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Expresion diferencial de genes en Pyropia columbina (Bangiales, Rhodophyta) bajo hidratacion y desecacion natural.

Differential gene expression in pyropia columbina (Bangiales, Rhodophyta) under natural hydration and desiccation conditions

INTRODUCTION

Red algae (Rhodophyta), the most ancient lineage of photosynthetic eukaryotes (Baldauf et al., 2000; Yoon et al., 2004), are distributed worldwide and include several commercially important species. Porphyra and Pyropia spp. are rhodophytes that represent an annual value of over US$1.3 billion (Blouin et al., 2011). In Chile, Pyropia columbina (Montagne) W.A. Nelson (formerly Porphyra columbina (Sutherland et al., 2011) is one of the economically important species, together with members of the rhodophycean genera Mazzaella, Gracilaria and Gelidium (Santelices, 1989; Hoffmann & Santelices, 1997; Buschmann et al., 2008), and it is found along the Chilean coast from 20[degrees] to 54[degrees]S (Hoffmann & Santelices, 1997; Guiry & Guiry, 2013). This species has a biphasic life history that includes a microscopic sporophyte generation (2n, conchocelis stage) alternating with a macroscopic generation of male and female gametophytes (n). The foliose gametophytes constitute the edible "Nori".

Water, and its intracellular balance, is a critical factor for all living organisms in both terrestrial and marine ecosystems. Mobile animals actively avoid desiccation, induced by water deficiency, while other organisms, such as resurrection plants--a small group of angiosperms that live in the most arid habitats of the world--are adapted to tolerate water losses of up to 90% (Gaff, 1987). This adaptation, in general terms, is based on the ability of an organism to equilibrate its internal water potential with the dry environment, and re-start normal functions when rehydrated (Alpert, 2000). Several studies using resurrection plants as model have been conducted to fully understand their impressive adaptation to desiccation (Scott, 2000). Recent advances in our understanding of the mechanisms of tolerance in these organisms have revealed changes at the morphological level, osmolites and protein synthesis, and a decline in ROS (reactive oxygen species) production and photosynthesis rate (Ingram & Bartels, 1996; Hoekstra et al., 2001; Bernacchia & Furini, 2004; Vicre et al., 2004; Dinakar et al., 2012). More specifically, it has been observed that the plant hormone ABA is accumulated in desiccated resurrection plants, which induces the expression of several proteins related to desiccation (Bartels et al., 1990; Dinakar et al., 2012). Also, leaves of resurrection plants tend to curl to reduce water loss and minimize oxidative damage due to desiccation (Vicre et al., 2004; Farrant et al., 2007; Toldi et al., 2009). Oxidative damage is attenuated or avoided by increasing antioxidant activity of some enzymes (e.g., ascorbate peroxidase, glutathione reductase, superoxide dismutase, among others) and levels of antioxidant compounds (e.g., anthocyanins) (Farrant et al., 2007; Toldi et al., 2009; Dinakar et al., 2012). Additionally, these plants reduce photosynthetic activity to minimize photo-oxidative damage that could lead to increased ROS levels. Thus, it seems clear that diverse metabolic pathways are involved in attenuating the oxidative stress condition caused by desiccation.

In the Chilean coastal ecosystems, P. columbina grows abundantly along the upper intertidal zone (Alveal, 1970; Santelices, 1989; Hoffmann & Santelices, 1997), where it is exposed to a wide range of environmentally stressful conditions, mainly desiccation-driven stress induced by low tides and air exposure (Contreras-Porcia et al., 2011a). P. columbina is well adapted to daily extremes, which range from exposure to water (full hydration) during high tides to long exposure to air (maximum desiccation) during low tides. Natural exposure to these extreme regimes have been described in other organisms exposed to desiccation. It is already known that Porphyra and Pyropia species have high tolerance to desiccation, and quickly recover photosynthetic activity once rehydrated after a period of desiccation (e.g., Smith & Berry, 1986; Kim et al., 2008; Contreras-Porcia et al., 2011a; Gao & Wang, 2012). Basic physiology of these organisms, including the mechanisms to tolerate environmentally stressful conditions, remains poorly studied. However, it is known that, in P. columbina, desiccation induces losses ca. 96% of the water content and enhanced significantly the production of ROS (Contreras-Porcia et al., 2011a). The quick return of ROS to their basal levels during high tide is explained by an efficient activation of the antioxidant system. In comparison, species inhabiting the lower intertidal zone [e.g.,

Mazzaella laminarioides (Bory de Saint-Vincent) Fredericq, Ulva compressa Linnaeus and Lessonia spicata (Suhr) Santelices] are more sensitive to desiccation, and this seems related to the absence of efficient mechanisms to attenuate the over-production of ROS during rehydration (Contreras-Porcia et al., 2012; Lopez-Cristoffanini et al., 2013). Flores-Molina et al. (unpublished data) recently provided some physiological and biochemical bases that help explain the role of desiccation on species distribution across the intertidal zone. They reported that sensitive species displayed i) inactivation of antioxidant enzymes, ii) over-oxidation of biomolecules, and iii) inactivation of the photosystem II. For example, Ulva compressa (Chlorophyta, Plantae) and Scytosiphon lomentaria (Lyngbye) Link (Ochrophyta, Chromista) inhabiting the mid-intertidal zone have lower tolerance to desiccation than P. columbina. However, they tolerate desiccation better than Lessonia spicata (Ochrophyta, Chromista) and Gelidium rex Santelices & I.A. Abbott (Rhodophyta, Plantae), both lower intertidal species.

Given the ecological and economic relevance of Pyropia and that some basic biochemical and physiological information on the mechanisms involved in tolerance to desiccation is known, we focused this work in determining the genes/proteins that are differentially expressed in P. columbina during the hydration-desiccation cycle, using Suppression Subtractive Hybridization (SSH) and ESTs determined by pyrosequencing (454 Life Sciences, Roche). The results of this study will help to elucidate the genetic basis underlying the high tolerance to desiccation displayed by this species, and will broaden our knowledge of the molecular biology/ecology of this organism and other macroalgal species.

MATERIALS AND METHODS Ethics statement

No specific permits were required for the described field studies. The study area is unrestricted to public access and use, and is not privately owned or designated as a protected area (reserves or parks). No protected or endangered species were involved in this study. Fronds of P. columbina were collected along 200-300 m of coastline during high (naturally hydrated plants, 100% relative water content (RWC)) and low tide (naturally desiccated plants, ca. 4% RWC) in Maitencillo beach, Valparaiso (32[degrees]39.5'S, 71[degrees]26.6'W). After collection, fronds were quickly rinsed (15-20 s) in 0.22 urn-filtered seawater, manually cleaned and frozen on site with liquid nitrogen.

RNA extraction

Total RNA was isolated from 20-30 g of fresh tissue of naturally hydrated and desiccated, pooled fronds of P. columbina. Tissue, frozen in liquid nitrogen, was homogenized in 25 mL of lysis buffer containing 4 M guanidinium thiocyanate, 25 mM EDTA, 200 mM sodium acetate, 2% polyvinylpyrrolidone (PVP-40) and 1% 2-mercaptoethanol. The homogenate was incubated for 10 min at 70[degrees]C with constant agitation in the presence of 20% sarcosin, and then centrifuged for 5 min at 16000 g. The RNA present in the supernatant solution was purified and re-extracted using an RNeasy mini Kit (Qiagen, Hilden, Germany), according to the manufacturer protocol. RNA quality and yield was assessed by spectrophotometry (NanoDropTM 1000 Spectrophotometer, Thermo Scientific, DE, USA) and denaturing agarose gel electrophoresis. Finally, the mRNA was obtained from the total RNA extracted (ca. 290-300 ug) using DynaBeads (Invitrogen, Oregon, USA). Prior to RNA extraction, all material was treated in 0.1% DEPC water.

Preparation of the cDNA libraries by SSH and next-generation sequencing

The synthesis of cDNAs, from both natural conditions (i.e., natural hydration and desiccation stress), were obtained using a SMARTTM cDNA Library Construction Kit (Clontech, Mountain View, CA, USA), as described in Wellenreuther et al. (2004). Then, two subtracted cDNA libraries were constructed: one with genes expressed exclusively under hydration (UH) and the other by those expressed under desiccation stress (UD), as described in Diatchenko et al. (1996). cDNAs were then sequenced by 454-pyrosequencing (Margulies et al., 2005). cDNAs were ligated to 454 self-made adaptors with Multiplex Identifier Adaptors (MIDs) for the GS FLX Titanium Chemistry following Roche's technical bulletin TCB 09004 introducing SfiI-sites. The 454 libraries were immobilized on beads and clonally amplified using a "GS FLX Titanium LV emPCR Kit". The libraries were then sequenced using a "GS FLX Titanium Sequencing Kit XLR70" and a "GS FLX Titanium PicoTiterPlate Kit". All kits were purchased from Roche and used according to the manufacturer protocols.

Data assembly and bioinformatics analysis

Readings from both libraries were processed by selfwritten Perl scripts and assembled into coatings, i.e., representing putative transcripts using MIRA 3 assembly (Contreras-Porcia et al., 2011b). The expressed sequences tags (ESTs) determined from both libraries were subjected to separated bioinformatics analyses. ESTs were analyzed for sequence similarities using the BLASTX program (NCBI, MD, USA). Reading frames with the highest sequence similarity scores were used to analyze protein identity using the BLASTP program. Threshold values were set above 50 for high-scoring segment pairs, with a minimum significance at least of e < 10-4 and an identity higher than 30%. ESTs coding for known proteins were classified into functional categories by the KO (KEGG Orthology) database for ortholog grouping and hierarchical classification of genes, according their functionality (Kanehisa et al., 2004) by using the BLAST2GO software (Gotz et al., 2008). Finally, the putative subcellular localization of the proteins was determined using the TARGETP (http://www.cbs.dtu.dk/services/TargetP Emanuelsson et al., 2000), WOLFPSORT (http://psort.ims.u tokyo.ac.jp), ChloroP (http://www.cbs.dtu.dk/services/ ChloroP Emanuelsson et al., 1999) and PSORTb (http://www.psort.org/psortb Yu et al., 2010) servers.

RESULTS

EST sequencing and assembly

A total of 8,054 sequence reads were obtained from the library enriched with hydration-responsive exclusive transcripts (UH library, Table 1). Moreover, 8,432 sequence reads were obtained from the library enriched with desiccation-responsive exclusive transcripts (UD library). In the UH library, 49.2% of ESTs ranged from 200 to 500 bp, with an average size of 423 bp. In the UD library, 46% of the ESTs ranged from 200 to 500 bp, with an average size of 385 bp. The rest of the transcripts in both libraries ranged between 100 to 190 bp. The sequences of both libraries are available on the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/ embl) with accession numbers HE858615 to HE859412 for UH and HE859413 to HE859937 for UD libraries.

Gene ontology from naturally hydrated and desiccated P. columbina transcriptome

Almost 59% of the total ESTs (i.e., 491) from UH displayed no similarity with sequences available in databases; whereas the remaining 41% (i.e., 347) were similar to registered proteins (Table 1). Similarities (68.8% of them) concentrated in sequences reported for Arthropoda (e.g., Culex quinquefasciatus), Chordata (e.g. Danio rerio) and Mollusca (e.g., Littorina saxatilis). Another 18% of the total ESTs were similar to proteins from the kingdom Plantae, mainly Rhodophyta (e.g., Porphyra purpurea) and Tracheophyta (e.g., Vigna unguiculata), and 10.7% to the kingdom Bacteria, mainly Proteobacteria (e.g., Haemophilus influenzae) and Cyanobacteria (e.g., Microcystis aeruginosa). Only 2.5% of the sequences were similar to proteins from Protozoa (i.e., Dictyostelium discoideum), Fungi (i.e., Saccharomyces cerevisiae) and Chromista (i.e., Pylaiella littoralis). Finally, analysis of the amino terminal sequences showed that 51.8% of the identified proteins were potentially assignable to the cytosol, 28.6% to mitochondria, 13% to the chloroplast, and 4.4% to the nucleus (Table 1).

In the UD library, 72% of the total ESTs (i.e., 412) displayed no similarity with previously reported sequences (Table 2). Of the remaining 28% (i.e., 160 ESTs) showing similarity with proteins registered in databases: 37% were similar to those reported for Plantae, mainly Rhodophyta (e.g., Porphyra purpurea) and Tracheophyta (e.g., Arabidopsis thaliana), 29.4% with proteins from Bacteria, mainly Proteobacteria (e.g., Burkholderia multivorans) and Cyanobacteria (e.g., Thermosynechococcus elongatus), 21% with proteins from Animalia, mainly Arthropoda (e.g., Aedes aegypti), Chordata (e.g., Mauremys mutica) and Mollusca (e.g., Crassostrea virginica), and 7.5% with proteins from Chromista, mainly Ochrophyta (e.g., Ectocarpus siliculosus). Only 5% of the sequences were similar to proteins from Protozoa (i.e., Dictyostelium discoideum) and Fungi (i.e., Leptosphaeria maculans). Finally, analysis of the amino terminal sequences showed that 47% of the identified proteins were potentially assignable to cytosol, 21-23% to both mitochondria and chloroplast, and 7% to the nucleus (Table 2).

