**3. Results and discussion: From gel to molecular/ecological interpretation**

Proteomic analyses have proved to be an important molecular approximation that enables comparisons between species and/or cell variants, and understanding of cell function and stress tolerance (e.g. metals, high salinity, high temperatures, among others) (e.g. Contreras et al., 2010; Kim et al., 2005; Ritter et al., 2010). Due to the particularity of the cellular components (e.g. high content of polysaccharides) of this group of organisms, protein extraction has been the principal problem. However, as stated in the previous sections, some protocols have proved capable of producing high quality protein extracts for 2-DE electrophoresis (microalgae: Mahong et al., 2012; Wang et al., 2003 and macroalgae: Contreras et al., 2008). A high quality protein extract will yield high-resolved 2-DE patterns. Therefore, with a suitable protocol the use of a proteomic approximation appears to be of high importance for understanding various physiological responses in this group of organisms. However, it is imperative to highlight that proteomic works in micro and principally in macroalgae, are considerably lower in comparison with vascular plants and animals. Then, our effort in this chapter was concentrated in describing those important works utilizing as model the algal assemblage.

One of the first proteomic studies in microalgae characterized the chloroplastic ribosomal proteins of wild-type and erythromycin-resistant mutants of *Chlamydomonas reinhardtii* (Mets & Bogorad, 1972). In the mutant *ery-M2d* a protein of the 52S subunit was missing when compared to the wild-type. Nevertheless, low intensity proteins spots with the same

The work described by Yotsukura et al. (2010) presents a similar protocol to the one described by Contreras et al. (2008). Here, proteins are extracted from the brown alga *Saccharina japonica* (Chromista, Ochrophyta)*,* important kelp described principally on the coastal areas of northern Japan. In this protocol, the protein extraction was improved by using phenol as the principal component in the lysis buffer (detailed protocol in Appendix B). This protocol was also used in *Ecklonia cava* (Chromista, Ochrophyta), other important kelp found on the coast along the Sea of Japan, and also good quality 2-D patterns were obtained (Yotsukura et al., 2012). The use of phenol in the protein extraction described by Contreras et al. (2008) has been recently used in the red alga *Porphyra columbina* in order to identify the proteins that are overinduced during desiccation stress tolerance responses. A highly resolved 2-DE protein was obtained using this method (Fig. 1), with minor modifications (detailed protocol in Appendix B), such as an important rinse of the protein pellet due principally to the over-production of phycocyanin and phycoerythrin. Thus, the phenol protocol developed by Contreras et al.

The first dimension of the 2-DE in the works mentioned above used approximately 200-500 µg of extracted proteins. However, for the isoelectric focusing (IEF) the protocols varied depending on the algal species used. For example, in *Bostrychia radicans* and *B. moritziana*, the voltage was linearly increased from 150-3,500 V during 3 h, followed by a constant 3,500 V, with focusing complete after 96 V. In *Scytosiphon gracilis* and *Ectocarpus siliculosu*s, on the other hand, the strips are actively rehydrated for 15 h in IEF buffer containing the proteins and focused at 20ºC with the following successive steps: a liner increase from 0 to 250 V for 15 min, a gradient phase from 250 V to 10,000 V for 4 h, and the a hold phase at 10,000 V for a total of 60 kVh. Using this protocol, the IEF for *Porphyra columbina* has some modifications,

**3. Results and discussion: From gel to molecular/ecological interpretation**  Proteomic analyses have proved to be an important molecular approximation that enables comparisons between species and/or cell variants, and understanding of cell function and stress tolerance (e.g. metals, high salinity, high temperatures, among others) (e.g. Contreras et al., 2010; Kim et al., 2005; Ritter et al., 2010). Due to the particularity of the cellular components (e.g. high content of polysaccharides) of this group of organisms, protein extraction has been the principal problem. However, as stated in the previous sections, some protocols have proved capable of producing high quality protein extracts for 2-DE electrophoresis (microalgae: Mahong et al., 2012; Wang et al., 2003 and macroalgae: Contreras et al., 2008). A high quality protein extract will yield high-resolved 2-DE patterns. Therefore, with a suitable protocol the use of a proteomic approximation appears to be of high importance for understanding various physiological responses in this group of organisms. However, it is imperative to highlight that proteomic works in micro and principally in macroalgae, are considerably lower in comparison with vascular plants and animals. Then, our effort in this chapter was concentrated in describing those important

