Transcriptome Analysis in Haematococcus pluvialis: Astaxanthin Induction by High Light with Acetate and Fe2.

Haematococcus pluvialis is a commercial microalga, that produces abundant levels of astaxanthin under stress conditions. Acetate and Fe2+ are reported to be important for astaxanthin accumulation in H. pluvialis. In order to study the synergistic effects of high light stress and these two factors, we obtained transcriptomes for four groups: high light irradiation (HL), addition of 25 mM acetate under high light (HA), addition of 20 µM Fe2+ under high light (HF) and normal green growing cells (HG). Among the total clean reads of the four groups, 156,992 unigenes were found, of which 48.88% were annotated in at least one database (Nr, Nt, Pfam, KOG/COG, SwissProt, KEGG, GO). The statistics for DEGs (differentially expressed genes) showed that there were more than 10 thousand DEGs caused by high light and 1800-1900 DEGs caused by acetate or Fe2+. The results of DEG analysis by GO and KEGG enrichments showed that, under the high light condition, the expression of genes related to many pathways had changed, such as the pathway for carotenoid biosynthesis, fatty acid elongation, photosynthesis-antenna proteins, carbon fixation in photosynthetic organisms and so on. Addition of acetate under high light significantly promoted the expression of key genes related to the pathways for carotenoid biosynthesis and fatty acid elongation. Furthermore, acetate could obviously inhibit the expression of genes related to the pathway for photosynthesis-antenna proteins. For addition of Fe2+, the genes related to photosynthesis-antenna proteins were promoted significantly and there was no obvious change in the gene expressions related to carotenoid and fatty acid synthesis.

Directed Evolution of Dunaliella salina Ds-26-16 and Salt-Tolerant Response in Escherichia coli.

Identification and evolution of salt tolerant genes are crucial steps in developing salt tolerant crops or microorganisms using biotechnology. Ds-26-16, a salt tolerant gene that was isolated from Dunaliella salina, encodes a transcription factor that can confer salt tolerance to a number of organisms including Escherichia coli (E. coli), Haematococcus pluvialis and tobacco. To further improve its salt tolerance, a random mutagenesis library was constructed using deoxyinosine triphosphate-mediated error-prone PCR technology, and then screened using an E. coli expression system that is based on its broad-spectrum salt tolerance. Seven variants with enhanced salt tolerance were obtained. Variant EP-5 that contained mutation S32P showed the most improvement with the E. coli transformant enduring salt concentrations up to 1.54 M, in comparison with 1.03 M for the wild type gene. Besides, Ds-26-16 and EP-5 also conferred E. coli transformant tolerance to freezing, cold, heat, Cu2+ and alkaline. Homology modeling revealed that mutation S32P in EP-5 caused the conformational change of N- and C-terminal α-helixes. Expression of Ds-26-16 and EP-5 maintained normal cellular morphology, increased the intracellular antioxidant enzymatic activity, reduced malondialdehyde content, and stimulated Nitric Oxide synthesis, thus enhancing salt tolerance to E. coli transformants.

Enhanced astaxanthin production from Haematococcus pluvialis using butylated hydroxyanisole.

Haematococcus pluvialis is a promising natural source of high-value antioxidant astaxanthin under stress conditions. Biotic or abiotic elicitors are effective strategies for improving astaxanthin production in H. pluvialis. Butylated hydroxyanisole (BHA) was identified as an effective inducer for H. pluvialis LUGU. Under a treatment of 2mgL(-1) BHA (BHA2), astaxanthin content reached a maximum of 29.03mgg(-1) dry weight (DW) (2.03-fold of that in the control) after 12day of the mid-exponential growth phase. Subsequently, H. pluvialis LUGU was subjected to BHA2 at different growth phases because an appropriate time node for adding elicitors is vital for the entire production to succeed. As a result, the highest astaxanthin content (29.3mgg(-1) DW) was obtained in cells on day 14 (BHA2 14) of the late-exponential growth phase. Furthermore, the samples treated with BHA2 14 and the control group were compared in terms of the transcriptional expression of seven carotenogenesis genes, fatty acid composition, and total accumulated astaxanthin. All selected genes exhibited up-regulated expression profiles, with chy, crtO, and bkt exhibiting higher maximum transcriptional levels than the rest. Oleic acid content increased 33.15-fold, with acp, fad, and kas expression being enhanced on the day when astaxanthin was produced rapidly.