Functional categorization

The identified genes/proteins were classified into thirteen functional categories according their functionality (Fig. 1). For example, most ESTs from hydrated fronds (Table 1) matched with proteins involved in protein synthesis, processing and degradation (ca. 24%, peptidylprolyl cis-trans isomerases, ubiquitin and proteasome proteins), respiration and mitochondria (ca. 14%), antioxidant function, chaperone and defense factors (ca. 9%, e.g., peroxiredoxin (PRX), arachidonate 5-lipoxygenase, a glutathione S-transferase, and several cytochrome P450 and HSPs), cell motility (ca. 9%) and basal metabolism (ca. 8%; e.g., glyceraldehyde 3-phosphate dehydrogenase and L-lactate dehydrogenase). However, the transcriptome induced by desiccation (Fig. 1) revealed that ESTs with higher representation are involved in protein synthesis, processing and degradation (ca. 15%, e.g., Clp-protease), photosynthesis and chloroplast structure (ca. 14.4%; e.g., ferredoxin NADP+ reductase), respiration and mitochondria (ca. 13.1%), proteins involved in cell wall metabolism (ca. 10.6%, cell-wall hydrolases, glycosyl transferases and chitin deacetylases) and antioxidant activity, chaperone and defense factors (7.5%; thioredoxin (TRX), catalase and HSPs) (Table 1). Finally, with an important number of sequences it was not possible to find clear functional similarity with known proteins (ca. 11%).

DISCUSSION

The hydration-desiccation cycle in P. columbina generates an unbalance in the intracellular redox potential (Contreras-Porcia et al., 2011a), a situation that must be controlled by a coordinated cascade of responses that are induced differentially in each condition. Our results show that, during hydration, predominantly expressed genes were those involved in protein metabolism. Among them, we highlight several ribosomal proteins, translation initiation factors and elongation factors, proteases, the proteasome system and several ubiquitins (Table 1). Of particular relevance is the ubiquitin-proteasome system, present in all eukaryotes, which is activated in response to several abiotic stress factors and participates in tolerance mechanisms by removing unfolded proteins and proteins damaged during oxidative stress (Dreher & Callis, 2007; Pena et al., 2007; Kurepa et al., 2008). In this functional group three types of peptidylprolyl cistrans-isomerases (PPIases) were identified and found to be similar to those from Arthropoda and Mollusca. These proteins catalyze the cis-trans isomerisation of prolines (C5H9N[O.sub.2]), and have been described in the processes of cellular signalling (with a calmodulin-binding domain), regulation of gene transcription, and acting as chaperones and folding catalysts. Specifically, in the gastropod Conus novaehollandiae these proteins facilitate the oxidative folding of several neurotoxic peptides (Safavi-Hemami et al., 2010). In plants, these proteins are involved in flowering (Wang et al., 2010) and in controlling cell proliferation, since PPIase expression increases in the presence of cytokinin (Vittorioso et al., 1998). PPIases are induced by wounding, heat and salt stress (Vucich & Gasser, 1996; Kurek et al., 1999), and have been directly involved in membrane protein folding (i.e., chloroplast and mitochondria) (Breiman et al., 1992). Thus, the quick re-establishment of the normal condition after desiccation in P. columbina could be in part explained by both: i) the re-folding of structural proteins with important functions such as those involved in transcriptional regulation, and ii) by the removal of oxidized proteins. In fact, levels of oxidised proteins measured during the hydration-desiccation cycle were consistently lower in P. columbina compared to those in sensitive species (Contreras-Porcia et al., 2011a, Flores-Molina et al., unpublished data).

Proteins that form part of the energy metabolism were more highly represented during hydration [i.e., Cytochrome C Oxidase subunits, NADH Dehydrogenase subunits, NADH-Ubiquinone Oxidoreductase subunits, F1F0-ATP Synthase subunits and ADP/ATP carrier proteins (Table 1)] than during desiccation stress (Table 2). Theoretically, maintaining the tolerance mechanisms required to buffer the effects of a stress imposes energy costs (e.g., Zagdanska, 1995). Acclimation to oxidative stress depends on a high availability of NADPH and ATP, since most of the intracellular metabolic reactions require energy. For example, the ubiquitin--mediated system for intracellular protein degradation is ATP-dependent in all organisms (Ciechanover et al., 1984). Additionally, the structure of the chromosomes plays a critical role in transcriptional regulation where the chromatin remodeling is also ATP-dependent (Luo & Dean, 1999). In the case of P. columbina it seems reasonable to hypothesize the occurrence of a higher energy production by the mitochondrial system during hydration, as many metabolic reactions are necessary to maintain a homeostatic redox state and, as a result, a healthy physiological condition during the hydration-desiccation cycle. Moreover, the decline in ATP production resulting from a general metabolic slow down during desiccation could be beneficial in preventing ROS production triggered by the electronic alterations induced by low water potential. In vascular plants under water stress, ROS production has been detected in apoplast, xylem vessels, chloroplasts and mitochondria (Mittler et al., 2004; Toldi et al., 2009). Therefore, it is possible that desiccation in P. columbina induces ROS production in the organelles, a hypothesis that needs to be experimentally demonstrated. Additionally, the mitochondrial metabolism should be measured in order to demonstrate a decay-activation sequence of this system during the desiccation-hydration cycle.

In the context of ROS attenuation by the antioxidant system during the hydration-desiccation cycle, several antioxidant enzymes should be expressed. In fact, a peroxiredoxin (PRX) typical 2-Cys, an arachidonate 5-lipoxygenase, a glutathione S-transferase, and several cytochrome P450 and HSPs (heat shock proteins, 27, 40, 70, 80, 90 types) were detected during hydration. Moreover, during desiccation, enzymes such as thioredoxin (TRX), catalase and low variants of HSPs (70 and 90, gene sequences different from hydrated fronds) were expressed. PRXs are involved in detoxification of hydrogen peroxide, alkylhydroperoxides and peroxinitrites (Hall et al., 2009). These enzymes react at low peroxide concentrations and may become inactive at higher concentrations. In plants, PRX transcripts increase in response to different abiotic stresses such as salinity, drought, and heavy metals (Wood et al., 2003; Dietz, 2011). Their expression in algae has been poorly studied, although some studies indicate they are regulated by light, oxygen, copper, desiccation and redox state (Goyer et al., 2002; Contreras-Porcia et al., 2011a, 2001b; Lovazzano et al., 2013; see section 3 in Contreras-Porcia & Lopez-Cristoffanini, 2012). In vascular plants, several PRXs have been described based on their catalytic mechanisms and subcellular localization (Baier & Dietz, 1997). In this study, PRX expresses mainly when fronds are hydrated, is localized exclusively in the chloroplast, and its reduction is TRX-dependent. The chloroplast TRX activity was also recorded during desiccation (Table 2). Therefore, it seems likely that P. columbina PRX play an important role in buffering oxidative stress and in post-desiccation detoxification of lipoperoxides in the chloroplast. However, other attenuation systems might also be operating in both environmental conditions in order to normalize the redox state imbalance. For example, a chlorophycean ferredoxin was identified during desiccation (Table 2). This is a small protein that plays a key role in electron distribution in the chloroplast (Schurmann & Buchanan, 2008) by regulating the chloroplast metabolic network through the TRX system, and contributes directly to ascorbate antioxidant protection (i.e., antioxidant compound and ascorbate peroxidase substrate) and PRX regeneration (Ceccoli et al., 2011; Dietz, 2011).

Another enzyme detected in hydrated fronds was a lipoxygenase (LOX, Table 1), a dioxygenase which peroxidates polyunsaturated fatty acids (Gigon et al., 2004). Lipid molecules produced by lipid degrading enzymes, such as oxylipins, can act as secondary messengers of stress-response signal transduction pathways (Blee, 2002; Vellosillo et al., 2007). However, a hyper-stimulation of lipoxygenase activity could induce an accumulation of lipope-roxides, which leads to cell damage and organelle dysfunction. For example, Contreras et al. (2009) demonstrated that hyper-activity of LOX led to an over-production of lipoperoxides, and at the end, to cell death in sensitive species under copper-induced oxidative stress. The particular expression of this enzyme during hydration could explain the exceptional control of the lipid peroxidation in P. columbina (Contreras-Porcia et al., 2011a) in comparison with several other algae (FloresMolina et al., unpublished data). Moreover, the involvement of PRX during the hydration-desiccation cycle may additionally explain the effective ROS and lipoperoxide attenuation in this species.

Several cytochrome P450 variants were found in P. columbina under natural conditions of hydration and desiccation. These enzyme variants are present in all living species and catalyze the oxygenation of a high variety of substrates (Anzenbacher & Anzenbacherova, 2003). It has been demonstrated that P450s are induced by abiotic and biotic stress (Narusaka et al., 2004; Stolf-Moreira et al., 2011). Some P450s have been identified as ABA 8'hydroxylase that degrades ABA (abscidic acid) during the hydration-desiccation cycle (Kushiro et al., 2004; Shinozaki & YamaguchiShinozaki, 2007). In vascular plants, ABA is overproduced during desiccation (as in P. columbina, Guajardo et al. pers. comm.), causes stomatal closure, and induces stress related genes. However, via the ABA 8'hydroxylase-P450, ABA concentration is reduced to basal levels during the transition from desiccation to hydration (Kushiro et al., 2004). ABA has been recently identified in several algal species (i.e., Tarakhovskaya et al., 2007; Yokoya et al., 2010), although its role in regulating the expression of genes associated with tolerance to abiotic/biotic stress has not being explored. In P. columbina the functional role of P450 could open new avenues to learn on tolerance pathways involved in managing environmental stressors, such as the ABA involvement in transcription of regulatory networks of desiccation stress signals and gene expression. Moreover, ABA in P. columbina could be involved in the up-regulation of several compounds like sugar, prolines and polyamines (e.g., putrescine, spermidine and spermine), which are known to increase their expression under water stress in vascular plants and algae (Guill & Tuteja, 2010; Alcazar et al., 2011; Kumar et al., 2011). Thus, it is also possible to hypothesize regarding the participation of the ABAindependent pathways in the regulatory response to dehydration stress. Indeed, a Clp-protease was identified during desiccation (Table 2), and the clp gene not only was induced by dehydration, but was also up-regulated during natural senescence (Nakashima et al., 1997). Analysis of the clp gene in transgenic plants indicates that the clp promoter contains cis-acting element(s) involved not only in ABA independent stress-responsive gene expression but also in senescence-activated gene expression (Simpson et al., 2003). Thus, several tolerance pathways, previously unknown in algae and other organisms, could be synergistically activated under particular environmental stress conditions.

Proteases are indispensable for the normal functioning of cells and tissues in all living organisms. However, their activities need to be correctly regulated (Habib & Fazili, 2007). During the hydration-desiccation cycle in P. columbina, several peptidases were identified (Tables 1 and 2). Even though the expression of these proteins is required for protein metabolism, an over-expression can be potentially harmful. During hydration, a metallocarboxypeptidase inhibitor (CPI) was identified, that forms a less active or fully inactive enzyme. In vascular plants, CPIs are activated by metals, mechanical wounds or insect injury (Villanueva et al., 1998; Habib & Fazili, 2007; Harada et al., 2010), and its expression is ABA-regulated (Villanueva et al., 1998). Thus, this type of protease in P. columbina could be part of the ABA genes that are regulated during the hydration-desiccation cycle needed to maintain cellular integrity under water deficiency.

ABC (ATP binding cassette) transporter sequences were identified in desiccated fronds. These transporters are involved in translocation of a wide variety of compounds across cell membranes, including ions, carbohydrates, lipids, xenobiotics, drugs, and heavy metals (Ehrmann et al., 1998; Sipos & Kuchler, 2006; Contreras et al., 2010). These transporters have been reported during metal tolerance in vascular plants, carrying metal complexes from cytosol to the vacuole (Clemens, 2001). Also, in humans they appear associated with the protection of placental tissue by preventing cellular accumulation of cytotoxic compounds (Aye & Keelan, 2013). The participation of these transporters in desiccated P. columbina was unexpected, despite information which recently demonstrated that tolerance to desiccation in the free-living soil bacterium Rhizobium legumenosarum was associated with ABC-transporter activity (Vanderlinde et al., 2010). In the bacterium, a mutation in the ATP-binding component of a previously uncharacterized ATP transporter (Young et al., 2006) decreased the tolerance to desiccation due to low exopolysaccharides levels in the cell wall envelope of the mutant. That study demonstrated the crucial role of polysaccharides and their transport in organisms tolerant to desiccation. Therefore, our results suggest, for the first time in algae, the involvement of ABC-transporters in desiccation tolerance, possibly through cell wall stabilization during desiccation stress.