One of the first proteomic studies in microalgae characterized the chloroplastic ribosomal proteins of wild-type and erythromycin-resistant mutants of *Chlamydomonas reinhardtii* (Mets & Bogorad, 1972). In the mutant *ery-M2d* a protein of the 52S subunit was missing when compared to the wild-type. Nevertheless, low intensity proteins spots with the same

(2008) could be used in macroalgae species from different taxonomic groups.

principally in a total operational voltage of 70 kVh.

works utilizing as model the algal assemblage.

Fig. 1. 2-D proteome of *Porphyra columbina* under natural desiccation. First dimension was performed on a linear gradient IPG strip of pH 4-7 using 600 µg of total proteins. The 12.5% SDS-PAGE gel was stained with colloidal Coomassie blue.

*pI* but different molecular weight were found, indicating that the *ery-M2* gene is involved in determining the properties of that protein. Hanson et al. (1974) performed 2-DE gels in order to characterize cytoplasmic and chloroplastic ribosomal proteins. Their results showed that the number of proteins in both small and large subunits was higher in cytoplasmic than in chloroplastic ribosomes, indicating that cytoplasmic ribosomes are more complex. Another study did a comparison between the ribosomal proteins of 8 species including *C. reinhardtii*  (Götz & Arnold, 1980). The results showed that the number of proteins in both subunits was similar among all species, and that the *Polytoma papillatun* (Plantae, Chlorophyta) proteins were the most similar to those of *C. reinhardtii* in terms of protein homology.

Piperno et al. (1977) analysed the flagella proteins of wild-type and paralyzed mutants of *C. reinhardtii* in order to identify the mutated protein that incapacitates the mobility in mutant strains. In the flagella of *pf* 14, which completely lack radial spokes and associated spokeheads, 12 polypeptides were missing. Also in *pf* 1 flagella, where spokes are clearly present but spokeheads appear to be absent, 6 polypeptides were missing. Then, protein electrophoretic studies confirmed the phenotypical characteristics displayed by both paralyzed mutants, where the missing proteins may be involved in spokes and spokeheads correct morphology. Another work in *C. reinhardtii,* used a proteomic approach to analyse photosynthetic thylakoid membrane proteins isolated from wild-type and mutant strains (Hippler et al., 2001). The two mutant strains were Δ*ycf4* (PSI-deficient) and *crd1* (which is conditionally reduced in PSI and LHCI under copper-deficiency). In this work more than 30 different LHCP spots were identified using a tandem quadrupole mass spectrometer,

Proteomics in Seaweeds: Ecological Interpretations 33

mechanisms to cope with the stress induced by the high concentration of metals. For example, in the work developed by Contreras et al. 2010, the copper-tolerance capacity of the brown algae species *Scyotsiphon gracilis* was evaluated by means of the 2-DE approximation. In this work, using the protocol previously described by Contreras et al. 2008 in the Appendix B, 19 over-expressed proteins were identified, including a chloroplast peroxiredoxin, a cytosolic phosphomannomutase, a cytosolic glyceraldehyde-3-phosphate dehydrogenase, 3 ABC transporters, a chaperonine, a subunit of the proteasome and a tRNA synthetase, among others (Table 1). The possible involvement of these over-expressed proteins in buffering oxidative stress and avoiding metal uptake in *S. gracilis* exposed to copper excess is discussed considering this proteomic information. For example, the peroxiredoxine (PRX) is an enzyme involved in the detoxification of hydrogen peroxide and fatty acid hydroperoxides (Dietz et al., 2006). In plants, *prx* transcripts increase in response to different abiotic stresses such as salinity, drought and metals (Dietz, 2003; Wood et al., 2003). Furthermore, PRX in the microalga *C. reinhardtii* and the red macroalga *Porphyra purpurea* (Plantae, Rhodophyta) have shown high similarity with plant PRXs (Baier and Dietz, 1997; Goyer et al., 2002). The expression of PRX in *C. reinhardtii* seems regulated by light, oxygen and redox state (Goyer et al., 2002). Thus, the PRX identified in *S. gracilis* may play an important role in oxidative stress buffering and in lipoperoxides detoxification. In fact, we have recently demonstrated the active participation of this enzyme in copper tolerant species in comparison with sensitive ones, where the over-expression of this enzyme is localized in the cortical cells (Lovazzano et al., personal communication). The proteomic information obtained by Contreras et al. 2010 in *S. gracilis* opens the opportunity of understanding many biological/physiological processes in algae. Using this information and those obtained using a biochemistry approximation, it is possible to strongly suggest a cross-talk between different pathways to re-establish the cellular homeostasis distorted by copper-associated oxidative stress in this species as well as in other tolerant ones (Fig. 2). Thus, the differential ability of each species to deal with oxidative stress resulting from the high copper levels, explains the persistence of tolerant species and the