A strategy for promoting astaxanthin accumulation in Haematococcus pluvialis by 1-aminocyclopropane-1-carboxylic acid application.

The green algae Haematococcus pluvialis is a freshwater unicellular microalga belonging to Chlorophyceae. It is one of the best natural sources of astaxanthin, a secondary metabolite commonly used as an antioxidant and anti-inflammatory agent. Due to the importance of astaxanthin, various efforts have been made to increase its production. In this study, we attempted to develop a strategy for promoting astaxanthin accumulation in H. pluvialis using 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene (normally known as an aging hormone in plants). Our results demonstrated that ACC could enhance the growth of H. pluvialis, thereby promoting astaxanthin accumulation. Therefore, ACC has an indirect influence on astaxanthin production. We further verified the effect of ACC with a direct treatment of ethylene originated from banana peels. These results indicate that ethylene could be applied as an indirect method for enhancing growth and astaxanthin biosynthesis in H. pluvialis.

Arsenate toxicity and metabolism in the halotolerant microalga Dunaliella salina under various phosphate regimes.

Microalgae play an important role in arsenic (As) biogeochemical cycles as they are capable of accumulating and metabolizing this metalloid efficiently. This study aimed to investigate the toxicity, accumulation and transformation of arsenate (As(v)) in Dunaliella salina, an exceptionally halotolerant microalga, under various phosphate (PO4(3-)) regimes. The results of the 72-h toxicity test showed that D. salina was tolerant to As(v). In addition, the toxicity of As(v) was mitigated by an increased PO4(3-) supply. D. salina resisted the adverse effects of As(v) through the suppression of As uptake, enhancement of As reduction, methylation in the cell and excretion from the cell. Our study revealed that D. salina reduced As(v) toxicity using different strategies, i.e., reduction of As uptake upon acute As stress (24 h) and increase of As efflux following chronic As exposure (9 day). Moreover, PO4(3-) strongly affected the adsorption, uptake and transformation of As(v) in D. salina. As(v) reduction, DMA production and As excretion were enhanced under P-limited conditions (0.112 mg L(-1)) or upon higher As(v) exposure (1120 µg L(-1)). Furthermore, PO4(3-) had a significant influence on the As removal ability of D. salina. A high As removal efficiency (>95.6%) was observed in the 5-day cultures at an initial As concentration of 11.2 µg L(-1) and PO4(3-) of 0.112 and 1.12 mg L(-1). However, only 10.9% of total As was removed under 11.2 mg L(-1) PO4(3-) after 9 days of incubation. The findings of this study illustrate the pivotal roles of extracellular PO4(3-) in As(v) toxicity and metabolism, and the results may be relevant for future research on the minimization of As contamination in algal products as well as on the enhancement of As removal from the environment.

A method linking the toxic effects at community-level with contaminant concentrations.

In this study, we developed a method to quantify and link the toxic effects in community-level ecosystems with concentrations of petroleum hydrocarbons. The densities of Platymonas helgolandica var. tsingtaoensis, Isochrysis galbana, and Brachionus plicatilis in single-species tests and customized ecosystems were examined in response to a concentration gradient of petroleum hydrocarbons ranging from 0 to 8.0mgL(-1). A three-population ecological model with interspecies competition-grazing relationships was used to characterize population sizes with concentrations of petroleum hydrocarbons. A threshold concentration of the simplified plankton ecosystem of 0.376mgL(-1) for petroleum hydrocarbons was calculated from the proposed model, which was higher than the no-effect concentration of 0.056mgL(-1) derived from the single-species toxicity tests and the predicted no-effect concentration of 0.076mgL(-1) calculated from the species sensitivity distribution. This finding indicates that interspecies competition and grazing reduced the toxic effect of petroleum hydrocarbons at the community level. The sensitivity analysis for model parameters demonstrates that plankton population biomasses are highly sensitive to filtration rates. Antagonism between interspecies interactions and petroleum hydrocarbon toxicity was attributed to the reduced filtration rate and zooplankton grazing pressure. The proposed method is a simple means to address the concern regarding the impacts of ecological interactions on ecological risk assessments of pollutants.

Quantitative proteomic analysis of Dunaliella salina upon acute arsenate exposure.