Several HSP were identified during hydration and desiccation stress. These proteins are recognized in prokaryotes and eukaryotes during responses to different physiological and environmental stress conditions (Feder & Hofmann, 1999). HSPs induction leads to a state of resistance for subsequent stress in the cell by preventing protein aggregation (Feder & Hofmann, 1999) and suppressing apoptosis (e.g., HSP90 see Beere et al., 2000 and Ravagnan et al., 2001). HSP70 blocks apoptosis by binding apoptosis activating factor-1 (Apaf-1), thereby preventing the formation of the apoptosome complex (Ravagnan et al., 2001). In fish hepatocytes, HSPs induction is involved in stress tolerance by modulating the action of key proteins and kinases in the signal transduction pathways (Padmini & Usha-Rani, 2011). In the seaweeds Fucus serratus and F. vesiculosus, a small HSP has also been recorded in response to abiotic stress, but its specific role remains undetermined (Pearson et al., 2010).

During desiccation sequences of cell-wall hydrolases, glycosyl transferases, and chitin deacetylases (CDA)--all involved in cell wall metabolism--were identified. CDA, a type of carbohydrate esterase, hydrolyzes the acetamide group in the Nacetylglucosamine polymers derived from glucose (e.g., chitin), and promotes the formation of glucosamine units (e.g., chitosan). The substrates for this enzyme come from the carbon (fructose 6phosphate) and nitrogen (glutamine) metabolism (Ghormade et al., 2010). CDA was first discovered in extracts of the fungus Mucor rouxii (Araki & Ito, 1975). It was further reported associated with cell wall synthesis. CDA has also been reported in association with spore formation in yeast and attack-defense systems in plant-pathogen interactions. In P. columbina an over-activation of this enzyme is likely to occur, due to the induction, under desiccation stress, of a fructose-1,6-phosphatase (Table 2) that can overproduce fructose 6-phosphate, an important precursor of the CDA substrate. Thus, the potential induction of CDA in desiccated P. columbina may help in remodeling and maintaining cell wall integrity during growth, survival, and pathogenesis.

CONCLUSIONS

Despite their crucial ecological role as primary producers, molecular information on stress responses in intertidal macroalgae remains limited. Pyropia columbina, such as others Porphyra and Pyropia species, is a good model for unravelling some of the biological and molecular responses associated with desiccation and other environmental conditions that may cause oxidative stress. In this context, two subtracted EST libraries were constructed in order to understand the metabolic pathways active during the hydration-desiccation cycle in this species. Results showed that a significant portion of the transcripts had no known homologues in algae or other organisms as far as sequence data were available. These sequences are interesting since they could represent genes unique to this species. On the other hand, several genes/proteins not previously described in algae were differentially expressed in both environmental conditions. This information contributes to a better understanding the molecular mechanisms involved in tolerance to desiccation. However, a confirmation of the expression profiles of the reported genes by qPCR is needed to characterize, for example, temporality of the gene expression profile during the hydrationdesiccation cycle. Additionally, our study provides for the first time, a set of candidate genes for further examination of the physiological responses to other environmental stressors. This genetic background will broaden our understanding on physiological differences may contribute, or even determine, the ecological features of macroalgae inhabiting the intertidal rocky zone.

DOI: 103856/vol41-issue5-fulltext-13

ACKNOWLEDGEMENTS

Funding provided by FONDECYT 11085019 and partially by FONDECYT 1120117, to LCP, and Marine Genomics Europe Technology Platforms Bid no. 43, to LCP, JC, MK and RR. Additional funding comes from FONDAP 1501-0001 (CONICYT), to the Center for Advanced Studies in Ecology & Biodiversity (CASEB) Program 7, and to JC.

Authors' Contributions: LCP, JC, and RR conceived and designed the project. MK and RR were responsible for library construction and sequencing. CLC, CL, MFM, EG, DT, and AN performed functional annotation analyses. LCP wrote the manuscript. All authors edited the manuscript. All authors read and approved the manuscript.

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Received: 18 April 2013; Accepted: 8 October 2013

Loretto Contreras-Porcia (1), Camilo Lopez-Cristoffanini (1,2), Carlos Lovazzano (1) Maria Rosa Flores-Molina (3), Daniela Thomas (1), Alejandra Nunez (1), Camila Fierro (1), Eduardo Guajardo (1) Juan A. Correa (2), Michael Kube (4) & Richard Reinhardt (5)

(1) Departamento de Ecologia y Biodiversidad, Facultad de Ecologia y Recursos Naturales Universidad Andres Bello, Republica 470, Santiago, Chile

(2) Departamento de Ecologia, Center for Advanced Studies in Ecology and Biodiversity (CASEB) Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile Postal code 6513677, Santiago, Chile

(3) Instituto de Ciencias Marinas y Limnologicas, Facultad de Ciencias, Universidad Austral de Chile 4 P.O. Box 567, Valdivia, Chile

(4) Department of Crop and Animal Sciences, Faculty of Agriculture and Horticulture Humboldt Universitat zu Berlin, Lentzeallee 55/57, 14195 Berlin, Germany

(5) Max-Planck Institute for Molecular Genetics, Ihnestr. 63-73, Berlin, Germany

Corresponding author: Loretto Contreras-Porcia (lorettocontreras@unab.cl)

Table 1. Functional category, identity and potential cellular
destination of proteins, and accession number of identified ESTs in P.
columbina under natural hydration (UH library). E value: the best
(lowest) Expect value (E value) of all alignments from that database
sequence, D*: Putative destination.

Functional category            Putative Identity

1. Signal transduction         Phosphatidylinositol 3-kinase 1

                               MAP kinase activated protein-kinase-2

                               Low-density lipoprotein receptor-
                               related protein 1B-like

                               Low-density lipoprotein receptor-
                               related protein 2 precursor

                               Mitogen-activated protein kinase
                               kinase kinase 15

                               Neurochondrin

                               Neurochondrin-like protein

                               Serine/threonine kinase

                               Protein serine/threonine kinase,
                               putative

                               Ras-related protein, isoform B

                               Serine/threonine kinase receptor-
                               associated protein

                               Serine/threonine protein kinase

                               Serine/threonine-protein phosphatase 5

                               SNF4/AMP-activated protein kinase
                               gamma subunit, isoform M

                               Troponin C type IIIa

                               Calmodulin

                               Cyclic AMP phosphoprotein

                               GAF sensor signal transduction
                               histidine kinase

                               Guanine nucleotide binding protein
                               beta polypeptide 2- like 1

                               Putative guanine nucleotide binding
                               protein beta polypeptide 2-like 1
                               protein

                               Troponin T

                               Calreticulin

                               Adenylyl cyclase associated protein

                               Transcription factor BTF3 homolog 4

2. Transcription, splicing,    Transcription factor
and replication
                               TFIIH basal transcription factor
                               complex P44 subunit

                               RNA polymerase beta subunit

                               Homeotic protein spalt-major

                               Nascent polypeptide associated
                               complex protein alpha subunit

                               Similar to nuclear histone binding
                               protein
                               orf544 reverse transcriptase

                               Reverse transcriptase al1

                               Reverse transcriptase homolog

                               RNA-directed DNA polymerase

                               RNA-directed DNA polymerase

                               RNA-directed DNA polymerase
                               (reverse transcriptase)

                               RNA-directed DNA polymerase
                               (reverse transcriptase)

                               Retrotransposon protein

                               Retrotransposon protein

                               RNA-binding protein 8A

                               rRNA intron-encoded homing
                               endonuclease

                               Ribonuclease R

                               rRNA promoter binding protein
                               ATP-dependent helicase

                               rRNA intron-encoded homing
                               endonuclease

                               Metal-dependent RNase

3. Basal metabolism            Mitochondrial Malate dehy
                               drogenase 2

                               Glyceraldehyde 3-phosphate
                               dehydrogenase

                               Glyceraldehyde 3-phosphate
                               dehydrogenase

                               Pyruvate dehydrogenase

                               Medium chain acyl-coenzyme A

                               dehydrogenase

                               L-lactate dehydrogenase-like

                               Acetyl-CoA carboxylase beta subunit

                               NAD-dependent
                               epimerase/dehydratase

                               Phosphogluconate dehydrogenase

                               Phosphoglycerate mutase

                               Mitochondrial Succinyl-CoA ligase
                               [ADP-forming] subunit beta

                               Succinyl-CoA ligase [GDP-forming]
                               subunit alpha

                               Transcript antisense to ribosomal RNA
                               protein 1(Tar1p)

                               Trehalose-6-phosphate synthase

                               Trehalose-6-phosphate synthase 1

                               2,5-diketo-D-gluconic acid reductase
                               A

                               3,4-dihydroxy-2-butanone-4-
                               phosphate synthase

                               3,4-dihydroxy-2-butanone-4-
                               phosphate synthase

                               3,4-dihydroxy-2-butanone-4-
                               phosphate synthase

                               Amylo-1, 6-glucosidase, 4-alpha-
                               glucanotransferase

                               Arginine kinase

                               Arginine kinase

                               Putative enolase

                               Putative enolase

                               Glutamate dehydrogenase

                               Glycerol kinase

                               Short-chain dehydrogenase

4. Antioxidant,                Peroxiredoxin (PRX) family, typical
chaperone and defense          2-Cys
factors
                               Arachidonate 5-lipoxygenase

                               Glutathione S-transferase sigma 5

                               28 kDa heat- and acid-stable
                               phosphoprotein

                               Apolipoprotein D

                               Probable Bax Inhibitor 1

                               Ferritin heavy chain-like

                               Ferritin heavy chain-like protein

                               Small heat shock protein IbpA

                               Heat shock protein 23 beta

                               Heat shock protein 27

                               Heat shock protein 27

                               Heat shock protein 27

                               Heat shock protein 27

                               Heat shock protein 40

                               Heat shock protein 70

                               Heat shock protein 70

                               Heat shock protein 70

                               Heat shock protein 70

                               Heat shock protein 70

                               Heat shock protein 70B

                               Chloroplast Heat shock protein 70

                               Heat shock protein 70

                               Heat shock protein 70- type chaperone
                               (chloroplast)

                               Heat shock protein 70C

                               Heat shock protein 82

                               Heat shock protein 90

                               Heat shock protein 90

                               Stress-induced-phosphoprotein 1

                               Cytochrome P450 4C1-like

                               Cytochrome P450 like_TBP

                               Putative cytochrome P450 like protein
                               precursor

5. Protein synthesis,          20S proteasome alpha subunit
processing and
degradation
                               26S protease regulatory subunit 8

                               26S proteasome non-ATPase
                               regulatory subunit

                               26S proteasome non-ATPase
                               regulatory subunit 14

                               26S proteasome regulatory complex
                               subunit RPN5/PSMD12

                               Proteasome subunit beta type 3

                               Proteasome subunit alpha type 3

                               Eukaryotic translation initiation
                               factor 3

                               Eukaryotic translation initiation factor
                               3 subunit 10

                               Putative 23S ribosomal RNA

                               Putative ribosomal protein S3

                               Ribosomal protein S8

                               30S ribosomal protein S10

                               Ribosomal protein RPS3a

                               40S ribosomal protein S4

                               Ribosomal protein S9

                               Ribosomal protein S13

                               Ribosomal protein S15 isoform A

                               40S ribosomal protein S16

                               Putative 40S ribosomal protein RPS16

                               40S ribosomal protein S30

                               40S ribosomal protein SA-like

                               Ribosomal protein RPL14

                               Ribosomal protein L32

                               60S ribosomal protein L6

                               60S ribosomal protein L10-3

                               60S ribosomal protein L11

                               60S ribosomal protein L12

                               Ribosomal protein RPL17

                               Large subunit ribosomal protein 23

                               Putative 60S ribosomal protein RPL27

                               Ribosomal protein L10a, component
                               of cytosolic 80S ribosome and 60S
                               large subunit

                               Putative 23S ribosomal RNA

                               Putative ribosomal protein S3

                               Putative ribosomal protein S3

                               Ubiquitin C

                               Ubiquitin C

                               Putative ubiquitin C variant 1

                               Predicted protein (ubiquitin isoform 1)

                               Ubiquitin-conjugating enzyme E2

                               E3 Ubiquitin-protein ligase UHRF1-
                               Like

                               Hypothetical protein 382

                               Molybdopterin biosynthesis protein

                               Peptidase membrane zinc

                               metallopeptidase

                               Protein disulfide isomerase

                               Serine peptidase 2

                               Serine protease

                               Serine-type endopeptidase

                               Putative serine-type endopeptidase

                               Signal peptidase complex catalytic
                               subunit SEC11A

                               Signal peptidase complex subunit 3

                               Signal recognition particle subunit
                               Srp68

                               Signal sequence receptor, beta-like

                               T-complex protein 1 subunit epsilon

                               Translation initiation factor

                               Protein translation factor

                               Translation initiation factor 2 gamma
                               subunit

                               Histidine-tRNA synthetase

                               Histidyl-tRNA synthetase

                               60S acidic ribosomal protein P0

                               60S acidic ribosomal protein P2

                               Transcript antisense to ribosomal RNA
                               protein 1 (Tar1p)

