**4. Conclusions**

Micro and macroalgae contain high levels of compounds that interfere with protein extraction. These compounds lead to precipitation of insoluble polymers where the proteome obtainment is almost impossible. However, many efforts have been made in the last years to minimize the coprecipitation of those compounds, and thus now important proteomic protocols are available. For example in macroalgae, it is highlighted the use of phenol during the protein extraction, resulting in consistent electrophoresis runs in several species, conciliating suitable quality and reliability for 2-DE gels and its downstream analysis. The advantage of using phenol as an extracting agent resides in its capacity to disrupt membranes, leaving most of the water-soluble molecules totality in the aqueous phase.

Compared to animals and vascular plants, there is limited information about the use of 2-DE in either micro or macroalgae, both at technical and proteomic level. In fact, low number of published information in the proteomic context can be registered in micro and macroalgae. For example, in this group of organisms only about 42 works can be founded in the www.ncbi.nlm.nih.gov data base, using as search the concepts proteomic or proteome. On the other hand, in vascular plants it is possible to find about 3,100 works in the same data base and ca. 13,100 in animals. Thus, insignificant information exists nowadays in the proteome involvement in algal species, independent of the taxonomic status, ecological importance and economic value of this group of organisms.

2-DE in algal species has allowed the identification of several pathways involved in tolerance mechanisms, associated principally to different abiotic factors. For example,

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

Micro and macroalgae contain high levels of compounds that interfere with protein extraction. These compounds lead to precipitation of insoluble polymers where the proteome obtainment is almost impossible. However, many efforts have been made in the last years to minimize the coprecipitation of those compounds, and thus now important proteomic protocols are available. For example in macroalgae, it is highlighted the use of phenol during the protein extraction, resulting in consistent electrophoresis runs in several species, conciliating suitable quality and reliability for 2-DE gels and its downstream analysis. The advantage of using phenol as an extracting agent resides in its capacity to disrupt

membranes, leaving most of the water-soluble molecules totality in the aqueous phase.

Compared to animals and vascular plants, there is limited information about the use of 2-DE in either micro or macroalgae, both at technical and proteomic level. In fact, low number of published information in the proteomic context can be registered in micro and macroalgae. For example, in this group of organisms only about 42 works can be founded in the www.ncbi.nlm.nih.gov data base, using as search the concepts proteomic or proteome. On the other hand, in vascular plants it is possible to find about 3,100 works in the same data base and ca. 13,100 in animals. Thus, insignificant information exists nowadays in the proteome involvement in algal species, independent of the taxonomic status, ecological

2-DE in algal species has allowed the identification of several pathways involved in tolerance mechanisms, associated principally to different abiotic factors. For example,

may be similar among different species of microalgae.

importance and economic value of this group of organisms.

**4. Conclusions** 

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 and latitudinal scale.