Dunaliella salina is resistant to arsenic (As) and can accumulate a large amount of this highly toxic metalloid in cells. To study the mechanisms of As tolerance, a label-free, LC-MS/MS-based proteomic approach was applied for the first time to identify and quantify differentially expressed proteins from D. salina exposed to 11.2 mg L(-1) arsenate (As(V)) for 72 h. The intracellular As content reached 19.8 mg kg(-1), leading to a significant increase of lipid peroxidation in cells and a 7.4% growth reduction of this microalga. Sixty-five proteins were differentially expressed (p < 0.05), with 45 significantly induced and 20 declined. These proteins were involved in energy metabolism, protein synthesis and folding, ROS scavenging and defense, phosphate transport and membrane trafficking, and amino acid synthesis. Taken together, this study provides novel insights on the As(V) detoxification in D. salina.

Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematococcus pluvialis.

Haematococcus pluvialis is one of the most promising natural sources of astaxanthin. However, inducing the accumulation process has become one of the primary obstacles in astaxanthin production. In this study, the effect of ethanol on astaxanthin accumulation was investigated. The results demonstrated that astaxanthin accumulation occurred with ethanol addition even under low-light conditions. The astaxanthin productivity could reach 11.26 mg L(-1) d(-1) at 3% (v/v) ethanol, which was 2.03 times of that of the control. The transcriptional expression patterns of eight carotenogenic genes were evaluated using real-time PCR. The results showed that ethanol greatly enhanced transcription of the isopentenyl diphosphate (IPP) isomerase genes (ipi-1 and ipi-2), which were responsible for isomerization reaction of IPP and dimethylallyl diphosphate (DMAPP). This finding suggests that ethanol induced astaxanthin biosynthesis was up-regulated mainly by ipi-1 and ipi-2 at transcriptional level, promoting isoprenoid synthesis and substrate supply to carotenoid formation. Thus ethanol has the potential to be used as an effective reagent to induce astaxanthin accumulation in H. pluvialis.

The diverse toxic effect of SiO2 and TiO2 nanoparticles toward the marine microalgae Dunaliella tertiolecta.

Nanoparticles (NPs) are widely used in many industrial applications. NP fate and behavior in seawater are a very important issue for the assessment of their environmental impact and potential toxicity. In this study, the toxic effects of two nanomaterials, silicon dioxide (SiO2) and titanium dioxide (TiO2) NPs with similar primary size (~20 nm), on marine microalgae Dunaliella tertiolecta were investigated and compared. The dispersion behavior of SiO2 and TiO2 NPs in seawater matrix was investigated together with the relative trend of the exposed algal population growth. SiO2 aggregates rapidly reached a constant size (600 nm) irrespective of the concentration while TiO2 NP aggregates grew up to 4 ± 5 µm. The dose-response curve and population growth rate alteration of marine alga D. tertiolecta were evaluated showing that the algal population was clearly affected by the presence of TiO2 NPs. These particles showed effects on 50 % of the population at 24.10 [19.38-25.43] mg L(-1) (EC50) and a no observed effect concentration (NOEC) at 7.5 mg L(-1). The 1 % effect concentration (EC1) value was nearly above the actual estimated environmental concentration in the aquatic environment. SiO2 NPs were less toxic than TiO2 for D. tertiolecta, with EC50 and NOEC values one order of magnitude higher. The overall toxic action seemed due to the contact between aggregates and cell surfaces, but while for SiO2 a direct action upon membrane integrity could be observed after the third day of exposure, TiO2 seemed to exert its toxic action in the first hours of exposure, mostly via cell entrapment and agglomeration.

Spectroscopic analyses of chemical adaptation processes within microalgal biomass in response to changing environments.