                               Venom serine carboxypeptidase
                               precursor

                               Cathepsin L1-like isoform 1 (cysteine
                               protease)

                               Cathepsin L2 cysteine protease

                               Cysteine proteinase cathepsin F

                               Elongation factor 1 gamma

                               Putative elongation factor 1 gamma

                               Elongation factor 1-beta

                               Elongation factor 1 beta

                               Elongation factor 1-beta

                               Elongation factor-1 gamma

                               Elongation factor-1 gamma

                               Elongation factor-1 gamma

                               Elongation factor-1 gamma

                               Elongation factor-1 gamma

                               Elongation factor-2

                               Peptidyl prolyl cis-trans isomerase B

                               Peptidyl-prolyl cis-trans isomerase

                               Peptidyl-prolyl isomerase-1

                               Phenylalanyl-tRNA synthetase beta
                               chain

                               Ribosome biogenesis protein Nsa2
                               homolog

                               Metallocarboxypeptidase inhibitor

6. Cell motility               Intermediate filament protein

                               Myomesin

                               Myosin light chain

                               Myosin 1 light chain

                               Myosin heavy chain isoform 3

                               Paramyosin, short form

                               Paramyosin, short form

                               Tropomodulin

                               Tropomodulin, isoform K

                               Tropomyosin-2 isoform 1

                               Actin

                               Actin

                               Actin-2, partial

                               Actin 4

                               Actin 57B

                               Actin 88F

                               Actin

                               Actin E2

                               Alpha actinin, isoform A

                               Beta-actin

                               Beta-actin

                               Beta-actin

                               Actin 87E

                               Actin type 1

                               Actin, cytoplasmic 2-like isoform 1

                               Alpha-tubulin
                               Ankyrin-1-like

                               Dynein heavy chain

                               Stretchin-Mlck, isoform L

                               Up-regulated during skeletal muscle
                               growth protein 5--like

                               Inhibitor of apoptosis protein

7. Cell growth and death       Translationally-controlled tumor
                               protein

                               Cathepsin D

                               Death-associated small cytoplasmic
                               leucine-rich protein

                               CHK1 checkpoint-like protein

8. Membrane transporters       Transmembrane channel-like 2

                               Protein-export membrane protein
                               Secd/Secf

                               RND superfamily resistance-
                               nodulation-cell division: proton (H+)
                               antiporter

                               Sugar transporter

                               Transport protein

9. Vesicular transport and     Putative Ras-related protein Rab-2A
metabolism
                               Rtnl1, isoform C

                               Transport protein Sec61 subunit alpha
                               2

10. Cell wall metabolism       Heparanase precursor

                               Cell wall-associated hydrolase

                               Cell wall-associated hydrolase

                               Cell wall-associated hydrolase

                               Cell wall-associated hydrolase

                               Cell wall-associated hydrolase

                               Cell wall-associated hydrolase

                               Cell wall-associated hydrolase

                               Glycoside hydrolases

                               Glycosyl transferase family protein

                               30S ribosomal protein S13

11. Photosynthesis and         Ribosomal protein L3
chloroplast proteins
                               Ribosomal protein L13

                               Ribosomal protein L35

                               Photosystem I P700 chlorophyll A
                               apoprotein A1

                               Photosystem I P700 chlorophyll A
                               apoprotein A1

                               Photosystem I P700 chlorophyll A
                               apoprotein A1

                               Photosystem II cytochrome C550

                               Photosystem II D2 protein

                               Photosystem II protein D1

                               Photosystem II protein D1

                               Photochlorophyllide reductase subunit B

                               Photochlorophyllide reductase subunit L

                               Photochlorophyllide reductase subunit N

                               Phycobilisome linker polypeptide

                               Phycoerythrin beta subunit

                               Phycoerythrin beta subunit

                               Putative rubisco expression protein

                               Allophycocyanin subunit alpha

                               ATP synthase CF1 delta subunit

                               ATP synthase CF0 A subunit

                               ATP synthase CF0 subunit I

                               Beta-Ig-H3/fasciclin

                               Clp protease ATP binding subunit

12. Respiration and            Cytochrome P450 likeTBP
mitochondrial proteins
                               Glycine cleavage system protein H

                               Mitochondrial processing peptidase
                               beta subunit

                               Cytochrome B-C1 complex subunit 9-
                               like

                               NADH dehydrogenase [ubiquinone] 1
                               beta subcomplex subunit 8
                               mitochondrial

                               NADH dehydrogenase subunit 1

                               NADH dehydrogenase subunit 2

                               NADH dehydrogenase subunit 2

                               NADH-ubiquinone oxidoreductase 39
                               kDa subunit

                               NADH-ubiquinone oxidoreductase Fe-
                               S protein 2 (Ndufs2)

                               Cytochrome oxidase subunit I

                               Cytochrome oxidase subunit I

                               Cytochrome oxidase subunit I

                               Cytochrome oxidase subunit I

                               Cytochrome oxidase subunit II

                               Cytochrome oxidase subunit II

                               Cytochrome oxidase subunit II

                               Cytochrome oxidase subunit II

                               Cytochrome oxidase subunit II

                               Cytochrome C oxidase subunit 3

                               Cytochrome C oxidase subunit III

                               Cytochrome C oxidase subunit III

                               Cytochrome C oxidase subunit IV

                               Cytochrome C oxidase subunit
                               Va/COX6
                               Cytochrome C oxidase polypeptide
                               VIa
                               Cytochrome C oxidase subunit 6a
                               polypeptide 1
                               F1F0-ATP synthase subunit
                               C/ATP9/proteolipid
                               F1F0-ATP synthase subunit
                               epsilon/ATP15
                               F1F0-ATP synthase subunit
                               OSCP/ATP5
                               Mitochondrial matrix protein P32
                               Mitochondrial succinate
                               dehydrogenase cytochrome B subunit
                               ADP / ATP carrier protein

                               ADP / ATP translocase
                               ATP synthase alpha subunit precursor
                               ATP synthase alpha subunit precursor

                               ATP synthase b
                               ATPSyn-beta

                               Mitochondrial ATP synthase delta
                               subunit
                               ATP synthase delta subunit, isoform A

                               ATP synthase, H+ transporting,
                               mitochondrial F1 complex, O subunit
                               Cytochrome B

                               Cytochrome B

                               Cytochrome B

                               Cytochrome C

                               Cytochrome C1

                               Cytochrome C1

                               ATP synthase F0 subunit 6

                               H+ transporting ATP synthase
                               subunit e
                               Predicted ubiquinol-cytochrome C
                               reductase core protein I
                               Voltage-dependent anion-selective
                               channel
13. Others                     Putative senescence-associated protein
                               Putative senescence-associated protein

                               Putative senescence-associated protein

                               Senescence-associated protein
                               Kazal domain-containing peptide
                               Putative secreted protein 10 kDa
                               AGAP009754-PA (angiotensin-
                               converting enzyme 4)
                               Another B-box affiliate, isoform A

                               AGAP008459-PA (cuticular protein 9,
                               low complexity family)
                               AGAP009872-PA (cuticular protein
                               133, RR-1 family)
                               Putative pupal cuticle protein
                               Fibroleukin-like

                               Cuticular protein analogous to
                               peritrophins 3-A1
                               Vitellogenin
                               Vitellogenin 1b
                               Vitellogenin 1a
                               Vitellogenin 2a
                               Vitellogenin A1

                               Vitellogenin 2b
                               Vitellogenin B

                               Vitellogenin C

                               Vitellogenin C

                               Vitellogenin precursor

                               Vitellogenin C
                               CDGSH iron sulfur domain-containing
                               protein 2-like protein
                               Opsin-1

                               Rhodopsin
                               Lethal(2)essential for life protein,
                                 l2efl
                               Similar to fibropellin Ia

                               Cob(I)yrinic acid a,c-diamide
                               adenosyltransferase

Functional category            Species and accession number

1. Signal transduction         Culex quinquefasciatus /
                                 XP_001847784.1
                               Glossina morsitans morsitans /
                                 ABC25082.1
                               Xenopus (Silurana) tropicalis /
                               XP 002937402.1

                               Danio rerio / NP_001181916.1

                               Apis mellifera / XP_003250315.1

                               Drosophila melanogaster / NP_649658.1

                               Harpegnathos saltator / EFN88263.1

                               Culex quinquefasciatus / XP_001844678.1

                               Aedes aegypti / XP_001660772.1

                               Drosophila melanogaster / NP_726881.1

                               Glossina morsitans morsitans /
                               ADD19360.1

                               Aedes aegypti / XP_001655486.1

                               Culex quinquefasciatus / XP_001850926.1

                               Drosophila melanogaster /
                               NP_001097854.1

                               Apis mellifera / NP_001011651.1

                               Pyropia yezoensis / ABN41559.1

                               Scophthalmus maximus / ABJ98640.1

                               Microcoleus vaginatus FGP-2
                               / ZP_08492202.1

                               Lethenteron camtschaticum/
                               BAE93065.1

                               Ovis aries / ABY75292.1

                               Pediculus humanus corporis /
                               XP_002424248.1
                               Eisenia andrei / ABI74618.1

                               Gossypium herbaceum subsp. africanum /
                                 ADZ16114.1
                               Salmo salar / ACI69109.1

2. Transcription, splicing,    Brugia malayi / XP_001891758.1
and replication
                               Culex quinquefasciatus /
                               XP_001845822.1

                               Pyropia yezoensis / YP_536931.1

                               Camponotus floridanus /
                               EFN74164.1
                               Glossina morsitans morsitans /
                               ADD19571.1

                               Strongylocentrotus purpuratus /
                               XP_791433.2
                               Porphyra purpurea / NP_049292.1

                               Saccharomyces cerevisiae S288c /
                                 NP_009310.1
                               Pylaiella littoralis / BAG06161.1

                               Moorea producens 3L / ZP 08427361.1

                               Microcystis aeruginosa NIES-843
                               / YP_001659893.1
                               Microcoleus vaginatus FGP-2 /
                               ZP_08493544.1

                               Arthrospira maxima CS-328 /
                               ZP_03271771.1

                               Oryza sativa Indica Group /
                               ABR26094.1

                               Oryza sativa Indica Group /
                               ABR26094.1
                               Apis mellifera / XP_395245.2

                               Thalassiosira pseudonana
                               CCMP1335 / XP_002294430.1

                               Weeksella virosa DSM 16922 /
                               YP_004238302.1

                               Brugia malayi / XP_001891797.1
                               Zobellia galactanivorans
                               / CAZ98388.1

                               Medicago truncatula /
                               XP_003614389.1

                               Streptococcus pyogenes M49 591 /
                               ZP_00366321.1

3. Basal metabolism            Culex quinquefasciatus /
                               XP_001849862.1

                               Simulium nigrimanum /
                               ACZ28396.1

                               Tribolium castaneum /
                               XP 974181.1

                               Aedes aegypti / XP_001648922.1

                               Pachycara brachycephalum /

                               AEC32921.1

                               Apis mellifera / XP_394662.4

                               Porphyra purpurea / NP_053808.1

                               Zea mays / NP_001148959.1

                               Tipula abdominalis /ACH95392.1

                               Porphyra purpurea / NP_053989.1

                               Camponotus floridanus /
                               EFN67099.1

                               Anolis carolinensis /
                               XP_003221906.1

                               Saccharomyces cerevisiae S288c /
                               NP_690845.1

                               Aedes aegypti / XP_001657813.1

                               Culex quinquefasciatus /
                               XP_001850996.1

                               Rhodococcus erythropolis SK121 /
                               ZP_04383522.1

                               Haemophilus influenzae HK1212 /
                               ZP_06222660.1

                               Haemophilus influenzae HK1212 /
                               ZP_06222660.1

                               Haemophilus influenzae HK1212 /
                               ZP_06222660.1

                               Danio rerio / NP_001166124.1

                               Philaethria wernickei /
                               ACZ26817.1

                               Glossina morsitans morsitans /
                               ADD19663.1

                               Lutzomyia longipalpis /
                               ABV60328.1

                               Myrmecocystus depilis /
                               ABY55706.1

                               Bombyx mori /NP_001040245.1

                               Candidatus Liberibacter
                               americanus str. Sao Paulo /
                               ACD87749.1

                               Aedes aegypti / XP_001663676.1

4. Antioxidant,                Pyropia yezoensis / YP_536954.1
chaperone and defense
factors
                               Strongylocentrotus purpuratus /
                               XP_001198535.1

