columns) under two different temperatures (2 ◦ C 2 ◦ C and 7 ◦ C, the JNK cascade was active in all cases. No increased and 7 ◦ C). Statistically significant differences (P < 0.05) between treatments are indi- phosphorylation of any of the bands was induced by temperature cated by different letters. stress in PAR, but conversely, the intensity of detected phosphor- ylation was reduced at 7 ◦ C compared with that at 2 ◦ C (Fig. 4A and B). However, following UV treatment, the degree of phosphor- lasted 1 h, and this MAPK is known to be involved in cell division ylation greatly increased. In all cases, band intensity was greater control and differentiation, and the effects of these stress conditions in the PA treatment than in the P treatment alone, and following are expected to occur in a longer time period. PAB treatment rather than PA alone. In addition, a higher degree Due to the use of polyclonal antibodies raised against the phos- of phosphorylation was always detected at 7 ◦ C both in PA and phorylated forms of both p38 and JNK of mammalian cells, it had PAB treatments, for both the 36 (Fig. 4A) and the 42 (Fig. 4B) kDa to be established whether these results were an artefact and the bands. However, the differences between PA and PAB treatments revealed bands were due to unspecific cross-reactivity. To demon- were greater at 7 ◦ C than at 2 ◦ C, indicating a possible cumula- strate that these bands really corresponded to MAPKs, two parallel tive effect between UVR and temperature. This interactive effect analyses were carried out. Firstly, the possibility of unspecific between temperature and UV stress was confirmed by two-way cross-reactivity with the antibodies raised against the phospho- ANOVA (P < 0.05) for both proteins. rylated forms of the MAPKs from mammalian cells was analysed As for the p38-like MAPK (Fig. 5), slight phosphorylation of a by incubating at least one lane of each treatment directly with 40 kDa protein was detected in L. solidungula in P, at both 2 ◦ C and the secondary antibodies (without incubating the membranes with 7 ◦ C. Similarly, as for the JNK-like MAPK, UV illumination induced the primary antibodies). Unspecific bands were discarded from further activation of the p38-like MAPK, this protein being more further analysis. Secondly, binding between the proteins on the phosphorylated in the PAB treatment than the PA treatment. Fur- PVDF membrane and the primary phospho-antibody was specif- thermore, the degree of phosphorylation was much higher at 7 ◦ C ically blocked by using specific blocking peptides. The use of the than at 2 ◦ C in both PA and PAB treatments. Similar to Ph-JNK, the Author's personal copy 34 M.L. Parages et al. / Environmental and Experimental Botany 91 (2013) 30–37 Fig. 5. High temperature under UVR induce p38-like protein phosphorylation. Western blots for the detection of the phosphorylated forms of the p38 MAPK-like of Laminaria solidungula by P (open columns), PA (grey columns) and PAB (black columns) treatments. The specific polyclonal antibodies rose against the phospho- p38 of mammalian cells cross-reacted with a 40 kDa protein. The graph depicts the mean ± SE (n = 3). Each lane contains 100 ␮g protein extract. Statistically significant differences (P < 0.05) between treatments are indicated by different letters. Thus, the response to stress was different in both species (Fig. 8). In S. latissima, both the p38-like and the JNK-like MAPKs showed higher phosphorylation at 2 ◦ C than at 7 ◦ C, whereas the reverse occurred in L. solidungula, where phosphorylation was more pro- nounced at 7 ◦ C than at 2 ◦ C. No induction of phosphorylation of any of these MAPKs was detected after increasing the tempera- ture from 2 ◦ C to 7 ◦ C following P treatment. These results coincide with the maintenance of high Fv /Fm values in both species after the change in temperature (Fig. 2). A significant increase in the phos- phorylation of both kinases was always detected after exposure to UVR. UVA + UVB treatments induced higher phosphorylation than UVA alone in L. solidungula, especially at 7 ◦ C. For S. latissima, this response occurred only with JNK, both at 2 ◦ C and 7 ◦ C, while no dif- Fig. 4. UVR and high temperature induce the phosphorylation of JNK-like proteins in Laminaria solidungula. Western blots of L. solidungula extracts for the detection ferences in the p38 phosphorylation state were observed between of the phosphorylated forms of the MAPK-like JNK in P (open columns), PA (grey PA and PAB treatments at either temperature. A clear interactive columns) and PAB (black columns) treatments, revealing the presence and induction effect between temperature and UVR stress was detected in the of two different bands of 36 kDa (A) and 42 kDa (B). The graph depicts the mean ± SE phosphorylation of MAPK-like proteins in L. solidungula, however, (n = 3). Each lane contains 100 ␮g protein extract. Statistically significant differences no significant interaction was found for S. latissima. (P < 0.05) between treatments are indicated by different letters. 