Using the method described by Contreras et al. 2008, it was also possible to evaluate differential tolerance in *Ectocarpus siliculosus* strains, originated from habitats with contrasting histories of copper levels (Ritter et al., 2010). Here, the authors showed a differential stress tolerance between 50 and 250 µg L-1 of copper. This difference was also observed at the level of the 2-DE proteome profile. For example, in the tolerant strains from a copper contaminated site (i.e. Chañaral, Chile) a specific expression of PSII Mn-stabilizing protein, fucoxanthine chlorophyll a-c binding protein and vanadium-dependent bromoperoxidase proteins, among others, was observed. Thus, the occurrence of the differential proteome profile among the strains could be strongly suggested by the persistence copper driving force in the evolution of *Ectocarpus siliculosus* from the copper contaminated sites (Ritter et al., 2010). In other brown macroalgae such as *Ecklonia cava*, it was possible to observe the effects of temperature on the proteomic profile (Yotsukura et al., 2012). Here, the authors define that the differential protein expression induced by temperature could be considered as an important biomarker of the health individuals in the

In *Saccharina japonica* it was possible to observe differences at the level of the proteome under seasonal variation and pH conditions (Yotsukura et al., 2010; 2012). Under seasonal

absence of sensitive ones at copper contaminated zones.

culture conditions.


Table 1. Proteins differentially expressed in *S. gracilis* exposed to copper excess. The analysis by MSMS allowed to obtain various protein peptides which were identified by BLASTP (NCBI). (a) Changes in expression level compared with controls. (b) Number of peptides analyzed by LC/MS/MS. (c) NCBI access number of the species with the highest identity obtained by BLASP.

thereby, permitting proteins with transmembrane domains to be separated with high resolution. Here, the results showed that LHCI spots were present on Δ*ycf4* and absent on *crd1* mutants.

Proteomics approaches have been helpful in understanding tolerance to naturally or anthropologically occurring environmental factors (e.g. high light, thermal stress and heavy metals respectively) in different species. Due to anthropological activities (e.g. industry and mining), heavy metals such as cadmium (Cd) and copper (Cu) are accumulating in the environment (Vermeer & Castilla, 1991; Medina et al., 2005). At high concentrations these metals are a source of abiotic stress, and can be highly toxic to organisms. In this matter, proteomic approaches are of high utility because they may provide new information regarding

nº of peptides

tRNA synthetase over 24 *Helicobacter pylori* (P56126) 5.9, 60

Proteosome, subunit over 24 *Oryza sativa* (Q10KF0) 5.6, 53

carboxylase large chain over <sup>22</sup>*Porphyra yezoensis* (Q760T5) 9.6, 65

dehydrogenase 1 over <sup>22</sup>*Gracilaria verrucosa* (P30724) 6.2, 43 Peptidase/Protease over 19 *Methanothermobacter* (O27355) 6.3, 42 tRNA binding protein over 23 *Anaplasma* (Q2GJX4) 6.2, 38