                               Locusta migratoria /AEB91977.1

                               Harpegnathos saltator /
                               EFN84845.1

                               Culex quinquefasciatus /
                               XP_001848365.1

                               Harpegnathos saltator /
                               EFN76935.1

                               Ailuropoda melanoleuca /
                               XP_002929594.1

                               Phlebotomus papatasi /
                               ABV44737.1

                               Trichinella pseudospiralis /
                               ABJ55915.1

                               Ceratitis capitata / ACG58884.1

                               Drosophila melanogaster /
                               NP_524000.1

                               Drosophila bipectinata /
                               AEB40325.1

                               Drosophila parabipectinata /
                               AEB40323.1

                               Ceratitis capitata / ACD76913.1

                               Bactrocera dorsalis/ADO30472.1

                               Porphyra purpurea / NP_053925.1

                               Pyropia haitanensis / Q06W39.1

                               Pyropia haitanensis / ACF71814.1

                               Ostreococcus lucimarinus /
                               XP_001417572.1

                               Raphidiopsis brookii D9 /
                               ZP_06305357.1

                               Dunaliella salina / ACJ24805.1

                               Pyropia yezoensis / ABF54971.1

                               Gracilaria tenuistipitata var. liui /
                               YP_063608.1

                               Guillardia theta / AAC35702.1

                               Ascaris suum / ADY47984.1

                               Philodina roseola / ACC43981.1

                               Spodoptera exigua / ACQ78181.1

                               Thitarodes pui/ ADA61011.1

                               Ciona intestinalis /
                               XP_002128875.1

                               Acyrthosiphon pisum /
                               XP_001945361.2

                               Citrullus lanatus / BAD26579.1

                               Phillyrea latifolia / CAK18871.1

5. Protein synthesis,          Scylla paramamosain /
processing and                 ACY66486.1
degradation
                               Acromyrmex echinatior /
                               EGI70168.1
                               Aedes aegypti / XP_001662445.1

                               Aedes albopictus / ADB43603.1

                               Glossina morsitans morsitans /
                               ADD19088.1

                               Culex quinquefasciatus /
                               XP_001843139.1

                               Oncorhynchus mykiss /
                               ACO07671.1

                               Culex quinquefasciatus /
                               XP_001851152.1

                               Culex quinquefasciatus /
                               XP_001844367.1

                               Vigna unguiculata / CAO02532.1

                               Vigna unguiculata / CAO02550.1

                               Polaribacter irgensii 23-P /
                               ZP_01119260.1

                               Polaribacter sp.MED152 /
                               ZP_05108264.1

                               Eurythoe complanata /
                               ABW23199.1

                               Mytilus edulis / ABA55738.1

                               Haliotis discus discus /
                               ABX26127.1

                               Xenopus (Silurana) tropicalis /
                               NP_001016602.1

                               Lysiphlebus testaceipes /
                               AAX62477.1

                               Glossina morsitans morsitans /
                               ADD20564.1

                               Novocrania anomala /
                               ACD65104.1

                               Aedes aegypti / XP_001653913.1

                               Nasonia vitripennis /
                               XP_001608082.1

                               Arenicola marina / ABW23170.1

                               Lepidochitona cinerea /
                               ACR24953.1

                               Harpegnathos saltator /
                               EFN79475.1

                               Zea mays / NP_001149336.1

                               Glossina morsitans morsitans /
                               ADD20563.1

                               Zea mays / ACG35173.1

                               Eurythoe complanata /
                               ABW23221.1

                               Pristionchus pacificus /
                               ABR87202.1

                               Novocrania anomala /
                               ACD65128.1

                               Chlamydomonas reinhardtii /
                               XP_001699807.1

                               Vigna unguiculata / CAO02532.1

                               Vigna unguiculata / CAO02550.1

                               Vigna unguiculata / CAO02550.1

                               Schistosoma japonicum /
                               CAX79699.1

                               Equus caballus / NP_001075331.1

                               Taeniopygia guttata / ACH45550.1

                               Physcomitrella patens subsp.
                               patens / XP 001764949.1

                               Zea mays / NP_001140410.1

                               Meleagris gallopavo /
                               XP 003213379.1

                               Pyropia yezoensis / YP_537017.1

                               Porphyra purpurea / NP_053945.1

                               Pelodictyon phaeoclathratiforme

                               BU-1 / YP_002018463.1

                               Dictyostelium discoideum AX4 /
                               XP_635206.1

                               Radix peregra / ABL67951.1

                               Aedes aegypti / XP_001655711.1

                               Culex quinquefasciatus /
                               XP_001846627.1

                               Aedes aegypti / XP_001663439.1

                               Danio rerio / NP_001002521.1

                               Bombyx mori /NP_001091763.1

                               Glossina morsitans morsitans /
                               ADD18348.1

                               Saccoglossus kowalevskii /
                               XP_002740523.1

                               Harpegnathos saltator /
                               EFN84860.1

                               Anopheles gambiae str. PEST /
                               XP_316499.2

                               Griffithsia japonica / AAM93956.1

                               Allacma fusca / CAG29667.1

                               Porphyra purpurea / NP_053958.1

                               Moorea producens 3L /
                               ZP_08428961.1

                               Haliotis diversicolor / ABY87386.1

                               Spodoptera frugiperda /
                               AAL62467.1

                               Saccharomyces cerevisiae S288c /
                               NP_690845.1

                               Apis mellifera / NP_001152775.1

                               Danio rerio / XP_001341714.2

                               Pinctada fucata / ADC52431.1

                               Glossina morsitans morsitans /
                               ADD19167.1

                               Caenorhabditis brenneri /
                               ACE00305.1

                               Ixodes scapularis /
                               XP_002410199.1

                               Culex tarsalis / ACJ64291.1

                               Simulium nigrimanum /
                               ACZ28393.1

                               Artemia salina / P12262.3

                               Trichinella spiralis /
                               XP_003375107.1

                               Bothriocyrtum californicum /
                               ABG88914.1

                               Aliatypus plutonis / ABG88916.1

                               Deinopis spinosa / ABG88956.1

                               Aptostichus sp. 4 NAA-2006 /
                               ABG88917.1

                               Harbansus paucichelatus /
                               AAR01294.1

                               Conus novaehollandiae /
                               ADC80506.1

                               Culex quinquefasciatus /
                               XP_001846632.1

                               Gryllus firmus / ACD69575.1

                               Porphyra purpurea / NP_053956.1

                               Ictalurus punctatus /
                               NP_001187984.1

                               Medicago truncatula /
                               XP_003616487.1

6. Cell motility               Biomphalaria glabrata /
                               AAZ39528.1

                               Aedes aegypti / XP_001655591.1

                               Gryllotalpa orientalis /
                               AAW22542.1

                               Palaemonetes varians /
                               ACR54116.1

                               Daphnia pulex / EFX87106.1

                               Harpegnathos saltator /
                               EFN75172.1

                               Camponotus floridanus /
                               EFN63022.1

                               Culex quinquefasciatus /
                               XP_001867000.1

                               Drosophila melanogaster /
                               NP_001189319.1

                               Bombyx mori /NP_001103782.1

                               Pyropia yezoensis / BAB64309.1

                               Apis mellifera / XP_003251465.1

                               Ascaris suum / ADY47269.1

                               Tetranychus urticae / ACN53544.1

                               Glossina morsitans morsitans /
                               ADD19431.1

                               Drosophila melanogaster /
                               NP_524367.1

                               Timema tahoe / ADX66580.1

                               Drosophila virilis / AAK25829.1

                               Drosophila melanogaster /
                               NP_477484.2

                               Diabolocatantops pinguis /
                               ACV32627.1

                               Diabolocatantops pinguis /
                               ACV32627.1

                               Channa punctata / AEA50896.1

                               Glossina morsitans morsitans /
                               ADD19714.1

                               Ostrea edulis / CAL69229.1

                               Callithrix jacchus /
                               XP_002758560.1

                               Boltenia villosa / AAM76122.1
                               Apis mellifera / XP_397331.2

                               Culex quinquefasciatus /
                               XP_001843519.1

                               Drosophila melanogaster /
                               NP_001188951.1

                               Acyrthosiphon pisum /
                               XP_003247952.1

                               Bombyx mori /AAN46650.1

7. Cell growth and death       Cyprinus carpio /ABC59222.1

                               Pteria penguin / AEI58895.1

                               Bombyx mori /NP_001138799.1

                               Perca flavescens / ADX97237.1

8. Membrane transporters       Taeniopygia guttata /
                               XP 002196817.1

                               Psychroflexus torquis ATCC
                               700755 / ZP_01252230.1

                               Lactobacillus rhamnosus LMS2-1
                               / ZP_04439763.1

                               Haliscomenobacter hydrossis
                               DSM 1100 / YP_004445152.1

                               Paenibacillus sp. HGF5 /
                               ZP_08281960.1

9. Vesicular transport and     Aedes aegypti / XP_001650005.1
metabolism
                               Drosophila melanogaster /
                               NP_787988.1
                               Culex quinquefasciatus /
                               XP_001850184.1

10. Cell wall metabolism       Rattus norvegicus / NP_072127.1

                               Escherichia sp. 3253FAA /
                               ZP_04532936.1

                               Burkholderia multivorans ATCC
                               17616 / YP_001949468.1

                               Microscilla marina ATCC 23134/
                               ZP_01689674.1

                               Roseobacter sp. AzwK-3b /
                               ZP_01900972.1

                               Escherichia sp.3253FAA/
                               ZP_04532936.1

                               Helicobacter canadensis /
                               ZP_07804970.1

                               Streptomyces sp. e14 /
                               ZP_06708251.1

                               Aedes aegypti / XP_001650341.1

                               Acidiphilium cryptum JF-5 /
                               YP_001233531.1

                               Pyropia yezoensis / ABJ91310.1

11. Photosynthesis and         Porphyra purpurea / NP_053924.1
chloroplast proteins
                               Porphyra purpurea / NP_053902.1

                               Pyropia yezoensis / YP_536952.1

                               Arthrospira platensis str. Paraca /
                               ZP_06383599.1

                               Bangia fuscopurpurea /
                               AAM62007.1

                               Porphyra purpurea / NP_053894.1

                               Porphyra purpurea / NP_053809.1

                               Pyropia yezoensis / YP_537038.1

                               Pyropia yezoensis / YP_536893.1

                               Porphyra purpurea / NP_053822.1

                               Porphyra purpurea / NP_053888.1

                               Porphyra purpurea / NP_053797.1

                               Porphyra purpurea / NP_053798.1

                               Cyanothece sp. PCC 7822 /
                               YP_003889235.1

                               Porphyra purpurea / NP_053977.1

                               Ceramium boydenii / AAM88398.1

                               Rhodomonas salina /
                               YP_001293520.1

                               Microcystis aeruginosa NIES-843
                               / YP_001656041.1

                               Porphyra purpurea / NP_053853.1

                               Pyropia yezoensis / YP_536928.1

                               Vigna unguiculata / CAO02552.1

                               Acidiphilium cryptum JF-5 /
                               YP_001235357.1

                               Pyropia yezoensis / YP_537014.1

12. Respiration and            Nicotiana tabacum /BAA10929.1
mitochondrial proteins
                               Hydrogenivirga sp. 128-5-R1-1 /
                               ZP_02176878.1
                               Glossina morsitans morsitans /
                               ADD20239.1

                               Anolis carolinensis /
                               XP_003230444.1

                               Nasonia vitripennis /
                               NP_001161450.1

                               Littorina saxatilis / CAM57998.1

                               Fusiturris similis /
                               YP_003204746.1

                               Porphyra purpurea / NP_049304.1

                               Culex quinquefasciatus /
                               XP 001865472.1

                               Aedes aegypti / XP_001655316.1

                               Lucilia cuprina / CBX78584.1

                               Dicranomyia variabilis /
                               ABV71159.1

                               Littorina saxatilis / CAA10593.1

                               Echinolittorina novaezelandiae /
                               CAF22173.1

                               Geranomyia advena / ABV71230.1

                               Ceratitis ditissima / ADB55739.1

                               Dicranomyia stygipennis /
                               ABV71214.1

                               Dicranomyia swezeyi /
                               ABV71215.1

                               Littorina saxatilis / CAA10594.1

                               Potamopyrgus antipodarum /
                               ADB93443.1

                               Cymatium parthenopeum /
                               YP_003204809.1

                               Aedes albopictus / YP_194916.1

                               Drosophila simulans /
                               ABM88269.1

                               Simulium nigrimanum /
                               ACZ28403.1
                               Oncorhynchus mykiss /
                               ACO08369.1
                               Saccoglossus kowalevskii /
                               XP_002738468.1
                               Simulium nigrimanum /
                               ACZ28249.1
                               Glossina morsitans morsitans /
                               ADD20505.1
                               Glossina morsitans morsitans /
                               ADD18988.1
                               Bombyx mori /ABD36320.1
                               Culex quinquefasciatus /
                               XP_001859594.1
                               Ricinus communis /
                               XP_002517886.1
                               Apis mellifera /NP_001010975.1
                               Drosophila silvestris / ABY55748.1
                               Strongylocentrotus purpuratus /
                               NP 999743.1