4. Discussion interactive effect between temperature and UV stress was con- firmed by two-way ANOVA (P < 0.05). UVR is a source of potential stress for most organisms in nat- S. latissima showed a different behaviour; basal JNK and p38 ural environments (Bischof et al., 2006). The current trend of phosphorylation was also found in the P treatment (Figs. 6 and 7), stratospheric ozone depletion in the Arctic (Manney et al., 2011) and as before, no further phosphorylation occurred at 7 ◦ C in the represents a threat for many sessile organisms in this area. There- PAR treatment. However, as in L. solidungula, a clear increase in fore, solar UVR might affect Arctic marine organisms via a wide JNK phosphorylation was found under UV radiation, this increase range of harmful effects, from reduced productivity, to changes in being more apparent in S. latissima at 2 ◦ C than at 7 ◦ C (Fig. 6A and the structure of the communities (Heinrich et al., 2012). In addition B), whereas the reverse was the case for L. solidungula (Fig. 4). In all to UV effects, the Arctic is the region of the world where the highest cases, UVA + B treatment induced a higher degree of phosphoryla- temperature increase is expected (Zacher et al., 2011). tion than UVA alone, for both the 36 (Fig. 6A) and the 40 kDa bands It is widely accepted that MAPKs constitute the central core of (Fig. 6B). However, the interactive effect between temperature and the stress-signalling network of animal cells, and more recently, UV stress was not significant (two-way ANOVA, P > 0.05). their study has been extended to higher plants and algae. In this Finally, phosphorylation of a p38-like MAPK (Fig. 7) was also study, the presence and activation of MAPK-like proteins in two highly induced by UVR in S. latissima, and, again, activation was species of Arctic kelps is described, and a differential response much higher at 2 ◦ C than at 7 ◦ C. No differences between PA and between L. solidungula and S. latissima has been observed. S. latis- PAB treatments were observed at any temperature. As for Ph-JNK, sima is a widely-distributed kelp in the Arctic and in cold temperate the interactive effect between temperature and UV stress was not regions, and can be found from the cold coasts of Northern Spain significant (two-way ANOVA, P > 0.05). and Portugal to the coasts of the Arctic circle. In Kongsfjorden, it Author's personal copy M.L. Parages et al. / Environmental and Experimental Botany 91 (2013) 30–37 35 Fig. 7. Phosphorylation of p38 MAPK of Saccharina latissima is activated by UVR and low temperature. Western blots for the detection of the phosphorylated forms of the p38 of S. latissima by P (open columns), PA (grey columns) and PAB (black columns) treatments. The specific polyclonal antibodies generated against phospho- p38 of mammalian cells cross-reacted with a 42 kDa protein. The graph depicts the mean ± SE (n = 3). Each lane contains 100 ␮g protein extract. Statistically significant differences (P < 0.05) between treatments are indicated by different letters. grows in the mid-sublittoral zone. In contrast, L. solidungula is an Arctic endemic species that grows in the lower sublittoral zone, in a less fluctuating environment than that of S. latissima, in deeper waters and at lower temperature and lower UVR (Hop et al., 2012). Two MAPKs similar to p38 and JNK of mammalian cells are phos- phorylated in both S. latissima and L. solidungula in response to environmental stress. Both kelps are rapidly able to respond to different conditions, in some cases without further activation of the stress–response mechanism mediated by MAPKs. It has been shown that the basal level of active (phosphorylated) p-38- and JNK-like in both species is not altered when a change in tempera- Fig. 6. UVR and low temperature induce the phosphorylation of JNK-like proteins in Saccharina latissima. Western blots of S. latissima extracts for the detection of ture occurs (from 2 ◦ C to 7 ◦ C). However, when UVR is applied, rapid the phosphorylated forms of the MAPK-like JNK under P (open columns), PA (grey MAPK phosphorylation occurs. Results