RNA binding protein over 28 *Bacillus phage* (P06953) 6.4, 25 ABC transporter subunit over 40 *Theileria parva* (XP\_764551.1) 8, 20.6 RNA polymerase, subunit over 19 *Francisella tularensis* (Q5NHU3) 9.2, 17.5 Peroxiredoxin over 20 *Porphyra purpurea* (P51272) 9.5, 18 Chaperonine over 10 *Caulobacter crescentus* (P48222) 5.6, 8

ABC transporter subunit over 35 *Janibacter* sp. (ZP\_00996449.1) 8.6, 7.3 Table 1. Proteins differentially expressed in *S. gracilis* exposed to copper excess. The analysis by MSMS allowed to obtain various protein peptides which were identified by BLASTP (NCBI). (a) Changes in expression level compared with controls. (b) Number of peptides analyzed by LC/MS/MS. (c) NCBI access number of the species with the highest identity

thereby, permitting proteins with transmembrane domains to be separated with high resolution. Here, the results showed that LHCI spots were present on Δ*ycf4* and absent on

Proteomics approaches have been helpful in understanding tolerance to naturally or anthropologically occurring environmental factors (e.g. high light, thermal stress and heavy metals respectively) in different species. Due to anthropological activities (e.g. industry and mining), heavy metals such as cadmium (Cd) and copper (Cu) are accumulating in the environment (Vermeer & Castilla, 1991; Medina et al., 2005). At high concentrations these metals are a source of abiotic stress, and can be highly toxic to organisms. In this matter, proteomic approaches are of high utility because they may provide new information regarding

Transferase over 13 *Dechloromonas aromatica* 

Phosphomannomutase over <sup>24</sup>*Schizosaccharomyces pombe*

ATP synthase, subunit over <sup>13</sup>*Syntrophus aciditrophicus*

ATP binding protein over <sup>24</sup>*Methanocaldococcus jannaschi*

Transcriptional regulator over <sup>24</sup> *Mesorhizobium loti* 

Carbohydrate kinase over <sup>35</sup>*Salmonella enterica*

ABC transporter subunit over <sup>45</sup>*Desulfitobacterium hafniense* 

analized*<sup>b</sup>* Species, nº access*<sup>c</sup> pI*, Mw

(Q47F82)

(Q9UTJ2)

(Q2LQZ7)

(Q58049)

(YP\_152740.1)

(ZP\_01371968.1)

(CAD31581.1)

(KDa)

5.6, 65

 5.3, 53

6.0, 67

6.4, 38

3, 18.2

6.2, 8.2

 8.7, 29

Protein Expression

Ribulose biphosphate

Glyceraldehyde 3-phosphate

obtained by BLASP.

*crd1* mutants.

level*<sup>a</sup>*

mechanisms to cope with the stress induced by the high concentration of metals. For example, in the work developed by Contreras et al. 2010, the copper-tolerance capacity of the brown algae species *Scyotsiphon gracilis* was evaluated by means of the 2-DE approximation. In this work, using the protocol previously described by Contreras et al. 2008 in the Appendix B, 19 over-expressed proteins were identified, including a chloroplast peroxiredoxin, a cytosolic phosphomannomutase, a cytosolic glyceraldehyde-3-phosphate dehydrogenase, 3 ABC transporters, a chaperonine, a subunit of the proteasome and a tRNA synthetase, among others (Table 1). The possible involvement of these over-expressed proteins in buffering oxidative stress and avoiding metal uptake in *S. gracilis* exposed to copper excess is discussed considering this proteomic information. For example, the peroxiredoxine (PRX) is an enzyme involved in the detoxification of hydrogen peroxide and fatty acid hydroperoxides (Dietz et al., 2006). In plants, *prx* transcripts increase in response to different abiotic stresses such as salinity, drought and metals (Dietz, 2003; Wood et al., 2003). Furthermore, PRX in the microalga *C. reinhardtii* and the red macroalga *Porphyra purpurea* (Plantae, Rhodophyta) have shown high similarity with plant PRXs (Baier and Dietz, 1997; Goyer et al., 2002). The expression of PRX in *C. reinhardtii* seems regulated by light, oxygen and redox state (Goyer et al., 2002). Thus, the PRX identified in *S. gracilis* may play an important role in oxidative stress buffering and in lipoperoxides detoxification. In fact, we have recently demonstrated the active participation of this enzyme in copper tolerant species in comparison with sensitive ones, where the over-expression of this enzyme is localized in the cortical cells (Lovazzano et al., personal communication). The proteomic information obtained by Contreras et al. 2010 in *S. gracilis* opens the opportunity of understanding many biological/physiological processes in algae. Using this information and those obtained using a biochemistry approximation, it is possible to strongly suggest a cross-talk between different pathways to re-establish the cellular homeostasis distorted by copper-associated oxidative stress in this species as well as in other tolerant ones (Fig. 2). Thus, the differential ability of each species to deal with oxidative stress resulting from the high copper levels, explains the persistence of tolerant species and the absence of sensitive ones at copper contaminated zones.