Via photosynthesis, marine phytoplankton transforms large quantities of inorganic compounds into biomass. This has considerable environmental impacts as microalgae contribute for instance to counter-balancing anthropogenic releases of the greenhouse gas CO2. On the other hand, high concentrations of nitrogen compounds in an ecosystem can lead to harmful algae blooms. In previous investigations it was found that the chemical composition of microalgal biomass is strongly dependent on the nutrient availability. Therefore, it is expected that algae's sequestration capabilities and productivity are also determined by the cells' chemical environments. For investigating this hypothesis, novel analytical methodologies are required which are capable of monitoring live cells exposed to chemically shifting environments followed by chemometric modeling of their chemical adaptation dynamics. FTIR-ATR experiments have been developed for acquiring spectroscopic time series of live Dunaliella parva cultures adapting to different nutrient situations. Comparing experimental data from acclimated cultures to those exposed to a chemically shifted nutrient situation reveals insights in which analyte groups participate in modifications of microalgal biomass and on what time scales. For a chemometric description of these processes, a data model has been deduced which explains the chemical adaptation dynamics explicitly rather than empirically. First results show that this approach is feasible and derives information about the chemical biomass adaptations. Future investigations will utilize these instrumental and chemometric methodologies for quantitative investigations of the relation between chemical environments and microalgal sequestration capabilities.

Red light and carbon dioxide differentially affect growth, lipid production, and quality in the microalga, Ettlia oleoabundans.

Ettlia oleoabundans, a freshwater unicellular green microalga, was grown under different light qualities ± carbon dioxide-enriched air to determine the combined effects on growth and lipid production of this oleaginous species. Keeping total light intensity constant, when a portion of the cool white was replaced by red, volumetric lipid yield increased 2.8-fold mainly due to the greater yield of oleic acid, a desirable biodiesel precursor. Only 30 min of red light treatment was sufficient to increase lipid yield and quality to the same level as cultures provided red light for >14 days, indicating the potential role of red light in stimulating lipid production of this species. Carbon dioxide enrichment via air sparging enhanced exponential growth, carbon conversion efficiency, and nutrient consumption. Together, these results showed that light quality plays an important role in microalgal lipid production. Adjustment in light quality and gas delivery efficiency with carbon dioxide enrichment improved lipid yield and quality in this and possibly other oleaginous algal species.

Influence of PbS nanoparticle polymer coating on their aggregation behavior and toxicity to the green algae Dunaliella salina.

The potential hazards of nanoparticles (NPs) to the environment and to living organisms need to be considered for a safe development of nanotechnology. In the present study, the potential toxic effects of uncoated and gum Arabic-coated lead sulfide nanoparticles (GA-coated PbS NPs) on the growth, lipid peroxidation, reducing capacity and total carotenoid content of the hypersaline unicellular green algae Dunaliella salina were investigated. Coatings of PbS NPs with GA, as confirmed by Fourier transform infrared spectroscopy, reduced the toxicity of PbS NPs. Uncoated PbS NP toxicity to D. salina was attributed to higher algal cell-NP agglomerate formation, higher lipid peroxidation, lower content of total reducing substances and lower total carotenoid content. Low levels of Pb(2+) in the growth culture media indicate that PbS NP dissolution does not occur in the culture. Also, the addition of 100 µM Pb(2+) to the culture media had no significant (P>0.05) effect on algal growth. The shading of light (shading effect) by PbS NPs, when simulated using activated charcoal, did not contribute to the overall toxic effect of PbS NPs which was evident by insignificant (P>0.05) reduction in the growth and antioxidant capacity of the algae. When PbS NP aggregation in culture media (without algal cells) was followed for 60 min, uncoated form aggregated rapidly reaching aggregate sizes with hydrodynamic diameter of over 2500 nm within 60 min. Effective particle-particle interaction was reduced in the GA-coated NPs. Aggregates of about 440 nm hydrodynamic diameter were formed within 35 min. Afterwards the aggregate size remained constant. It is concluded that PbS NPs have a negative effect on aquatic algae and their transformation by GA capping affects NPs aggregation properties and toxicity.

Characterization and expression patterns of nitrate reductase from Dunaliella bardawil under osmotic stress and dilution shock.