show that in L. solidungula, columns) and PAB (black columns), reveal the presence and induction of two differ- the effect of UVA + UVB on MAPK phosphorylation is more pro- ent bands of 36 kDa (A) and 40 kDa (B). The graph depicts the mean ± SE (n = 3). Each nounced than for UVA treatment alone, and that it is reinforced lane contains 100 ␮g protein extract. Statistically significant differences (P < 0.05) at 7 ◦ C. Thus, at 7 ◦ C there is a possible cumulative effect of both between treatments are indicated by different letters. stressors (temperature and UVR), leading to the highest degree of phosphorylation detected in this species. It appears that this Fig. 8. Increased temperature and UV radiation lead to different behaviour of MAPK activation. Summary of the different behaviours of MAPK activation (phospho-p38 and phospho-JNK) in Saccharina latissima and Laminaria solidungula under the different combinations of light treatments and temperatures used in this study. Author's personal copy 36 M.L. Parages et al. / Environmental and Experimental Botany 91 (2013) 30–37 Arctic-endemic kelp is able to cope with increasing temperature species, which possess very effective response mechanisms against stress (in the range used in this study), without further activation stress. This extent has been confirmed for S. latissima by several of the MAPK-like cascades, but that regulation of the response to authors. This kelp is well adapted to low light conditions (Bartsch UV stress requires further activation of the MAPKs. et al., 2008), with high pigment concentration and photosynthetic S. latissima also responded to increasing temperature and UVR and morphological adaptations (Borum et al., 2002; Bartsch et al., by de novo phosphorylation of both MAPKs. As before, no fur- 2008). ther activation occurred when the temperature was altered in the Recently, a plant photoreceptor (UV RESISTENCE LOCUS8-UVR8) absence of UVR. However, UVR treatment induced de novo phos- has been proposed to regulate gene expression in response to UVB phorylation of both p38 and JNK MAPKs. In contrast to L. solidungula, (Brown et al., 2005; Brown and Jenkins, 2008; Jenkins, 2009; Wu higher band intensity was detected at 2 ◦ C than at 7 ◦ C when UVR et al., 2012). Brown et al. (2005) concluded that UVR8 is mainly was given, and differences between PA and PAB appeared only in localised to the nucleus, and Brown and Jenkins (2008) and Jenkins the phosphorylation of JNK. Two explanations for this differential (2009) defined two distinct pathways, one UVR8-dependent and behaviour are possible: firstly, the typical summer temperature another UVR8-independent, which stimulate different sets of of surface water in Kongsfjorden is around 5–6 ◦ C (Hanelt et al., genes in Arabidopsis leaf tissue in response to UVB; according 2001), while at 10–15 m deep in a stratified water column, the to these authors, responses to UVB are mediated by both non- temperature can be as low as 2 ◦ C. Thus, the differential response specific signalling pathways (involving DNA damage, reactive of L. solidungula and S. latissima might be the result of the dif- oxygen species production, as well as wound/defence signalling ferent temperatures at their growing sites, which could be a few molecules), as well as specific signalling pathways mediated by degrees lower at certain times of the year in the case of L. solidun- UVR8, that regulate photomorphogenic responses at low levels of gula. Thus, the selected temperature of 7 ◦ C represents an increase UVB. Thus, the role of signalling cascades in response to UVB such of 4–5 ◦ C for this species, whereas for S. latissima, the experimental as stress-activated MAPKs would be to mediate stress signal trans- temperature of 2 ◦ C represents a decrease of 4–5 ◦ C. Secondly, the duction through the above-mentioned non-specific pathways. differential response might reflect the fact that S. latissima is not UV dose-dependent responses will be further studies in future an Arctic endemic species, and therefore not as adapted to low- in situ experiments in the Arctic to evaluate better the ecological temperature as L. solidungula. According to Wiencke et al. (1994), consequences of the phosphorylation of these putative MAPKs. The S. latissima is an “Arctic cold-temperate species”, and L. solidungula short-term experiments here have helped to determine the pres- is an “endemic Arctic species”. Heinrich et al. (2012) have shown ence and response of p38-like and JNK-like MAPKs in two species of that optimal quantum yield of S. latissima did not change within the Arctic kelps. In an extended experimental set-up, ERK-like MAPKs temperature range 2–17 ◦ C at low irradiance, and that under stress would be also considered, due to their demonstrated involvement growth-related genes are mostly repressed, while genes involved in in cell division in microalgae (Jiménez et al., 2007; García-Gómez energy generation, heat shock, signal transduction and antioxidant et al., 2012) and differentiation in higher plants (Coronado et al., functions are induced. 