Using the method described by Contreras et al. 2008, it was also possible to evaluate differential tolerance in *Ectocarpus siliculosus* strains, originated from habitats with contrasting histories of copper levels (Ritter et al., 2010). Here, the authors showed a differential stress tolerance between 50 and 250 µg L-1 of copper. This difference was also observed at the level of the 2-DE proteome profile. For example, in the tolerant strains from a copper contaminated site (i.e. Chañaral, Chile) a specific expression of PSII Mn-stabilizing protein, fucoxanthine chlorophyll a-c binding protein and vanadium-dependent bromoperoxidase proteins, among others, was observed. Thus, the occurrence of the differential proteome profile among the strains could be strongly suggested by the persistence copper driving force in the evolution of *Ectocarpus siliculosus* from the copper contaminated sites (Ritter et al., 2010). In other brown macroalgae such as *Ecklonia cava*, it was possible to observe the effects of temperature on the proteomic profile (Yotsukura et al., 2012). Here, the authors define that the differential protein expression induced by temperature could be considered as an important biomarker of the health individuals in the culture conditions.

In *Saccharina japonica* it was possible to observe differences at the level of the proteome under seasonal variation and pH conditions (Yotsukura et al., 2010; 2012). Under seasonal

Proteomics in Seaweeds: Ecological Interpretations 35

Fig. 2. Cellular events involved in the mechanisms of copper stress tolerance in algae. Dotted arrows indicate routes not directly evidenced in brown algae. The alteration of the state redox, cell damage, and the metal may trigger the antioxidant machine [i.e. compounds and antioxidant enzymes (activation of cycle Halliwell-Asada (MDHAR, DHAR and GP), CAT, SOD, AP, TRX, PRX)] as the activations of protein/genes that form part of various metabolic pathways. Proteins such as HSP or CHA may be involved in the protein

protection as in the transport of the metal to proteins that use it as a cofactor, respectively. The sequestration of the metal by different proteins (i.e. MT, GST and PC) is an important homeostatic pathway of tolerance to the metal. The strengthening of the cell wall can increase the resistance to the entry of the metal to the cell. Copts, copper transporter; ROS,

dehydroascorbate reducatase; GP, gluthathione peroxidase; AP, ascorbate peroxidase; SOD,

of treated (10 µM Cd for 4 days) and untreated (control) cells (Kim et al., 2005). The protocol used in this work, as was discussed before, yielded deficient 2-DE gels, resulting in few

superoxide dismutase; CAT, catalase; TRX, tioredoxine; PRX, peroxiredoxine; PMM, phosphomannomutase; VIT-C, vitamin C or ascorbic acid; CHA, copper chaperone; GST, glutathione-s-transferase; PC, phytochelatin; MT, metallothionein; ASC, ascorbate; GSH, glutathione; HSP, heat shock protein; TOM 22; cox 17 transporter; Urm1 (modifier protein

reactive oxygen species; MDHAR, monodehydroascorbate reductase; DHAR,

type ubiquitin); NDK, nucleoside diphosphate kinase.

variation, the specific expression of different proteins was identified, among them the vanadium-dependent bromoperoxidase (Yotsukura et al., 2010). Comparatively, under different pH culture conditions the over-expression of several proteins was described such as: glyceraldehyde-3-phosphate dehydrogenase, actin, phosphoglycerate kinase, elongation factor Tu and ATP synthase subunit β, among others. Thus, different metabolic pathways could be induced in brown macroalgae according to the type of stress factor. In this context, the utilization of the 2-DE approximation has been extraordinarily important in unravelling the tolerance mechanisms associated with environmental variables from natural and anthropogenic sources. In fact, the identification of important enzymes, never before described in algae (i.e. Peroxiredoxine (Contreras et al., 2010) and vanadium-dependent bromoperoxidase (Ritter et al., 2010)), opens the opportunity to further understanding the biology of this group of organisms.

