Brown algae (Phaeophyta) make up most of the benthic biomass on temperate and polar rocky shores. Kelp forests are key ecosystems of such coastal areas and they constitute habitat and breeding grounds for a very large diversity of marine life forms - besides offering a physical protection by providing a wave energy-absorbing buffer for coasts. Brown algae belong to a lineage that has been evolving independently of other major photosynthetic lineages, in particular the green plants (Chlorophyta) and red algae (Rhodophyta). Instead, they are classified within the Stramenopiles and Chromalveolates together with diatoms, golden-brown algae and oomycetes (Baldauf 2003). As a consequence of this singular evolutionary history, brown algae exhibit many unusual, and often unique, features. These features are adaptations to the marine coastal environments in which brown algae are usually the dominant organisms in terms of biomass, in particular by forming extensive kelp forests structuring these ecosystems. The key role of kelp forests, effectively constituting an interface between the ocean, the atmosphere and land masses, in the biogeochemical cycle of halogens is well established (Carpenter et al. 2000, Kupper et al. 2008).
Like all other marine organisms, marine algae are subject to a virtually permanent onslaught of a broad spectrum of pathogens - including viruses (Muller and Knippers 2001), chytrid fungi (Gleason et al. 2011), oomycetes (Strittmatter et al. 2009) and endophytic / parasitic algae (Ellertsdottir and Peters 1997). While some algal pathogens have been known since the 19th century (e.g., Wright 1877), mechanistic aspects have been studied only for little more than a decade (e.g., Potin et al. 2002). A significant finding of recent years was that brown and red algae share key defense mechanisms with animals and higher plants (Kupper et al. 2001, Potin et al. 2002).
The mammalian response to microbial attack involves the activation of immune response genes, including those involved in the arachidonic acid cascades, leading to the synthesis of leukotrienes and prostaglandins (Funk 2001, Gachon et al. 2010). In comparison, plants synthesize compounds of direct antimicrobial toxicity such as reactive oxygen species (ROS), phytoalexins and pathogenesis-related proteins. Higher plants are also known to activate genes involved in the oxylipin cascade such as lipoxygenases, in a process that appears similar to the arachidonic acid cascade in mammals (Andreou et al. 2009). Many inducible defense genes are regulated by signalling pathways involving one or more low molecular weight signal molecules, such as jasmonic acid, salicylic acid, ethylene, and possibly H2O2 (Reymond and Farmer 1998, Orozco-Cardenas et al. 2001).
The oxidative burst was initially discovered in human macrophages (Baldridge and Gerard 1933). It is now recognized also as a key element of plant (Wojtaszek 1997, Mittler et al. 2004) and algal (Potin et al. 2002, Dring 2005, Cosse et al. 2007) defense, where it is considered both as a rapid defense response and an internal emergency signal. It consists of the rapid and massive production of ROS such as superoxide (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (?OH)which are likely produced via the interaction of plasma membrane-associated NAD(P)H oxidases (Wojtaszek 1997). Hence, pathogen attack triggers both the production of ROS and the oxidation cascade of fatty acids leading to the production of bioactive oxylipins in plants. In algal systems, the oxidative burst was initially discovered in response to injury in a red alga (Collen et al. 1994). Over the last decade, oxidative bursts in response to oligosaccharide elicitors were observed in a number of red and brown algal models, in most cases to control the growth of bacterial biofilms (Weinberger et al. 1999, Weinberger and Friedlander 2000
Most polyunsaturated fatty acids (PUFAs) in higher plants belong to the C18 family, whilst polyunsaturated C20 fatty acids seem to occur only in very few species. In contrast, algal lineages such as the Phaeophyceae (Hofmann and Eichenberger 1997) produce both C18 and C20 PUFAs in significant amounts. A number of algae have become industrial sources of biotechnologically important PUFAs such as arachidonic acid (C20:4) or eicosapentaenoic acid (C20:5) (Colombo et al. 2006, Spolaore et al. 2006).