                               Spodoptera exigua / ACL77780.1
                               Drosophila simulans /
                               XP_002105781.1
                               Aedes aegypti / XP_001655448.1

                               Drosophila melanogaster /
                               NP_524402.1
                               Xenopus (Silurana) tropicalis /
                               NP_001037877.1
                               Bolinus brandaris /
                               YP_003204844.1
                               Drosophila mojavensis /
                               DAA06245.1
                               Liriomyza trifolii /
                               YP_003734909.1
                               Glossina morsitans morsitans /
                               ADD19108.1
                               Tribolium castaneum /
                               EFA01361.1
                               Glossina morsitans morsitans /
                               ADD19528.1
                               Drosophila littoralis /
                               YP_002327406.1
                               Bombyx mori /NP_001091812.1

                               Oryctolagus cuniculus /
                               XP_002713430.1
                               Culex quinquefasciatus /
                               XP_001842637.1
13. Others                     Lilium longiflorum / ABO20851.1
                               Cupressus sempervirens /
                               ACA30301.1
                               Perkinsus marinus ATCC 50983/
                               XP_002771156.1
                               Chlorella variabilis / EFN58441.1
                               Anopheles darlingi / ACI30205.1
                               Argas monolakensis / ABI52743.1
                               Anopheles gambiae /
                               XP_001238056.2
                               Drosophila melanogaster /
                               NP_611390.2
                               Anopheles gambiae /
                               XP_001688982.1
                               Anopheles gambiae str. PEST/
                               XP_001689165.1
                               Aedes aegypti / XP_001661677.1
                               Meleagris gallopavo /
                               XP_003201937.1
                               Acyrthosiphon pisum /
                               NP_001156724.1
                               Lethocerus deyrollei /BAG12118.1
                               Culex tarsalis / ADH04225.1
                               Culex tarsalis / ADH04224.1
                               Culex tarsalis / ADH04226.1
                               Culex quinquefasciatus /
                               XP_001843136.1
                               Culex tarsalis / ADH04227.1
                               Ochlerotatus atropalpus /
                               AAV31927.1
                               Toxorhynchites amboinensis /
                               AAV31932.1
                               Anopheles albimanus /
                               AAV31933.1
                               Tenebrio molitor/AAU20328.2

                               Aedes polynesiensis / AAV31926.1
                               Camponotus floridanus /
                               EFN65337.1
                               Culex quinquefasciatus /
                               XP_001845697.1
                               Aedes aegypti / XP_001657619.1
                               Aedes aegypti / XP_001663499.1

                               Strongylocentrotus purpuratus /
                               XP_001199485.1
                               Selenomonas noxia ATCC43541 /
                               ZP 06604478.1

Functional category            E value     D*     Pyropia accession
                                                  number

1. Signal transduction         0.0000004   nuc    HE859298

                               0           cyt    HE859158

                               5E-14       cyt    HE858680

                               0           cyt    HE858962

                               0           cyt    HE859035

                               0           n.d.   HE859071

                               2E-16       n.d.   HE859267

                               0           nuc    HE859315

                               0           nuc    HE858859

                               0.0000004   nuc    HE859339

                               0           nuc    HE858825

                               0           cyt    HE859285

                               0           cyt    HE859130

                               0           cyt    HE858743

                               0           mit    HE859041

                               0           cyt    HE858656

                               1E-10       cyt    HE858976

                               5E-18       cyt    HE858774

                               0           cyt    HE858703

                               0           cyt    HE858908

                               0           cyt    HE859343

                               0           mit    HE859085

                               0           cyt    HE858826

                               0           nuc    HE858889

2. Transcription, splicing,    0           nuc    HE858778
and replication
                               0.0000002   nuc    HE858942

                               0           chl    HE858844

                               3E-12       n.d.   HE859115

                               8E-17       n.d.   HE858969

                               1E-11       nuc    HE858932

                               0           mit    HE858715

                               1E-12       mit    HE858909

                               0.00002     mit    HE858919

                               0.00006     mit    HE859330

                               0           mit    HE859084

                               0.00007     mit    HE859136

                               2E-11       mit    HE858655

                               5E-14       nuc    HE858617

                               3E-09       mit    HE859408

                               0           cyt    HE859164

                               8E-12       mit    HE858662

                               0           cyt    HE858827

                               0.00007     mit    HE859073
                               5E-14       n.d.   HE859151

                               0.00002     cyt    HE858668

                               0           cyt    HE858664

3. Basal metabolism            0           mit    HE858747

                               0.00004     cyt    HE858837

                               0           cyt    HE858850

                               0           mit    HE859341

                               0           mit    HE859282

                               0           cyt    HE858775

                               0           chl    HE859176

                               1E-13       cyt    HE859024

                               0           cyt    HE859302

                               0           chl    HE858842

                               0           mit    HE859064

                               2E-10       mit    HE858963

                               0.000007    mit    HE858643

                               0           cyt    HE858687

                               0.000002    cyt    HE858920

                               0.00002     cyt    HE859383

                               5E-16       cyt    HE858684

                               1E-13       cyt    HE858659

                               0           cyt    HE858708

                               0           cyt    HE858838

                               0           cyt    HE858779

                               0           cyt    HE859110

                               0           mit    HE858686

                               5E-08       mit    HE859204

                               0           mit    HE859167

                               0           mit    HE858676

                               1E-17       cyt    HE859135

4. Antioxidant,                0           chl    HE858809
chaperone and defense
factors
                               1E-13       cyt    HE858870

                               0           n.d.   HE858928

                               2E-13       cyt    HE858971

                               0           n.d.   HE858692

                               3E-14       n.d.   HE859251

                               0.000001    cyt    HE859190

                               0           cyt    HE858813

                               0           mit    HE858802

                               0.00001     cyt    HE858848

                               0.00007     cyt    HE859216

                               5E-15       cyt    HE858649

                               5E-08       cyt    HE858750

                               0           cyt    HE859304

                               6E-17       n.d.   HE858885

                               0           chl    HE858621

                               0           chl    HE858874

                               0           cyt    HE858890

                               0           n.d.   HE858770

                               9E-10       cyt    HE859296

                               3E-12       cyt    HE859318

                               0           chl    HE858982

                               0           chl    HE858640

                               0           chl    HE859033

                               0           cyt    HE858821

                               0           n.d.   HE859067

                               0           cyt    HE859242

                               0           n.d.   HE858745

                               0           cyt    HE858818

                               0           mit    HE859003

                               1E-12       mit    HE858931

                               0           mit    HE858629

5. Protein synthesis,          0           cyt    HE859122
processing and
degradation
                               0           cyt    HE858682

                               0           cyt    HE859333

                               0           cyt    HE858896

                               0           cyt    HE859277

                               0           cyt    HE859180

                               0           cyt    HE859121

                               0           cyt    HE859058

                               0           cyt    HE859324

                               4.93E-09    cyt    HE858807

                               8E-09       cyt    HE858624

                               0           cyt    HE858921

                               0.000005    cyt    HE859208

                               0           mit    HE858801

                               0           cyt    HE859005

                               0           mit    HE858760

                               0           cyt    HE859014

                               0           cyt    HE858833

                               0           cyt    HE859173

                               0           cyt    HE858899

                               2E-14       cyt    HE858808

                               0           nuc    HE859192

                               0           mit    HE859118

                               0           cyt    HE859166

                               0           cyt    HE858776

                               1E-11       cyt    HE858638

                               0           mit    HE858806

                               0           nuc    HE859020

                               3E-16       nuc    HE858940

                               0           cyt    HE859259

                               0           mit    HE858729

                               0           cyt    HE858749

                               3E-13       mit    HE858674

                               4E-13       mit    HE859345

                               8E-15       mit    HE859397

                               0           cyt    HE858679

                               0           cyt    HE858977

                               0           cyt    HE858934

                               4E-14       chl    HE858820

                               0           chl    HE858941

                               2E-08       cyt    HE858646

                               1E-14       cyt    HE858985

                               9E-10       cyt    HE859052

                               3E-12       cyt    HE858858

                               2E-16       cyt    HE858804

                               0           ext    HE858888

                               0.0000001   cyt    HE859141

                               8E-12       cyt    HE858654

                               4E-18       cyt    HE858699

                               7E-08       cyt    HE859034

                               0           cyt    HE859075

                               4E-10       cyt    HE858914

                               6E-14       cyt    HE858810

                               0           cyt    HE859168

                               0           cyt    HE859078

                               0           cyt    HE858711

                               0           cyt    HE859111

                               0           chl    HE858867

                               2E-10       chl    HE859222

                               0           cyt    HE859006

                               5E-18       cyt    HE859081

                               0.0000002   mit    HE859051

                               0           cyt    HE858876

                               0.0000005   mit    HE858698

                               0           mit    HE859305

                               0           cyt    HE859086

                               1E-13       cyt    HE859382

                               0           cyt    HE859183

                               0           cyt    HE858793

                               0           cyt    HE858987

                               4E-16       cyt    HE858954

                               2E-09       cyt    HE859353

                               0           cyt    HE859385

                               0           cyt    HE859214

                               3E-12       cyt    HE859387

                               0           cyt    HE859398

                               5E-12       cyt    HE859262

                               0           cyt    HE858895

                               0           cyt    HE859125

                               6E-12       cyt    HE859254

                               0           chl    HE859109

                               0           nuc    HE858869

                               0           cyt    HE858630

6. Cell motility               0           cyt    HE859009

                               1E-13       cyt    HE859273

                               0           cyt    HE858829

                               3E-16       cyt    HE859189

                               0           n.d.   HE859089

                               0           n.d.   HE858678

                               0           n.d.   HE859063

                               0           cyt    HE859137

                               0           cyt    HE858787

                               0           cyt    HE858718

                               1E-14       cyt    HE859049

                               0           cyt    HE858761

                               0           cyt    HE858791

                               0           cyt    HE859406

                               7E-13       cyt    HE858814

                               0           cyt    HE858619

                               0           cyt    HE858642

                               0           cyt    HE859379

                               0           cyt    HE859142

                               0           cyt    HE858722

                               0           cyt    HE858763

                               0           cyt    HE859079

                               0           cyt    HE858840

                               0           cyt    HE859022

                               0           cyt    HE858700

                               0           cyt    HE858975
                               0           cyt    HE859178

                               0.0002      cyt    HE859030

                               0.000003    cyt    HE858663

                               0.0006      cyt    HE858948

                               0           cyt    HE858854

7. Cell growth and death       1E-11       cyt    HE858712

                               3E-17       cyt    HE858983

                               0           cyt    HE858839

                               0.0001      cyt    HE859132

8. Membrane transporters       0.0002      n.d.   HE859194

                               0           n.d.   HE859043

                               0.000006    n.d.   HE859400

                               2E-08       n.d.   HE858852

                               0.001       n.d.   HE859386

9. Vesicular transport and     4E-15       gol    HE859113
metabolism
                               0           n.d.   HE859153

                               0           n.d.   HE859332

10. Cell wall metabolism       0.00004     cyt    HE858732

                               0           cyt    HE859007

                               0           cyt    HE858683

                               0           cyt    HE858990

                               5E-16       cyt    HE859054

                               3E-12       cyt    HE859275

                               0.00002     cyt    HE859290

                               4E-09       cyt    HE859401

                               0           cyt    HE858995

                               0.000008    cyt    HE858973

                               0           chl    HE859292

11. Photosynthesis and         0           chl    HE858764
chloroplast proteins
                               0           chl    HE858795

                               0.0000007   chl    HE859002

                               0           chl    HE859186

                               0           chl    HE858916

                               0           chl    HE859114

                               0           chl    HE858727

                               0.000001    chl    HE859365

                               0           chl    HE858641

                               0           chl    HE858733

                               0           chl    HE858824

                               0           chl    HE859157

                               0           chl    HE859287

                               0.001       chl    HE859143

                               3E-08       chl    HE858627

                               0           chl    HE858639

                               0           chl    HE859066

                               1E-12       chl    HE858886

                               5E-12       chl    HE858967

                               9E-11       chl    HE859243

                               7E-09       chl    HE858944

                               0.0000003   chl    HE858860

                               0           chl    HE858999

12. Respiration and            0           chl    HE858796
mitochondrial proteins
                               0.00003     chl    HE859272