In microalgae, several works have also been reported. For example, Wang et al. (2004) studied the proteome changes of *Haematococcus pluvialis* under oxidative stress induced by the addition of acetate and Fe2+ and exposure to excess of high light intensity. About 70 proteins were identified in which 19 were up-regulated (e.g. antioxidant enzymes and sugar synthesis proteins) and 13 were down-regulated (e.g. metabolism and cell growth proteins). Also, transient regulated proteins were identified in which 31 were up-regulated (e.g. antioxidant enzymes) and only 8 were down-regulated (e.g. chloroplastic proteins). In 2006, Förster et al. performed a proteome comparison among wild-type and two very high lightresistant mutants (*VHLR-S4* and *VHLR-S9*) under different high light stress. About 1500 proteins were detected in the gel and 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. The results revealed complex alterations in response to the stress, where total proteins varied drastically in the wild-type compared to the mutants. Nevertheless, the mutant *VHLR-S4* proved to have better adaptation to high light stress since a more controlled protein regulation was conducted (e.g. up-regulation of several chaperonins and down-regulation of energy metabolism proteins). Another work conducted in *H. pluvialis* analyzed the proteome under high irradiance, but combined with nitrogen starvation (Tran et al., 2009). In the gels, about 900 protein spots were detected of which 13 were down-regulated and 36 up-regulated. Among the up-regulated proteins, a glutathione peroxidase and a translocase from the outer mitochondrial membrane were matched to *C. reinhardtii*; therefore, these stress responses may be common among these microalgae. A study assessing a proteomic analysis on *C. reinhardtii* under a short-term exposure to irradiance revealed significant down regulation of several heat-shock proteins (HSPs) (Mahong et al., 2012) under differential times of exposition to this stress (0 h, 1.5 h, 3 h and 6 h of high light). Spot densities allowed the determination that early rearrangement of the light-harvesting antenna proteins occurs, where this was manifested by the up- and down-regulation of several protein spots identied as LHC-II polypeptides. Moreover, increased expression of proteins involved in carbohydrate metabolism was found, which could help accelerate the utilization of electrons generated, in order to minimize the risk of superoxide formation. Surprisingly, after 6 hours of high light several molecular chaperones were down-regulated and this could result in drastic effects on cell structure and function. Nevertheless, *C. reinhardtii* is normally light-sensitive which could be explained by the down-regulation of molecular chaperones.

In microalgae, the response of species to heavy metal contamination has also been evaluated. A proteomic analysis conducted on *N. oculata* showed differences between protein expression

variation, the specific expression of different proteins was identified, among them the vanadium-dependent bromoperoxidase (Yotsukura et al., 2010). Comparatively, under different pH culture conditions the over-expression of several proteins was described such as: glyceraldehyde-3-phosphate dehydrogenase, actin, phosphoglycerate kinase, elongation factor Tu and ATP synthase subunit β, among others. Thus, different metabolic pathways could be induced in brown macroalgae according to the type of stress factor. In this context, the utilization of the 2-DE approximation has been extraordinarily important in unravelling the tolerance mechanisms associated with environmental variables from natural and anthropogenic sources. In fact, the identification of important enzymes, never before described in algae (i.e. Peroxiredoxine (Contreras et al., 2010) and vanadium-dependent bromoperoxidase (Ritter et al., 2010)), opens the opportunity to further understanding the

