**1. Introduction**

20 Gel Electrophoresis – Advanced Techniques

Rossignol, T., Kobi, D., Jacquet-Gutfreund, L. & Blondin, B. (2009). The proteome of a wine

Salvadó, Z., Chiva, R., Rodríguez-Vargas, S., Rández-Gil, F., Mas, A., Guillamón, J. M.

Santamaría, P., Garijo, P., López, R., Tenorio, C., Gutiérrez, A. R. (2005). Analysis of yeast

Schuller, D., Valero, E., Dequin, S. & Casal, M. (2004). Survey of molecular methods for the

Schuller, D., Casal, M. (2007). The genetic structure of fermentative vineyard-associated

Trabalzini, L., Paffetti, A., Scaloni, A., Talamo, F., Ferro, E., Coratz, G., Bovalini, P., Santucci,

Zuzuarregui, A., Monteoliva, L., Gil, C., del Olmo, M. (2006). Transcriptomic and proteomic

*Applied Microbiology*, Vol. 107, pp. 47-55, ISSN 1364-5072

*FEMS Yeast Research*, Vol. 8, pp. 1137-1146, ISSN 1567-1356

*Leeuwenhoek*, Vol. 91, pp. 137-150, ISSN 0003-6072

103, pp. 49-56, ISSN 0168-1605

1567-1356.

ISSN 0168-1605

1, pp. 836-847, ISSN 0099-2240

yeast strain during fermentation: correlation with the transcriptome. *Journal of* 

(2008). Proteomic evolution of a wine yeast during the first hours of fermentation.

population during spontaneous alcoholic fermentation: Effect of the age of the cellar and the practice of inoculation. *International Journal of Food Microbiology*, Vol.

typing of wine yeast strains. *FEMS Microbiology Letters,* Vol. 231, pp. 19-26, ISSN

*Saccharomyces cerevisiae* populations revealed by microsatellite analysis. *Antonie van* 

A. (2003). Proteomic response to physiological fermentation stresses in a wild-type wine strain of *Saccharomyces cerevisiae*. *Biochemistry Journal*, Vol. 370, pp. 35-46 Viana, F., Gil, J. V., Vallés, S., Manzanares, P. (2009). Increasing the levels of 2-phenyl acetate

in wine through the use of a mixed culture of *Hanseniaspora osmophila* and *Saccharomyces cerevisiae*. *International Journal of Food Microbiology*, Vol. 135, pp. 68-74,

approach for understanding the molecular basis of adaptation of *Saccharomyces cerevisiae* to wine fermentation. *Applied and Environmental Microbiology*, Vol. 72, No. 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

Proteomics in Seaweeds: Ecological Interpretations 23

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

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.

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, as demonstrated by the formation of sporophytic thalli from monoecious fronds (*n*) during long daily periods of desiccation stress due to its position in the intertidal zone (Contreras-Porcia et al., 2012). Additionally, desiccation in *P. columbina* induces morphological and cellular alterations accompanied by a loss of ca. 96 % of the water content (Contreras-Porcia et al., 2011b). Specifically, under natural desiccation stress, the production of ROS (i.e. H2O2 and O2) in *P. columbina* is significantly induced (Contreras-Porcia et al., 2011b). However, during the high tide, ROS quickly returned to basal levels because *P. columbina* displays an efficient antioxidant system. In addition, at

increased (Sabatini et al., 2009).

2007; Lesser, 1996; Mishra & Jha, 2011; Vega et al., 2006).

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 byproducts 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

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

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 byproducts 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

(Oliveira, 1985).

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, as demonstrated by the formation of sporophytic thalli from monoecious fronds (*n*) during long daily periods of desiccation stress due to its position in the intertidal zone (Contreras-Porcia et al., 2012). Additionally, desiccation in *P. columbina* induces morphological and cellular alterations accompanied by a loss of ca. 96 % of the water content (Contreras-Porcia et al., 2011b). Specifically, under natural desiccation stress, the production of ROS (i.e. H2O2 and O2) in *P. columbina* is significantly induced (Contreras-Porcia et al., 2011b). However, during the high tide, ROS quickly returned to basal levels because *P. columbina* displays an efficient antioxidant system. In addition, at

Proteomics in Seaweeds: Ecological Interpretations 25

Flengsrub & Kobro, 1989; Mechin et al., 2003). These contaminants pose a significant difficulty for 2-DE, as they cause horizontal and vertical streaking, smearing and a reduction in the number of distinctly resolved protein spots. Thus, the selection of the most appropriate protein extraction method is necessary in order to obtain high quality extracts, and therefore, a high quality 2-DE pattern. For a better understanding and explanation of the current techniques and methodology in algae proteomic, this chapter has been divided

