Proteomics in Seaweeds: Ecological Interpretations

Abstract

Macro and micro-algae are fundamental components of coastal benthic ecosystems and are responsible for a large part of the coastal primary production (Lobban & Harrison, 1994). Adverse effects on these groups caused by natural or anthropogenic phenomena, can affect directly or indirectly organisms of higher trophic levels and the integrity of entire ecosystems. In this context, both the ecological and economic importance of many algal species justifies the need to expand our knowledge on the molecular biology of these organisms. The distribution and abundance of algal species occurring in the marine zone results from the interplay of biotic (i.e. competition and herbivore pressure) and abiotic (i.e. tolerance to extreme and fluctuating environments) factors (Abe et al., 2001; Burritt et al., 2002; Davison & Pearson, 1996; Pinto et al., 2003; van Tamelen, 1996). For example, the distribution of macroalgal species at the upper limit of the rocky intertidal zone is principally determined by abiotic factors such as UV radiation, light, salinity, temperature changes, nutrient availability and desiccation (e.g. Aguilera et al., 2002; Burritt et al., 2002; Cabello-Pasini et al., 2000; Contreras-Porcia et al., 2011a; Véliz et al., 2006). On the other hand, the microalgae diversity is maintained by a combination of variable forces - environmental oscillations (e.g. habitat instability), more severe disturbances and recovery from catastrophic forcing - backed by the powerful dispersive mobility of this group (Reynolds, 2006). The richness, relative abundance and occasional dominances of the phytoplankton in successive years, depends on water movements, thermal stress and carbon fluxes, but mainly on nutrient enrichment of the sea (Hodgkiss & Lu, 2004; Holm-Hansen et al., 2004; Reynolds, 2006; Wang et al., 2006; Zurek & Bucka, 2004). Superimposed on the natural abiotic oscillations, algae are also exposed to various other sources of stress, particularly those resulting from human industrial, urban and agricultural activities. Among these is copper mining, whose wastes have reportedly caused severe and negative effects on the coasts of England (Bryan & Langston, 1992), Canada (Grout & Levings, 2001; Marsden & DeWreede, 2000), Australia (Stauber et al., 2001) and Chile (Correa et al., 1999). Although copper is a micronutrient for plants and animals, occurring naturally in coastal seawater at levels at or below 1 µg L-1 (Apte & Day, 1998; Batley, 1995; Sunda, 1989), at higher concentrations it becomes highly toxic. The phenomenon of toxicity in algae is strongly influenced by the speciation of this metal (Gledhill et al., 1997), and within the cell it likely operates through the Haber-Weiss reaction, characterized by a heavy metal-catalyzed production of hydroxyl radicals from hydrogen peroxide (Baker & Orlandi, 1995). For example, in northern Chile, mine wastes originated at a copper mine pit are disposed of directly into the sea. The rocky intertidal zone along the impacted coasts shows a severe reduction in species richness, and the macroalgal assemblage is reduced to the opportunistic algae Ulva compressa (Plantae, Chlorophyta) and Scytosiphon lomentaria (Chromista, Ochrophyta) (Medina et al., 2005). This negative effect on the biota has been widely recognized as the result of the persistent high levels of copper in the water, by far the most important metal brought into the system by mine wastes (Medina et al., 2005). Many macroalgae species are absent, such as Lessonia nigrescens complex (Chromista, Ochrophyta), which are key components in structuring the intertidal zone (Ojeda & Santelices, 1984). As for microalgae, an example is a mine effluent that contained high levels of copper, which was disposed in a reservoir named Venda Nova in northern Portugal. There, a phytoplankton survey was carried out between the years 1981-1982. A shift in the dominant species was demonstrated when compared with an uncontaminated area, Alto Rabagão. More than 50% of the algal species developed lower populations. Also, at the most polluted zone, phytoplankton density, biomass and richness were strongly reduced (Oliveira, 1985). In macro and micro-algae it is possible to determine that under natural abiotic factors, a common cellular response could involve the over-production of reactive oxygen species (ROS) (Andrade et al., 2006; Contreras et al., 2005, 2007b, 2009; Contreras-Porcia et al., 2011a; Kumar et al., 2010; Lee & Shin, 2003; Liu et al., 2007; Rijstenbil, 2001). ROS are ubiquitous by- products of oxidative metabolism that are also involved in intracellular signalling processes (e g. Blokhina & Fagerstedt, 2010; Rhee, 2006). ROS are produced directly by the excitation of O2 and the subsequent formation of singlet oxygen, or by the transfer of one, two or three electrons to O2. This results in the formation of superoxide radicals, hydrogen peroxide or hydroxyl radicals, respectively (Baker & Orlandi, 1995). Oxidative damage to cellular constituents such as DNA/RNA, proteins and lipids may occur (e g. Contreras et al., 2009; Vranová et al., 2002) when ROS levels increase above the physiological tolerance range. However, a coordinated attenuation system can be activated in order to eliminate this ROS over-production, and therefore, the oxidative stress condition (e. g. Burritt et al., 2002; Ratkevicius et al., 2003; Rijstenbil, 2001). For example, in the coastal zones of northern Chile it has been demonstrated that the high copper levels in the seawater generate in sensitive species a high oxidative stress condition, which appears as the starting point for a series of molecular defense responses. In first place, the condition of oxidative stress has been demonstrated by the direct production of ROS and oxidized lipid in individuals living at an impacted site as well as in those transplanted from control sites to the impacted site (Contreras et al., 2005; Ratckevicius et al., 2003). Compared with high tolerant species such as Ulva and Scytosiphon, in low tolerant species such as L. nigrescens the ROS production by copper, specifically superoxide anions, is poorly attenuated, which is reflected in i) higher levels of oxidized lipids, ii) the generation of cellular alterations and iii) negative effects on early developmental stages of the life cycle (Andrade et al., 2006; Contreras et al., 2007a; 2009). Thus, ecophysiological differences are evident between diverse algal species. This is also true for microalgal species since there are species-specific responses to oxidative stress caused by high levels of copper. For example, it was demonstrated that 4 species of phytoplankton under high concentrations of copper only grew up to 80-95% of that observed in the control condition (Bilgrami & Kumar, 1997). Furthermore, a study including two microalgae species exposed to copper stress showed significant differences between them. In the high tolerant species, Scenedesmus vacuolatus, in comparison to the low tolerant species, Chlorella kessleri, the chlorophyll a/chlorophyll b ratio was partially reduced. Likewise, both the antioxidant enzyme activity and protein content were progressively increased (Sabatini et al., 2009). Another environmental factor that affects the abundance and distribution in macroalgae is desiccation. It is an important stress factor faced by living organisms because, as cells lose water, essential macromolecules are induced to form non-functional aggregates and organelles collapse (Alpert, 2006). Some animals (Clegg, 2005) and plants are well adapted to significant water losses, displaying full physiological recovery during rehydration (Alpert, 2006; Farrant, 2000). Compared to vascular plants or animals, in macroalgae the effects of desiccation on the physiology and the molecular mechanisms involved in its tolerance are poorly understood. For example, in one of the few reports available, the activation of different antioxidant enzymes, such as ascorbate peroxidase (AP) and glutathione reductase (GR) was recorded in the upper intertidal macroalga Stictosiphonia arbuscula (Plantae, Rhodophyta) (Burritt et al., 2002) as a response to desiccation-mediated oxidative stress. The remaining studies have focused on assessing the capacity to tolerate desiccation displayed by measuring the photosynthetic apparatus activity in Porphyra, Gracilaria, Chondrus, and Ulva species among others (Abe et al., 2001; Ji & Tanaka, 2002; Smith et al., 1986; Zou & Gao, 2002). Presently, the only study using molecular approaches to unravel the desiccation tolerance responses, found that genes encoding for photosynthetic and ribosomal proteins are up-regulated in Fucus vesiculosus (Chromista, Ochrophyta) (Pearson et al., 2001, 2010). Additionally, independent studies have shown that diverse physiological parameters are altered by desiccation including the lipid and protein levels (Abe et al., 2001), photosynthetic alterations (Fv/Fm) as well as cellular morphology and ontogenetic changes (e.g. Contreras-Porcia et al., 2011b; Varela et al., 2006). Moreover, in microalgae it has been shown that salt (i.e. changes in water osmolarity) and temperature stress can be highly stressful and may finally trigger a programmed cell death (PCD) (Kobayashi et al., 1997; Lesser, 1997; Takagi et al., 2006; Zuppini et al., 2010). In these species the effects of both types of stress have been widely studied, and have been reported to provoke photosynthetic alterations, ROS production and ultimately cell death (Liu et al. 2007; Lesser, 1996; Mishra & Jha, 2011; Vega et al., 2006). Recently, the red species Porphyra columbina Montagne (Plantae, Rhodophyta) was recognised among the macroalgae that are highly tolerant to natural desiccation stress. P. columbina is highly seasonal and grows abundantly along the upper intertidal zone (Hoffmann & Santelices, 1997; Santelices, 1989). This alga is well adapted to the extreme fluctuating regimes of water/air exposure, a