A complementary DNA (cDNA) of nitrate reductase (NR) from Dunaliella bardawil was isolated using RT-PCR and RACEs techniques. The full-length D. bardawil NR (DbNR) cDNA is 3,107 bp containing a putative open reading frame of 2,670 bp in length which encodes 889 amino acids with a calculated molecular weight (MW) of 98.37 kDa, a 34-bp 5'-untranslated region, and a 3'-untranslated region of 403 bp with a poly (A) tail. BLAST search showed that the nucleotide and putative protein sequence exhibit sequence identities of 92 and 79% with the corresponding gene from Dunaliella tertiolecta, respectively. Protein structural analysis showed a typical NR structure of DbNR with five structural distinctive domains which form three common subparts of eukaryotic NR (Euk-NR). Phylogenetic analysis based on the holo-DbNR and sulfite oxidase (SO) and cytochrome b reductase (CbR) subparts manifested that (1) DbNR has a closer relationship with those counterparts from algae and higher plants than from other species and (2) DbNR might have evolved from ancient SO and CbR in a "domain shuffling" pattern. The glycerol contents and transcriptional expression patterns of DbNR under salt stress and dilution shock treatments were also traced. The results implied an indirect role of NaCl on the induction of DbNR through an osmoregulation pathway.

Carotenoid genes transcriptional regulation for astaxanthin accumulation in fresh water unicellular alga Haematococcus pluvialis by gibberellin A3 (GA3).

The fresh water unicellular alga Haematococcus pluvialis is a promising natural source of astaxanthin. The present study investigated the transcriptional expression of carotenoid genes for astaxanthin accumulation in H. pluvialis using real-time fluorescence quantitative PCR (qRT-PCR). With treatments of 20 and 40 mg/L of gibberllin A3 (GA3), five genes ipi-1, ipi-2, psy, pds and bkt2 were up-regulated with different expression profiles. GA20 (20 mg/L of GA3) treatment had a greater effect on transcriptional expression of bkt2 than on ipi-1 ipi-2, psy and pds (> 4-fold up-regulation). However, GA40 (40 mg/L of GA3) induced more transcriptional expression of ipi-2, psy and bkt2 than both ipi-1 and pds. The expression of lyc, crtR-B and crtO for astaxanthin biosynthesis was not affected by GA3 in H. piuvialis. In the presence of GA3, astaxanthin biosynthesis genes of ipi-1, pds and bkt2 were up-regulated at transcriptional level, psy at post-transcriptional level, whereas ipi-2 was up-regulated at both levels. The study could potentially lead to a scale application of exogenous GA3 in astaxanthin production with H. pluvialis just like GAs perform in increasing crops production and it would provide new insight about the multifunctional roles of carotenogenesis in response to GA3.

Naphthenic acid biodegradation by the unicellular alga Dunaliella tertiolecta.

Naphthenic acids (NAs) are a major contributor to toxicity in tailings waste generated from bitumen production in the Athabasca Oil Sands region. While investigations have shown that bacteria can biodegrade NAs and reduce tailings toxicity, the potential of algae to biodegrade NAs and the biochemical mechanisms involved remain poorly understood. Here, we discovered that the marine alga Dunaliella tertiolecta is able to tolerate five model NAs (cyclohexanecarboxylic acid, cyclohexaneacetic acid, cyclohexanepropionic acid, cyclohexanebutyric acid and 1,2,3,4-tetrahydro-2-naphthoic acid) at 300mgL(-1), a level which exceeds that of any single or combination of NAs typically found in tailings ponds. Moreover, we show that D. tertiolecta can metabolize four of the model NAs. Analysis of NA-amended cultures of D. tertiolecta via low resolution gas chromatography-mass spectrometry allowed us to quantify decreasing NA levels, identify metabolites, and formulate putative mechanisms of biodegradation. Degradation of cyclohexanebutyric acid and cyclohexanepropionic acid proceeded via ß-oxidation and resulted in the transient accumulation of cyclohexaneacetic acid and cyclohexanecarboxylic acid, respectively. Cyclohexanecarboxylic acid was metabolized via 1-cyclohexenecarboxylic acid suggesting that further degradation may occur by step-wise ß-oxidation. When D. tertiolecta was inoculated in the presence of oil sands tailings water from the Athabasca region, biodegradation of single-ring NAs was observed relative to controls. This result corroborates the trend we observed with the single-ring model NAs.

Effects of salinities on the gene expression of a (NAD+)-dependent glycerol-3-phosphate dehydrogenase in Dunaliella salina.