It is important to highlight that due to the small size of microalgae, all of the protein extraction protocols for these organisms begin with a centrifugation step in order to pellet cells. This helps to concentrate cells, and consequently allows a correct extraction of the

One of the first proteomics studies on microalgae dates from the year 1972, in which Mets & Bogorad showed alterations in the chloroplast ribosomes proteins of erythromycin-resistant mutants of *Chlamydomonas reinhardtii* (Plantae, Chlorophyta) compared to the wild-type. The ribosomal protein extraction performed on this work was the LiCl-urea method described by Leboy et al. (1971) that was developed for *Escherichia coli* (as cited in Mets & Bogorad, 1972). Thus, the Mets & Bogorad work was a precursor to microalgae proteomic studies. Here, ribosomes are disrupted and freed of RNA by adding LiCl. Then, the samples are centrifuged to precipitate total RNA and the supernatant, which contains the proteins, is

Several studies in the same decade also focused their attention on characterizing ribosomal proteins (e.g. Götz & Arnold, 1980; Hanson et al., 1974). The Hanson et al. (1974) work based their protocols on the Mets & Bogorad (1972) research paper and also used *C. reinhardtii* as model species. Instead, in 1980 Götz & Arnold used a different ribosomal protein extraction after testing several protocols. The procedure chosen was the acetic-acid method in presence of MgCl2 according to Kaltschmidt & Wittmann (1972), method that was also first developed for *E. coli*. In this method, MgCl2 and glacial acetic acid are added to the ribosome suspension, and then the mixture is centrifuged to pellet RNA. For better mixture cleaning,

Not all studies from this decade focused their attention on ribosomal proteins as was the case of the work of Piperno et al. (1977), in which the protein mixture came from *Chlamydomona* flagella and axonemes. It is important to highlight this research since the extraction method used was very rustic. After the flagella and axoneme separation, the

Recent studies evaluate more complex protein mixtures, so the method chosen must be more accurate in extracting proteins with minimum contaminants and interferents. In fact, a work in *C. reinhardtii* that performed an analysis of all the thylakoid membranes proteins used a more complex protocol (Hippler et al., 2001) than the ones previously discussed in

in two sections: microalgae and macroalgae methodology.

the pellet can be extracted a second time in the same way.

proteins were dissolved only in SDS and kept for 2-DE analysis.

**2.1.2 Current proteomic studies** 

**2.1 Microalgae methodology** 

**2.1.1 Early proteomic studies** 

desired proteins.

retained.

biomolecular level, only a low production of oxidized proteins is recorded during desiccation, due to the efficient antioxidant system of this alga.

The results mentioned above, indicate that desiccation in *P. columbina* causes an overproduction of ROS, which is efficiently attenuated. Morphological and photosynthetic changes could be operating as tolerance mechanisms, due to the fact that these responses principally prevent biomolecular alterations, protein aggregation and cellular collapse. For example, it has been proposed that cell wall folding is a cellular strategy used to prevent tearing the plasmalemma from the cell wall during desiccation, ensuring cell integrity (Contreras-Porcia et al., 2011b). The activation of antioxidant enzymes and the photoinhibition of the photosynthetic apparatus help to explain the attenuation of ROS. Thus, ROS excess is buffered by the activation of several physiological and biochemical responses, which suggest a mechanism allowing this plant to tolerate desiccation (Contreras-Porcia et al., 2011b). The ecophysiological responses in this species help, in part, to account for its position and dominance at the highest level in the intertidal zone, and thereby, suggesting desiccation stress tolerance as a determinant trait for explaining that situation. In fact, our recent results demonstrate that the magnitude of the effects generated by desiccation in algae is related to the position of the species in the intertidal zone. Additionally, this work demonstrated the exceptional metabolism of *P. columbina* used to buffer this stress condition. Thus, the determinations of novel metabolic pathways are necessaries in order to fully understand the high desiccation tolerance in this species, for example at the proteomic level. In fact, in this time our forces are concentrated in resolving the proteomic profile of this species under natural hydration and desiccation stress.

Finally, the need to unravel the mechanisms associated with tolerance to different environmental factors by algal species opens the electrophoretic and proteomic approximations as important tools in comprehending and explaining the observed tolerances. However, little information regarding electrophoretic and proteomic analysis is available in algal species. Compared with other group of organisms (e.g. vascular plant or animals) protein extraction in macroalgae has been extraordinary difficult, due principally to the limited knowledge at biochemical and molecular levels. In this context, the present chapter aims to understand the different proteomic approaches utilized in this group of organisms in order to comprehend their ecophysiological behaviour.