Oxylipins and prostaglandins in particular are important intra- and intercellular messengers in all living organisms. Among the cellular signalling responses which ROS generation can trigger is the activation of fatty acids by the production of oxygenated PUFAs called oxylipins which play a key role against abiotic and biotic stress in plants and metazoans. In mammals, these compounds (which include leukotrienes and prostaglandins) are produced from the oxidation of C20 PUFAs. Prostaglandins mediate pivotal functions in immunity processes and cell development, allergic reactions and, in a broader sense, defensive stress responses (Funk 2001). Furthermore, in plants, their recruitment during wounding stress, pathogen and herbivore invasions has been fully confirmed in recent years (Farmer et al. 2003, Farmer and Schulze-Lefert 2005). In our previous work, we had shown that bacterial surface macromolecules (especially lipopolysaccharides) can trigger both an oxidative burst and induce the release of free fatty acids and synthesis of oxylipins in
Considering the paramount importance of prostaglandins in the defense of mammalian cells, the scope of this study was to screen 5 prostaglandins as potential defense elicitors in brown algae. In analogy to our previous studies, we monitored
Prostaglandins (PGE1, PGE2, PF2α, PGB1, PGA2) were obtained from FLUKA and Cayman Europe (Tallinn, Estonia), dissolved in dimethyl sulfoxide and stored at -20℃ until further use. These prostaglandins had been preselected among a multitude of compounds due to their established, stress-related roles in other organisms.
For experiments involving
For assessing the effects of prostaglandins on oxidative metabolism in
For all further studies following the finding of PGA2 as a potent inducer of the oxidative burst, a dose-response curve for the oxidative burst was measured using the aforementioned chemiluminescence assay, with final PGA2 concentrations of 0.1, 1, 3, 6 and 10 μM, respectively. For analyses of free fatty acids and oxylipins,
Overview of the primers used for monitoring the response of stress gene candidates in Ectocarpus siliculosus
Series II; Hewlett Packard, Palo Alto, CA, USA) coupled to mass spectrometry (HP 5971A; Agilent Technologies, Santa Clara, CA, USA) after appropriate derivatizations. Fatty acids were quantified by GC-MS as methyl esters from standard curves obtained by measuring the peak surfaces of authentic standards. Statistic significance for increases of PUFAs and oxylipins amounts after treatments compared to controls was tested by a Student t-test.
All experiments with
Treatments with PGA2 (obtained from FLUKA and Cayman Europe, as above) were performed at a final concentration of 10 μM in an experimental procedure developed by Zambounis, Strittmatter and Gachon (unpublished / personal communication). Briefly, algal cell cultures were treated as follows: PGA2 or ethyl ester (controls; 10 μL per 15 mL of culture) was added for 2 h each day during seven consecutive days. Following incubation the material was rinsed and placed in fresh half-strength modified Provasoli-enriched seawater in 650 mL filter cap suspension flasks (175 cm²; Greiner Bio-One). The experimental procedure included two biological and three technical replicates each, respectively. Cultures were harvested for RNA extraction by concentrating the culture on 70 μm nylon mesh filters (Cell Strainers™; BD Biosciences, Oxford, UK), quickly dried with an autoclaved paper towel, and immediately immersed in RNALater™ solution (Ambion, Austin, TX, USA), left for perfusion for 1 day at 4℃, followed by storage at -80℃.
Total RNA was extracted from approximately 70 mg (fresh weight) of tissue using the Qiagen RNeasy® Plant Mini-kit (Qiagen, Crawley, UK) and DNase-treated (RNAse-free DNAse I; Invitrogen, Paisley, UK), according to the manufacturer’s instructions with the following modifications: Two volumes (900 μL) of buffer RLC was added to the disrupted tissue (Step 2; see RNeasy® Plant Mini-kit protocol; Qiagen) which resulted in cleaner, higher yield RNA extraction. Quality and integrity assessments as well as quantification of the RNA were performed using a Bioanalyzer 2100™ (Agilent Technologies) with the corresponding Agilent RNA 6000 Nano Kit. From each RNA sample, 0.5 μg was reverse transcribed to cDNA using SuperScript® III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s instructions. Subsequently, first strand cDNAs were diluted with nuclease-free water to a final concentration of 1 ng μL-1.
Potential molecular responses of
Quantitative expression analysis was performed using a QUANTICA™ real-time PCR system (Techne-Barloworld, Stone, Staffordshire, UK). Quantitative polymerase chain reactions (qPCRs) were run in a final volume of 20 μL with 2 ng cDNA and 300 μM of each primer with MesaGreen™ reaction mix (Eurogentec, Southampton, UK). Reactions were run in triplicates. The cycle parameters were 10 min at 95℃, followed by 45 runs of 15 s at 95℃ and 1 min at 60℃. Dissociation curves were obtained by melting curve analysis from 65 to 95℃ with readings every 0.5℃ and 10 s hold between them.