In microalgae, several works have also been reported. For example, Wang et al. (2004) studied the proteome changes of *Haematococcus pluvialis* under oxidative stress induced by the addition of acetate and Fe2+ and exposure to excess of high light intensity. About 70 proteins were identified in which 19 were up-regulated (e.g. antioxidant enzymes and sugar synthesis proteins) and 13 were down-regulated (e.g. metabolism and cell growth proteins). Also, transient regulated proteins were identified in which 31 were up-regulated (e.g. antioxidant enzymes) and only 8 were down-regulated (e.g. chloroplastic proteins). In 2006, Förster et al. performed a proteome comparison among wild-type and two very high lightresistant mutants (*VHLR-S4* and *VHLR-S9*) under different high light stress. About 1500 proteins were detected in the gel and 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. The results revealed complex alterations in response to the stress, where total proteins varied drastically in the wild-type compared to the mutants. Nevertheless, the mutant *VHLR-S4* proved to have better adaptation to high light stress since a more controlled protein regulation was conducted (e.g. up-regulation of several chaperonins and down-regulation of energy metabolism proteins). Another work conducted in *H. pluvialis* analyzed the proteome under high irradiance, but combined with nitrogen starvation (Tran et al., 2009). In the gels, about 900 protein spots were detected of which 13 were down-regulated and 36 up-regulated. Among the up-regulated proteins, a glutathione peroxidase and a translocase from the outer mitochondrial membrane were matched to *C. reinhardtii*; therefore, these stress responses may be common among these microalgae. A study assessing a proteomic analysis on *C. reinhardtii* under a short-term exposure to irradiance revealed significant down regulation of several heat-shock proteins (HSPs) (Mahong et al., 2012) under differential times of exposition to this stress (0 h, 1.5 h, 3 h and 6 h of high light). Spot densities allowed the determination that early rearrangement of the light-harvesting antenna proteins occurs, where this was manifested by the up- and down-regulation of several protein spots identied as LHC-II polypeptides. Moreover, increased expression of proteins involved in carbohydrate metabolism was found, which could help accelerate the utilization of electrons generated, in order to minimize the risk of superoxide formation. Surprisingly, after 6 hours of high light several molecular chaperones were down-regulated and this could result in drastic effects on cell structure and function. Nevertheless, *C. reinhardtii* is normally light-sensitive which could be

biology of this group of organisms.

explained by the down-regulation of molecular chaperones.

In microalgae, the response of species to heavy metal contamination has also been evaluated. A proteomic analysis conducted on *N. oculata* showed differences between protein expression

Fig. 2. Cellular events involved in the mechanisms of copper stress tolerance in algae. Dotted arrows indicate routes not directly evidenced in brown algae. The alteration of the state redox, cell damage, and the metal may trigger the antioxidant machine [i.e. compounds and antioxidant enzymes (activation of cycle Halliwell-Asada (MDHAR, DHAR and GP), CAT, SOD, AP, TRX, PRX)] as the activations of protein/genes that form part of various metabolic pathways. Proteins such as HSP or CHA may be involved in the protein protection as in the transport of the metal to proteins that use it as a cofactor, respectively. The sequestration of the metal by different proteins (i.e. MT, GST and PC) is an important homeostatic pathway of tolerance to the metal. The strengthening of the cell wall can increase the resistance to the entry of the metal to the cell. Copts, copper transporter; ROS, reactive oxygen species; MDHAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reducatase; GP, gluthathione peroxidase; AP, ascorbate peroxidase; SOD, superoxide dismutase; CAT, catalase; TRX, tioredoxine; PRX, peroxiredoxine; PMM, phosphomannomutase; VIT-C, vitamin C or ascorbic acid; CHA, copper chaperone; GST, glutathione-s-transferase; PC, phytochelatin; MT, metallothionein; ASC, ascorbate; GSH, glutathione; HSP, heat shock protein; TOM 22; cox 17 transporter; Urm1 (modifier protein type ubiquitin); NDK, nucleoside diphosphate kinase.

of treated (10 µM Cd for 4 days) and untreated (control) cells (Kim et al., 2005). The protocol used in this work, as was discussed before, yielded deficient 2-DE gels, resulting in few