                               0           mit    HE858961

                               0.00001     mit    HE858900

                               4E-14       mit    HE858968

                               0           mit    HE858930

                               8E-12       mit    HE858904

                               0.0005      mit    HE859276

                               0           mit    HE858694

                               0           mit    HE859008

                               0           mit    HE858780

                               0           mit    HE858720

                               0           mit    HE858856

                               0           mit    HE859031

                               0           mit    HE858740

                               3E-10       mit    HE859328

                               0.000008    mit    HE858917

                               2E-10       mit    HE859117

                               0           mit    HE858884

                               0           mit    HE858669

                               0           mit    HE859331

                               0           mit    HE858725

                               0           mit    HE858714

                               0           mit    HE858693

                               4E-08       mit    HE858746

                               0           mit    HE859201

                               1E-14       mit    HE858739

                               4E-16       mit    HE858689

                               0           mit    HE858984

                               2E-11       mit    HE858994
                               4E-10       mit    HE859236

                               0           mit    HE858960

                               0           mit    HE858665
                               0           mit    HE859342
                               0           mit    HE858957

                               1E-10       mit    HE859072
                               0           mit    HE858786

                               0           mit    HE859029

                               0           mit    HE858979

                               4E-17       mit    HE859056

                               0           mit    HE858728

                               0           mit    HE858782

                               0.000003    mit    HE859193

                               0           mit    HE858933

                               0           mit    HE858766

                               0           mit    HE859010

                               0.0000003   mit    HE858843

                               2E-08       mit    HE859061

                               0           mit    HE859147

                               0           mit    HE859045

13. Others                     2E-15       n.d.   HE858777
                               0           mit    HE859069

                               0.0000003   n.d.   HE859266

                               0.00001     mit    HE859270
                               0.000001    n.d.   HE859175
                               7E-15       cyt    HE859146
                               0           cyt    HE859209

                               0           ext    HE859269

                               0.00001     ext    HE858653

                               0           ext    HE858951

                               2E-17       ext    HE858625
                               0           cyt    HE858705

                               0           ext    HE859090

                               0           mit    HE858626
                               0.00002     n.d.   HE858633
                               2E-13       cyt    HE859037
                               0           chl    HE858675
                               0.0000002   cyt    HE858849

                               0           cyt    HE858632
                               0.000001    ext    HE858657

                               4E-09       cyt    HE859170

                               0           mit    HE858622

                               0.000001    n.d.   HE859404

                               0           cyt    HE858631
                               0           mit    HE858716

                               0.000008    n.d.   HE859197

                               0           n.d.   HE858950
                               0           n.d.   HE858887

                               0.000003    cyt    HE858672

                               0.00002     n.d.   HE858754

Table 2. Functional category, identity and potential cellular
destination of proteins and accession number of identified ESTs in
Pyropia columbina under natural desiccation (UD library). Data from
the hydrated library (UH) is presented in Table 1. E value: the best
(lowest) Expect value (E value) of all alignments from that database
sequence, D*: Putative destination.

Functional category          Putative identity

1. Signal transduction       Calmodulin
                             Enkurin TRPC channel interacting
                             protein
                             Guanine nucleotide binding protein 3

                             Hexamerin 2 beta
                             Serine/threonine protein phosphatase 1
2. Transcription, splicing   RNA-directed DNA polymerase
and replication              (reverse transcriptase)
                             Reverse transcriptase

                             RNA-directed DNA polymerase
                             (reverse transcriptase)
                             DNA-directed RNA polymerase, beta
                             subunit
                             RNA polymerase alpha subunit
                             Argonaute2 (AGO2)
                             Predicted metal-dependent RNase

                             Group II intron reverse
                             transcriptase/maturase

                             Histone H3.3
                             Retrotransposon protein

3. Basal metabolism          Aconitate hydratase
                             Lipase
                             Fructose-1,6-biphosphatase F-II
                             Fructose-bisphosphate aldolase

                             Alpha-amylase B

                             3-oxoacyl-ACP reductase

                             C-4 sterol methyl oxidase 2
                             Amine oxidase

                             Scavenger receptor cysteine-rich
                             protein
                             Vacuolar H+-ATPase C chain
4. Antioxidant, chaperone    Thioredoxin
and defense factors
                             Catalase
                             Heme oxygenase
                             Cytochrome P450-Like TBP protein
                             Heat shock protein 70
                             dnaK gene product (Heat shock
                             protein 70)
                             Heat shock protein Hsp70

                             Molecular chaperones HSP70/HSC70,
                             HSP70
                             Heat shock protein Hsp70

                             Heat shock protein 90, partial
                             Heat shock protein 90

                             Molecular chaperones
                             GRP78/Bip/KAR2, HSP70
                             superfamily
5. Protein synthesis,        Ubiquitin
processing and               Ubiquitin protein
degradation
                             Ubiqutin

                             Ubiquitin

                             Ubiquitin-conjugating enzyme E2-17 kDa
                             Proteasome subunit beta type-6

                             Molybdopterin biosynthesis protein
                             30S ribosomal protein S18

                             Ribosomal protein L35
                             Protein translation factor
                             Eukaryotic translation elongation
                             factor 1 alpha 1
                             EF2, translation elongation factor 2
                             Elongation factor-like protein

                             Clp protease ATP binding subunit
                             Cysteine synthase
                             Der F 3 allergen

                             trpB gene product

                             Isoleucyl-tRNA synthetase

                             Prefolding-like protein

                             Protease inhibitor G11A6
                             Signal peptidase, catalytic subunit

6. Cell motility             Actin
                             Actin
                             Actin
                             Actin
                             Actin 1
                             Beta-actin
                             Tropomyosin
                             Troponin T-1

                             Beta-tubulin
                             Calponin
7. Cell growth and death     Cell division protein
                             Cell division protein FtsA

                             CHK1 checkpoint homolog

                             CHK1 checkpoint-like protein
                             CHK1 checkpoint-like Protein
8. Membrane transporters     ABC Transporter B family member 13
                             ABC Transporter

                             ATP-binding cassette (ABC)
                             superfamily
9. Vesicular transport and   Endoplasmic reticulum vesicle
metabolism                   transporter protein

                             GTPase SAR1
                             Small GTP-binding protein
10. Cell wall metabolism     Cell wall-associated hydrolase

                             Cell wall-associated hydrolase
                             Cell wall-associated hydrolase
                             Cell wall-associated hydrolase
                             Cell wall-associated hydrolase
                             Cell wall-associated hydrolase
                             Cell wall-associated hydrolase
                             Cell wall-associated hydrolase

                             Putative cell wall-Associated hydrolase

                             Cell wall-associated hydrolase
                             Cell wall-associated hydrolase

                             Cell wall-associated hydrolase

                             Cell wall-associated Hydrolase

                             Cell wall-associated hydrolase

                             Chitin deacetylase 7 precursor
                             Glycine cleavage system protein H

                             Glycosyl transferase

11. Photosynthesis and       Photosystem I P700 chlorophyll A
chloroplast proteins         apoprotein A1
                             Photosystem II protein D1
                             Photosystem II protein D2
                             Photosystem II protein D2
                             Ferredoxin NADP+ reductase
                             23S Ribosomal RNA
                             Ribosomal protein S3
                             Ribosomal protein L4
                             30S ribosomal protein S13

                             Ribosomal protein L2
                             30S ribosomal protein S12
                             40S ribosomal protein S16

                             50S ribosomal protein L3P
                             50S ribosomal protein L3

                             50S ribosomal protein L4
                             50S ribosomal protein L4
                             50S ribosomal protein L13

                             60S ribosomal protein L29-1
                             RNA polymerase beta subunit

                             ATP synthase beta subunit
                             ATP synthase CF1 delta subunit
                             Cell division protein
                             Ferredoxin, chloroplast precursor

                             Photochlorophyllide reductase subunit B
                             Photochlorophyllide reductase subunit B
                             HESB-like domain-containing protein 2
12. Respiration and          NADH dehydrogenase subunit 4L
mitochondrial proteins       NADH dehydrogenase subunit 7

                             Cytochrome C oxidase
                             Cytochrome C oxidoreductase subunit TT
                             II Cytochrome C oxidase subunit III
                             Cytochrome C oxidase subunit IV
                             Ribosomal protein S3
                             Ribosomal protein S5
                             40S ribosomal protein S13-2
                             40S ribosomal protein S15

                             50S ribosomal protein L16

                             50S ribosomal protein L16
                             conserved hypothetical protein

                             HNH endonuclease

                             Reverse transcriptase

                             Reverse transcriptase

                             Reverse transcriptase
                             F-Type H-ATPase beta subunit
                             Dehydration responsive protein
                             Putative microvillar-like protein 5

                             Tumor differentially expressed protein
13. Others                   Senescence-associated protein

                             Lipoprotein

                             Lipoprotein

                             Low-density lipoprotein receptor-
                             related protein
                             Apolipoprotein D isoform 2
                             Putative apolipoprotein D
                             Dermatopontin 2
                             Cysteine-rich venom protein
                             Venom allergen
                             Leucine rich protein
                             Pg1 protein

                             Short-chain collagen C4-like

                             Similar To DUF221 domain protein
                             Microvillar-like protein 5

                             Tep1
                             Viral A-type inclusion protein

                             Yip1 domain-containing protein

Functional category          Species and accession number

1. Signal transduction       Pyropia yezoensis / ABN41559
                             Saccoglossus kowalevskii /
                             NP_001158494
                             Dictyostelium discoideum AX4 /
                             XP_637356.1
                             Aedes aegypti / XP_001659531.1
                             Malus domestica / AAD56010.1
2. Transcription, splicing   Ktedonobacter racemifer DSM 44963
and replication              / ZP_06971690.1
                             Cyanothece sp. ATCC 51142 /
                             YP_001801525.1
                             Arthrospira maxima CS-328 /
                             ZP_03273642
                             Crocosphaera watsonii WH 8501 /
                             ZP_00516049.1
                             Porphyra purpurea / NP_053903.1
                             Albugo laibachii Nc14 /CCA27516.1
                             Streptococcus pyogenes M49 591 /
                             ZP_00366321.1
                             Streptococcus dysgalactiae subsp.
                             dysgalactiae ATCC 27957 /
                             EFY02523.1
                             Harpegnathos saltator / EFN83153.1
                             Oryza sativa Indica Group /
                             ABR26094.1
3. Basal metabolism          Camponotus floridanus / EFN71522.1
                             Aedes aegypti / XP_001652527.1
                             Griffithsia japonica / AAP80707.1
                             Sphingobacterium sp. 21 /
                             YP_004319198.1
                             Culex quinquefasciatus /
                             XP_001846541.1
                             Streptomyces avermitilis MA-4680 /
                             NP_824830.1
                             Nicotiana benthamiana /AAQ83692.1
                             Plasmodium yoelii yoelii 17XNL /
                             XP_730508.1
                             Culex quinquefasciatus /
                             XP_001866937.1
                             Pyropia tenera / JC7151
4. Antioxidant, chaperone    Hyperthermus butylicus DSM 5456 /
and defense factors          YP_001013307.1
                             Pyropia yezoensis / ADO23652.1
                             Porphyra purpurea / NP_053881.1
                             Lilium longiflorum / ABO20848.1
                             Pyropia yezoensis / YP_536996.1
                             Thermosynechococcus elongatus BP-
                             1 / NP_682523.1
                             Raphidiopsis brookii D9 /
                             ZP_06305357.1
                             Ectocarpus siliculosus / CBN79394.1

                             Cylindrospermopsis raciborskii CS-
                             505 / ZP_06308882.1
                             Thraustotheca clavata / AAX10950.1
                             Cellulophaga algicola DSM 14237 /
                             YP 004165199.1

                             Ectocarpus siliculosus / CBJ48460.1

5. Protein synthesis,        Oncorhynchus mykiss / ACO07546.1
processing and               Triticum aestivum / AAQ08322
degradation
                             Phanerochaete chrysosporium /
                             CAA83244
                             Drosophila persimilis /
                             XP_002023314.1
                             Zea mays / ACG37110.1
                             Phytophthora infestans T30-4 /
                             XPJ002901169.1
                             Porphyra purpurea / NP_053945.1
                             Gemella sanguinis M325 /
                             ZP_08260903
                             Pyropia yezoensis / YP_536952.1
                             Griffithsia japonica/ AAM93956
                             Chlamydomonas reinhardtii /
                             XP_001696568.1
                             Ectocarpus siliculosus / CBJ32863.1
                             Pseudoperkinsus tapetis /
                             ADE62443.1
                             Pyropia yezoensis / YP_537014.1
                             Porphyra purpurea / AAP97124.1
                             Dermatophagoides farinae /
                             AAP35076.1
                             Acaryochloris marina MBIC11017 /
                             YP_001519094.1
                             Lacinutrix sp. 5H-3-7-4 /
                             YP_004579101.1
                             Opisthacanthus cayaporum /
                             CAX51417.1
                             Mayetiola destructor / ABB70517.1
                             Chlamydomonas reinhardtii /
                             XP_001697614.1
6. Cell motility             Helicoverpa armigera / ADN84930.1
                             Pyropia yezoensis / BAB64309.1
                             Undaria pinnatifida / ADW66613.1
                             Pyropia yezoensis / BAG71158.1
                             Paxillus involutus / ABQ85639.1
                             Mauremys mutica / ADX86815.1
                             Crassostrea virginica / AAC61869.1
                             Drosophila melanogaster /
                             AAR24583.1
                             Phytophthora palmivora / AAW58084
                             Haliotis diversicolor / ABU53030
7. Cell growth and death     Pyropia yezoensis / YP_537009.1
                             Lacinutrix sp. 5H-3-7-4 /
                             YP_004578825.1
                             Xenopus (Silurana) tropicalis /
                             CAJ83813.1
                             Helicoverpa armigera/ABK29471.1
                             Perca flavescens / ADX97237.1
8. Membrane transporters     Arabidopsis thaliana /NP 174115
                             Thalassiosira pseudonana
                             CCMP1335 / XP_002288593
                             Ectocarpus siliculosus / CBJ32723.1