Glycerol-3-phosphate dehydrogenase (G3pdh) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. In this study, the effects of salinity changes on variation of cell shape and single cell glycerol content of Dunaliella salina were observed, and the effects of salinity changes on the gene expressions of a (NAD+)-dependent G3pdh (EC1.1.1.8) among G3pdh isozymes in D. salina were detected by real-time quantitative PCR. Results showed that the changes of shape and volume of D. salina cell cultured chronically at various salinities were minor, but when the salinity was changed rapidly, the variations of cell shape and cell volume of D. salina were significant, which were recovered basically after 2h except treating by high salinity. Also, it was found some lipid globules in the surface of D. salina cells when the salinity increased from 2.0 to 4.0-5.0 M NaCl rapidly. When D. salina was cultured chronically at various salinities, the accumulation of single cell glycerol increased with increased salinity, and D. salina also could rapidly decrease or increase single cell glycerol contents to adapt to hypoosmotic or hyperosmotic shock. The expression level of G3pdh in D. salina grown at various salinities was significantly inversely correlated to the salinity, but there was no significant correlation between the expression level of G3pdh and salinity after 2 h of treatment by hyperosmotic or hypoosmotic shock.

Different physiological responses influenced by salinity in genetically related Dunaliella salina isolates.

An isolate of Dunaliella salina (DUNS-1) and other two isolates (DUNS-2 and DUNS-3), collected from coastal lagoons with 14 and 30% (w/v) of NaCl, respectively, were analyzed under different saline conditions. Glycerol (380 mg l(-1)) and carotene (5.9 mg l(-1)) contents for DUNS-2 were 0.3 and 10 times higher than DUNS-3, even though both isolates were collected from the same lagoon and share a similar ribosomal DNA sequence.

Descriptive and mechanistic toxicity of conazole fungicides using the model test alga Dunaliella tertiolecta (Chlorophyceae).

Conazole fungicides are commonly used to prevent fungal growth on turf grass and agricultural crops. As many of these sites are adjacent to coastal waterways and estuaries, there exists the potential for nontarget effects of runoff on marine organisms. This study reports 96 h EC(50) values for four selected conazole fungicides (triadimefon = 5.98 mg/L; triadimenol = 5.51 mg/L; propiconazole = 2.33 mg/L; hexaconazole = 0.91 mg/L) to the model test alga Dunaliella tertiolecta. We further investigated possible mechanisms of toxicity by examining sublethal effects of exposure on cell morphology, osmoregulatory function, and lipid composition. These mechanistic studies revealed that conazole exposure does not inhibit synthesis of the cell's glycerol osmolyte, but does result in an overall increase in cellular volume and total lipid content. Both fungi and chlorophytes rely on ergosterol to maintain membrane structure and fluidity, and we provide evidence that the sterol-inhibiting conazoles may interfere with ergosterol biosynthesis in the cell membrane of Dunaliella. These findings suggest that green algae may be especially susceptible to nontarget effects of sterol-inhibiting fungicides in marine systems.

Semi-continuous cultivation of Haematococcus pluvialis for commercial production.

The objectives of the present study on the growth of Haematococcus pluvialis were to indicate the effects of a long-term semi-continuous cultivation, sterilization, carbon dioxide, and different culture media by using artesian well water. This investigation was an enterprise in order to commercialize the production economically. When the effect of CO(2) was investigated in basal culture medium, the influence of sterilization was also researched in Rudic's culture medium in vertical panel-type photobioreactors for 31 days of semi-continuous cultivation. The maximum cell concentration of 10.55 x 10(5) cells ml(-1), which corresponds to the growth rate of 0.271 day(-1) with the areal productivity of 3.531 g m(-2) day(-1), was found in non-sterilized RM medium on the 24th day of the third run of semi-continuous cultivation at a renewal rate of 50% in a vertical panel-type photobioreactor.

Proteomics of proteins associated with astaxanthin accumulation in the green algae Haematococcus lacustris under the influence of sodium orthovanadate.

An extensive proteomics analysis has identified proteins associated with astaxanthin accumulation in the green algae Haematococcus lacustris under oxidative stress induced by sodium orthovanadate (SOV). Measurement of total carotenoid accumulation per cell biomass showed an increase from 81 to 136 pg/cell after being exposed to 2.5 mM SOV, when compared to the control cells at day 3 of cultivation. A total of 83 proteins were differentially expressed in SOV-treated H. lacustris in comparison with control cells. They consisted of 34 down-regulated and 49 up-regulated proteins. Of these, 17 highly-expressed proteins were analyzed by MALDI-TOF-MS to identify the function of the differentially expressed proteins in response to oxidative stress in H. lacustris.