Relative variation in gene expression ratios was calculated as fold changes relative to the control experiments using REST 2009 software (Pfaffl et al. 2002).
Among the five prostaglandins (PGE1, PGE2, PF2α, PGB1, PGA2) tested in
Fig. 2 provides an overview of free saturated and unsaturated fatty acid contents in
Three oxylipins, previously identified as relevant markers of fatty acid oxidation in
In order to monitor the gene expression profiles obtained after PGA2 treatment in
based on our results in a parallel proteomics study of
Significantly, this study reports the first case of an oxidative burst triggered by a prostaglandin in an algal or plant-like organism. The finding is very remarkable even with regard to animal models, where prostaglandins are known as modulators rather than actual inducers of oxidative bursts (Vaidya et al. 1999). To our knowledge, only one case of an oxidative burst triggered by a prostaglandin has been reported in eukaryotes, that of 15-deoxydelta(12,14)-prostaglandin J2, an agonist of peroxisome proliferator activated receptor gamma, which induces superoxide anion production in human cells, with an efficacy similar to its action on ERK phosphorylation (Huang et al. 2002). In contrast, some prostaglandins have been reported to inhibit the oxidative burst (e.g., Ottonello et al. 1995). Also, the oxidative burst observed here is among the strongest in amplitude ever observed in a brown algal system: The PGA2-triggered oxidative burst is considerably stronger with up to 5 μmol H2O2 g-1 FW observed, in contrast to around 1 mol H2O2 g-1 FW for oligoguluronates (Kupper et al. 2001, 2002), lipopolysaccharides (Kupper et al. 2006), polyunsaturated free fatty acids and methyl jasmonate (Kupper et al. 2009). In contrast to arachidonic acid and methyljasmonate (MeJA) (Kupper et al. 2009), the oxidative burst response to PGA2 in
Whilst the oxidative burst triggered by arachidonic acid, MeJA (Kupper et al. 2009), oligoguluronates (Kupper et al. 2001, 2002) and lipopolysaccharides (Kupper et al. 2006) can be inhibited by DPI, this does not apply to the response to PGA2. This finding indicates that at least two different sources of ROS co-operate in oxidative burst responses in brown algae. This is consistent with certain results in higher plants (Bolwell et al. 1998, Bestwick et al. 1999) which suggest that both an NAD(P)H oxidase, a neutrophil-like superoxide-generating enzyme, susceptible to inhibition by DPI, a suicide substrate of flavin-containing oxidases (O’Donnell et al. 1993), and peroxidases should be involved in the oxidative burst.
Moreover, it is noteworthy that besides activating an oxidative burst, lipopolysaccharides (Kupper et al. 2006), polyunsaturated free fatty acids, MeJA (Kupper et al. 2009) and PGA2 (this study) also induce fatty acid oxidation in
Furthermore and like for the peroxisome-proliferator activated receptors α and γ (PPAR α and γ) in mammals (Kliewer et al. 1995), arachidonic acid could be recognized by the same target receptors in
[Table 2.] Effects of prostaglandin A2 (PGA2) on relative gene expression in Ectocarpus siliculosus
Effects of prostaglandin A2 (PGA2) on relative gene expression in Ectocarpus siliculosus
tors of cyclooxygenase and prostaglandin biosynthesis, indomethacin and aspirin (Jaworek et al. 2001), allene oxide synthase, aspirin (Pan et al. 1998) and of lipoxygenase (salicylhydroxamic acid) on the oxidative burst triggered by arachidonic acid in
Evidence is growing that if terrestrial plants recognize their own lipid-based signals, they may also be able to recognize those of herbivores. For instance, caterpillar regurgitant contains aminated eicosanoids (peptidolipids) that are perceived by higher plants and induce elevated defenses (Arimura et al. 2000, Turlings et al. 2000). In this respect, it is noteworthy that prostaglandins have been described as elicitors from the oral secretions of herbivores (Schultz and Appel 2004) recognized by plants. A similar interpretation might be considered for the role of prostaglandins in the defense of
The substantial knowledge about the important model
In conclusion, the discovery of an oxidative burst in an alga triggered by a prostaglandin is intriguing. Prostaglandins - in particular, PGA2 and its derivatives - are well established as feeding deterrents in corals (Gerhart 1984, Whalen et al. 2010): the findings presented in this study highlight new perspectives of PGA2 being a more widespread defense molecule and inducer in the marine environment, in very distantly related lineages.