Proteomics in Seaweeds: Ecological Interpretations 37

under copper stress the identification of proteins such as peroxiredoxine, enzyme involved in the detoxification of hydrogen peroxide and fatty acid hydroperoxide has Allowed to understand the differential degree of tolerance between Copper tolerant and sensitive species. In fact, using the proteomic protocol described in these species, which uses phenol in the protein extraction, a differential proteome profile in algal individuals between desiccation stress and normal hydration was founded. In this context, new tolerance mechanisms will be revealed using this approximation in order to understand the high desiccation tolerance that exists in this species in comparison with many others, including that from the same phylum. Thus, 2-DE approximation is an important tool that can be interconnected with those obtained to ecological level in order to understand mechanisms of stress tolerance, and therefore explanation of distribution patterns at local

*Chlamydomonas reinhardtii* **(Plantae, Chlorophyta)** (Förster et al., 2006). The figure

This protocol is an adaption of the one described by Mathesius and co-workers (Grotewold,

1. Collect *Chlamydomonas* cells by centrifugation at 5,000 x *g* for 5 min at 20° C. Determine

2. Grind pelleted *Chlamydomonas* cells to a fine powder in liquid nitrogen using a mortar and pestle after addition of 0.5 g of glass powder per 1 g fresh weight of pelleted

3. Suspend the ground material in -20°C cold acetone containing 10% w/v TCA and 0.007% w/v DTT. Sonicate this suspension on ice six times for 10 s each with intermittent 1-min breaks using an ultrasonicator. Centrifuge samples at 35,000 x *g* for

4. Wash the pellet twice by resuspension in -20°C acetone containing 0.07% w/v DTT, placing it at -20°C for 30 min and centrifuging at 12,000 x *g* for 15 min at 4°C. 5. Lyophilize the pellet for 3 min and resuspend in sample buffer containing 9 M urea, 4% w/v CHAPS, 1% w/v DTT, 0.8% v/v ampholytes (ones suited for the desired 2D-gel),

6. Sonicate samples twice in a sonic bath in an ice-water mixture for 5 min and centrifuge

7. Determine protein concentration of the sample (e.g. with a Bradford assay or a BCA

fresh weight of cell pellets. Samples can be stored at -80°C for later use.

and latitudinal scale.

**5. Appendix A: Microalgae methodology** 

correspond to the species mentioned.

2003) that are suited for plant material.

25 mM Tris base, 1 mM PMSF and 5 mM EDTA.

assay) and keep at –80°C until used for isoelectric focusing.

them at 19,000 x *g* for 15 min at 20°C.

cells.

15 min at 4°C.

proteins detected with only 11 of them with significant changes. Also, the Cd concentration was far from toxic levels suggesting that changes in the protein expression were not needed. This is a non-sequenced species, and therefore, cross-species protein identification was conducted in order to identify those expressed in *N. oculata*. The results showed that malate dehydrogenase and NADH-dehydrogenase were newly induced, whereas glyceraldehyde 3-phosphate dehydrogenase was suppressed. The induction of malate dehydrogenase could be a defense mechanism against Cd toxicity, since at least in *C. reinhardtii* this enzyme controls the malate valve system, which exports reducing power from the chloroplast. Another work assessing Cd toxicity evaluated the proteomic profiles of treated (150 µM Cd) and untreated (control) mutants lacking cell walls of *C. reinhardtii* (Gillet et al., 2006). These mutants are more sensitive to heavy metals due to the lack of a cell wall (Macfie et al., 1994 as cited in Gillet et al., 2006). It was observed that cadmium slowed down the growth rate, and furthermore, induced a 30-50% of growth inhibition. In this work, an elevated number of protein spots were detected and subsequently identified. In fact, 20 proteins were downregulated in response to Cd stress. Among the down-regulated proteins were those that are involved in amino acid and nitrogen metabolism, chloroplast function and molecule biosynthesis to minimize ROS production. The most variable protein was the RubisCo large subunit, where the protein spot in the control treatment was 15.3 times more intense than in the Cd treatment. It was observed that enzymes with antioxidant properties, chaperonins, and enzymes involved in ATP and carbohydrate metabolism were up-regulated. In addition, in both works chloroplast proteins were found to be down-regulated and proteins involved in antioxidant response to be up-regulated. Therefore, the Cd tolerance mechanism may be similar among different species of microalgae.