9. Vesicular transport and   Arabidopsis thaliana / NP_564162
metabolism

                             Triticum aestivum/ACD03831.1
                             Solanum lycopersicum / AAA80679
10. Cell wall metabolism     Microscilla marina ATCC 23134 /
                               ZPJ01689674.1
                             Escherichia sp 3_2_53FAA / ZP_04532936.1
                             Escherichia sp. 3_2_53FAA / ZP_04532936.1
                             Escherichia sp. 3_2_53FAA / ZP_04532936.1
                             Escherichia sp. 3_2_53FAA / ZP_04532936.1
                             Escherichia sp. 3_2_53FAA / ZP_04532936.1
                             Escherichia sp. 3_2_53FAA / ZP_04532936.1
                             Microscilla marina ATCC 23134 /
                               ZP_01689674.1
                             Campylobacter jejuni subsp. Jejuni
                               BH-01-0142 / ZP_03217789.1
                             Prevotella bryantii B14 / ZP_07059369.1
                             Burkholderia multivorans ATCC17616 /
                               YPJ001949468.1
                             Erysipelothrix rhusiopathiae ATCC 19414 /
                               ZP 08082416.1
                             Brucella abortus NCTC 8038 /
                               ZP_05822976.1
                             Brucella suis bv. 4 str. 40 /
                               ZP_05839398.1
                             Tribolium castaneum / NP_001104012.1
                             Hydrogenivirga sp. 128-5-R1-1 /
                               ZP_02176878.1
                             Rhodospirillum photometricum DSM 122 /
                               CCG06616.1

11. Photosynthesis and       Porphyra purpurea / NP_053894.1
chloroplast proteins
                             Pyropia yezoensis / YP_536893.1
                             Pyropia yezoensis / YP_537038.1
                             Porphyra purpurea / NP_053967
                             Pisum sativum / AAB59349.1
                             Vigna unguiculata / CAO02530.1
                             Vigna unguiculata / CAO02550.1
                             Pyropia yezoensis / YP_536993.1
                             Trichodesmium erythraeum IMS101 /
                               YP_722618.1
                             Chlamydomonas reinhardtii / ACS16390.1
                             Porphyra purpurea / NP_049316.1
                             Phytophthora infestans T30-4 /
                               XP_002899087.1
                             Cyanobacterium sp.UCYN-A / YP_003422083.1
                             Prochlorococcus marinus str. MIT 9515 /
                               YP 001012054.1
                             Lyngbya sp. PCC 8106 / ZP_01619314.1
                             Cyanothece sp. PCC 7424 / YP_002378953.1
                             Arthrospira platensis NIES-39 /
                               BAI90083.1
                             Glycine max / XP_003546330
                             Porphyra purpurea / NP_053860.1

                             Trebouxia photobiont / ABP98704.1
                             Porphyra purpurea /NP 053853.1
                             Porphyra purpurea /NP 05393 7.1
                             Micromonas sp. RCC299 /
                             XP_002508604.1
                             Porphyra purpurea /NP 053888.1
                             Pyropia yezoensis / YP 536960.1
                             Zea mays / NP_001149194
12. Respiration and          Porphyra purpurea /NP 049322.1
mitochondrial proteins       Coccomyxa subellipsoidea C-169 /
                             YP_004339024.1
                             Ephedra viridis / AAD01658.1
                             Porphyra purpurea /NP 049294.1
                             Tricula hortensis / YP_003434166.1
                             Aedes aegypti / XP_001663204.1
                             Porphyra purpurea /NP 049318.1
                             Griffithsia japonica / AAP80699.1
                             Arabidopsis thaliana /NP 567151.1
                             Oryza sativa Japonica Group /
                             BAD30388
                             Psychrobacter arcticus 273-4 /
                             YP_263793.1
                             Porphyra purpurea / NP_049319.1
                             Clostridium botulinum NCTC 2916/
                             ZP_02955128.1
                             Arthrospira maxima CS-328 /
                             ZP_03274502.1
                             Synechococcus sp. PCC 7335 /
                             ZP_05039776.1
                             Arthrospira platensis NIES-39 /
                             BAI90930.1
                             Porphyra purpurea / NP_049296.1
                             Ectocarpus siliculosus / CBJ32298.1
                             Corchorus olitorius /BAJ11784.1
                             Phlebotomus perniciosus /
                             ADJ57676.1
                             Haplopelma schmidti / ACH48223.1
13. Others                   Cupressus sempervirens /
                             ACA30301.1
                             Lactobacillus rhamnosus GG /
                             CAR86203.1
                             Lactobacillus rhamnosus GG /
                             CAR86203.1
                             Ascaris suum / ADY39765.1

                             Apis mellifera / XP_623787.2
                             Aedes aegypti / XP_001660232.1
                             Biomphalaria glabrata / AAZ80785.1
                             Aedes aegypti / XP_001655503.1
                             Aedes aegypti /XP_001655382
                             Arachis hypogaea / ABH09321.1
                             Staphylococcus aureus subsp. aureus
                             MN8 / ZP_06947416
                             Amphimedon queenslandica /
                             XP_003391538.1
                             Leptosphaeria maculans / CBX98851
                             Phlebotomus perniciosus /
                             ADJ57676.1

                             Anopheles arabiensis / ACG68562.1
                             Trichomonas vaginalis G3 /
                             XP_001319569.1
                             Dictyostelium discoideum /
                             XP_639001

Functional category          E value       D *    Pyropia accession
                                                  number

1. Signal transduction       8E-11         cyt    HE859451
                             2E-11         cyt    HE859515

                             0             cyt    HE859861

                             0             cyt    HE859661
                             0             cyt    HE859472
2. Transcription, splicing   8E-13         nuc    HE859542
and replication
                             9E-11         nuc    HE859708

                             0             nuc    HE859482

                             0             nuc    HE859717

                             0.000000003   nuc    HE859795
                             0             cyt    HE859580
                             8E-17         nuc    HE859892

                             8E-11         nuc    HE859734

                             0             nuc    HE859642
                             0             nuc    HE859423

3. Basal metabolism          0             mit    HE859793
                             0             ext    HE859755
                             0.0000002     cyt    HE859780
                             0             n.d.   HE859506

                             0             ext    HE859517

                             0.000003      ext    HE859599

                             4E-11         mit    HE859431
                             8E-13         cyt    HE859420

                             0.00002       n.d.   HE859437

                             0             n.d.   HE859667
4. Antioxidant, chaperone    0.00002       mit    HE859628
and defense factors
                             0.000004      mit    HE859586
                             0.00002       chl    HE859574
                             0             cyt    HE859591
                             0             chl    HE859563
                             0             chl    HE859424

                             0             chl    HE859837

                             0             mit    HE859570

                             0             chl    HE859573

                             0             nuc    HE859869
                             0             cyt    HE859588

                             0.00E00       cyt    HE859467

5. Protein synthesis,        0.0004        mit    HE859794
processing and               0             cyt    HE859488
degradation
                             0.000001      cyt    HE859630

                             0.0008        cyt    HE859858

                             0             cyt    HE859634
                             6E-11         cyt    HE859840

                             0             chl    HE859454
                             2E-10         n.d.   HE859511

                             0.000001      chl    HE859735
                             0             cyt    HE859466
                             0             cyt    HE859552

                             0             cyt    HE859801
                             0             cyt    HE859728

                             0             cyt    HE859649
                             0             cyt    HE859626
                             0.00002       n.d.   HE859604

                             5E-10         n.d.   HE859936

                             0             cyt    HE859750

                             0             cyt    HE859714

                             0.00001       cyt    HE859462
                             0             cyt    HE859469

6. Cell motility             0             cyt    HE859471
                             7E-13         cyt    HE859826
                             0             cyt    HE859564
                             0             cyt    HE859659
                             9E-17         cyt    HE859928
                             0             cyt    HE859680
                             0             mit    HE859676
                             0.00001       cyt    HE859762

                             0             cyt    HE859549
                             0             cyt    HE859480
7. Cell growth and death     2E-17         chl    HE859522
                             0             cyt    HE859665

                             0.0008        cyt    HE859790

                             1E-11         cyt    HE859593
                             2E-10         cyt    HE859702
8. Membrane transporters     9E-16         n.d.   HE859505
                             0.000001      n.d.   HE859535

                             0.00001       cyt    HE859694

9. Vesicular transport and   0.000006      cyt    HE859464
metabolism

                             0.0002        mit    HE859693
                             0.000008      cyt    HE859512
10. Cell wall metabolism     1E-17         cyt    HE859576

                             0             cyt    HE859695
                             0.000000003   cyt    HE859906
                             2E-11         cyt    HE859921
                             3E-13         cyt    HE859926
                             2E-12         cyt    HE859929
                             2E-15         cyt    HE859930
                             0             cyt    HE859413

                             9E-11         cyt    HE859421

                             0             cyt    HE859434
                             0             cyt    HE859495

                             0             cyt    HE859498

                             2E-14         mit    HE859566

                             0             cyt    HE859629

                             7E-10         cyt    HE859430
                             7E-15         cyt    HE859890

                             1E-11         n.d.   HE859904

11. Photosynthesis and       0             chl    HE859902
chloroplast proteins
                             0             chl    HE859757
                             0             chl    HE859806
                             0             chl    HE859500
                             0.00006       chl    HE859729
                             0.000000002   chl    HE859509
                             5E-12         chl    HE859706
                             0             chl    HE859613
                             0             chl    HE859559

                             0             chl    HE859868
                             6E-16         cyt    HE859584
                             2E-13         cyt    HE859787

                             0.00003       chl    HE859842
                             5E-15         chl    HE859881

                             3E-12         chl    HE859818
                             0.000001      chl    HE859828
                             0             chl    HE859578

                             0             cyt    HE859481
                             0             chl    HE859802

                             9E-11         chl    HE859691
                             1E-14         chl    HE859731
                             5E-16         chl    HE859460
                             0             chl    HE859587

                             2E-12         chl    HE859810
                             0             chl    HE859555
                             0             chl    HE859536
12. Respiration and          0.000007      mit    HE859443
mitochondrial proteins       3E-14         mit    HE859913

                             0.000000004   mit    HE859770
                             0             mit    HE859672
                             0             mit    HE859617
                             7E-16         mit    HE859683
                             4E-12         mit    HE859602
                             0             mit    HE859456
                             0             mit    HE859742
                             0             mit    HE859501

                             0.000005      mit    HE859479

                             8E-15         mit    HE859664
                             0.00000003    mit    HE859899

                             3E-15         mit    HE859427

                             4E-12         mit    HE859447

                             3E-10         mit    HE859590

                             0             mit    HE859800
                             0             mit    HE859457
                             1E-13         mit    HE859843
                             0.000006      mit    HE859901

                             0.000002      mit    HE859663
13. Others                   0             cyt    HE859606

                             0             n.d    HE859514

                             9E-11         n.d    HE859908

                             2E-13         n.d.   HE859807

                             0.00000003    n.d.   HE859474
                             5E-10         n.d.   HE859648
                             0.00001       n.d.   HE859882
                             0.0000005     n.d.   HE859698
                             0             n.d.   HE859494
                             2E-12         n.d    HE859468
                             0             n.d.   HE859493

                             0             cyt    HE859623

                             0             n.d.   HE859532
                             0.00004       cyt    HE859418

                                                  number
                             5.00E-11      cyt    HE859562
                             0.000001      cyt    HE859685

                             0             n.d.   HE859496
COPYRIGHT 2013 Pontificia Universidad Catolica de Valparaiso, Escuela de Ciencias del Mar
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Article Details
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Title Annotation:articulo en ingles
Author:Contreras-Porcia, Loretto; Lopez-Cristoffanini, Camilo; Lovazzano, Carlos; Rosa Flores-Molina, Maria
Publication:Latin American Journal of Aquatic Research
Date:Nov 1, 2013
Words:14070
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