2002). Moreover, the use of specific inhibitors of MAPK phosphor- As can be seen in Figs. 4–7, activation of the MAPKs in response ylation would help to understand the extent to which survival of to stress was more pronounced in L. solidungula than in S. latissima, the macrophytes might be compromised under stress. as shown by the analysis of the band intensity diagrams (samples from both species were run in parallel on the same gels, and the 5. Conclusions same protein concentration was loaded in each lane). This indi- cates that activation of the regulatory system was higher under In this study, we demonstrate that activation of JNK and p38- the experimental conditions for L. solidungula than for S. latissima. like MAPKs in the Arctic kelps S. latissima and L. solidungula rapidly This is in agreement with many previous reports in which the abil- occurs in response to environmental changes. Phosphorylation of ity of S. latissima to cope with UVR has been reported (see Roleda these MAPKs occurs under environmental stress imposed by UVR et al., 2007, for a review). Clayton and Wiencke (2004) have shown and increased temperature. In general, UVB radiation was respon- that the effect of UVR on zoospore germination in S. latissima is sible for a higher induction of these kinases, whereas the effect almost negligible, whereas Aguilera et al. (2004a) demonstrated of temperature was only apparent in light treatments other than that growth of this species was scarcely affected by UVR (and no PAR. Both macrophytes appear to be able to cope with the tem- differences appeared between PA and PAB treatments). Bischof and perature change tested in this study, without further activation of Hanelt (2004) concluded that S. latissima might show a genetically- the MAPK pathways, when UVR was removed. We consider that fixed ability to cope with UV, and that specimens from shallow these results contribute to the understanding of the complicated areas do not suffer the negative effects of short exposure to UVR as metabolic reactions implicated in the response to stress in these do specimens from deeper areas. In addition, Aguilera et al. (2004b) organisms. Further experiments will help to clarify the relation- demonstrated that S. latissima has much higher superoxide dis- ship between MAPK phosphorylation in these kelps and response mutase, glutathione reductase, ascorbate peroxidase and catalase to stress (as has been already shown for other micro- and macroal- activities than L. solidungula. Conversely, Michler et al. (2002) and gae), to gather information on the possible changes in the structure Aguilera et al. (2004a) showed that L. solidungula is more sensitive of the kelp communities dominated by these species in a future to UVR than other kelps from the upper and mid-sublittoral zones, scenario of temperature and UVR increase. and that its growth rate was more affected under PAB treatment than PA treatment. According to Roleda et al. (2007), the response to UV stress might be one of the key factors that determines the Acknowledgements observed zonation pattern of Laminariales in the field. Previous studies over several days’ duration, with intertidal C.J. and M.L.P. have been supported by the Ministry of Sci- macrophytes from the Strait of Gibraltar (Parages, 2012) suggest ence and Innovation of Spain (Grants No. CGL05-01071/BOS and that participation of both the p38-like and the JNK-like MAPK pro- CTM2011-24007) and by the European Center for Arctic Envi- teins in L. solidungula and S. latissima might be crucial for survival, ronmental Research, Arcfac Project 026129-2009-147. We thank and that both Arctic kelps show a high degree of resilience, as the diving group of the Alfred Wegener Institute headed by Max occurs in intertidal macrophytes. In our opinion, life in such a selec- Schwanitz for collection of algal specimens. This study was per- tive environment as the Arctic might have selected very resilient formed at the Ny-Ålesund international research and monitoring Author's personal copy M.L. 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