Multiple ice‐binding proteins of probable prokaryotic origin in an Antarctic lake alga,Chlamydomonas sp. ICE‐MDV (Chlorophyceae)

Ice‐associated algae produce ice‐binding proteins (IBPs) to prevent freezing damage. The IBPs of the three chlorophytes that have been examined so far share little similarity across species, making it likely that they were acquired by horizontal gene transfer (HGT). To clarify the importance and source of IBPs in chlorophytes, we sequenced the IBP genes of another Antarctic chlorophyte, Chlamydomonas sp. ICE‐MDV (Chlamy‐ICE). Genomic DNA and total RNA were sequenced and screened for known ice‐associated genes. Chlamy‐ICE has as many as 50 IBP isoforms, indicating that they have an important role in survival. The IBPs are of the DUF3494 type and have similar exon structures. The DUF3494 sequences are much more closely related to prokaryotic sequences than they are to sequences in other chlorophytes, and the chlorophyte IBP and ribosomal 18S phylogenies are dissimilar. The multiple IBP isoforms found in Chlamy‐ICE and other algae may allow the algae to adapt to a greater variety of ice conditions than prokaryotes, which typically have a single IBP gene. The predicted structure of the DUF3494 domain has an ice‐binding face with an orderly array of hydrophilic side chains. The results indicate that Chlamy‐ICE acquired its IBP genes by HGT in a single event. The acquisitions of IBP genes by this and other species of Antarctic algae by HGT appear to be key evolutionary events that allowed algae to extend their ranges into polar environments.     

Validation of housekeeping genes for gene expression studies in an ice alga <Emphasis Type="Italic">Chlamydomonas</Emphasis> during freezing acclimation

Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L.     

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Antarctic ice algae Chlamydomonas sp. ICE-L

A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length cDNA of CiHsp70 was 2,232 bp, consisting of a 5′-terminal untranslated region (UTR) of 76 bp, a 3′-terminal UTR of 203 bp with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17 amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or 93‰ NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment.     

A marine Chlamydomonas sp. emerging as an algal model

The freshwater microalga Chlamydomonas reinhardtii, which lives in wet soil, has served for decades as a model for numerous biological processes, and many tools have been introduced for this organism. Here, we have established a stable nuclear transformation for its marine counterpart, Chlamydomonas sp. SAG25.89, by fusing specific cis‐acting elements from its Actin gene with the gene providing hygromycin resistance and using an elaborated electroporation protocol. Like C. reinhardtii, Chlamydomonas sp. has a high GC content, allowing reporter genes and selection markers to be applicable in both organisms. Chlamydomonas sp. grows purely photoautotrophically and requires ammonia as a nitrogen source because its nuclear genome lacks some of the genes required for nitrogen metabolism. Interestingly, it can grow well under both low and very high salinities (up to 50 g · L^‐1) rendering it as a model for osmotolerance. We further show that Chlamydomonas sp. grows well from 15 to 28°C, but halts its growth at 32°C. The genome of Chlamydomonas sp. contains some gene homologs the expression of which is regulated according to the ambient temperatures and/or confer thermal acclimation in C. reinhardtii. Thus, knowledge of temperature acclimation can now be compared to the marine species. Furthermore, Chlamydomonas sp. can serve as a model for studying marine microbial interactions and for comparing mechanisms in freshwater and marine environments. Chlamydomonas sp. was previously shown to be immobilized rapidly by a cyclic lipopeptide secreted from the antagonistic bacterium Pseudomonas protegens PF‐5, which deflagellates C. reinhardtii.     

Growth limitation of three Arctic sea ice algal species: effects of salinity, pH, and inorganic carbon availability

The effect of salinity, pH, and dissolved inorganic carbon (TCO₂) on growth and survival of three Arctic sea ice algal species, two diatoms (Fragilariopsis nana and Fragilariopsis sp.), and one species of chlorophyte (Chlamydomonas sp.) was assessed in controlled laboratory experiments. Our results suggest that the chlorophyte and the two diatoms have different tolerance to fluctuations in salinity and pH. The two species of diatoms exhibited maximum growth rates at a salinity of 33, and growth rates at a salinity of 100 were reduced by 50% compared to at a salinity of 33. Growth ceased at a salinity of 150. The chlorophyte species was more sensitive to high salinities than the two diatom species. Growth rate of the chlorophyte was greatly reduced already at a salinity of 50 and it could not grow at salinities above 100. At salinity 33 and constant TCO₂ concentration, all species exhibited maximal growth rate at pH 8.0 and/or 8.5. The two diatom species stopped growing at pH > 9.5, while the chlorophyte species still was able to grow at a rate which was 1/3 of its maximum growth rate at pH 10. Thus, Chlamydomonas sp. was able to grow at high pH levels in the succession experiment and therefore outcompeted the two diatom species. Complementary experiments indicated that growth was mainly limited by pH, while inorganic carbon limitation only played an important role at very high pH levels and low TCO₂ concentrations.     

On the use of high‐throughput sequencing for the study of cyanobacterial diversity in Antarctic aquatic mats

The study of Antarctic cyanobacterial diversity has been mostly limited to morphological identification and traditional molecular techniques. High‐throughput sequencing (HTS) allows a much better understanding of microbial distribution in the environment, but its application is hampered by several methodological and analytical challenges. In this work, we explored the use of HTS as a tool for the study of cyanobacterial diversity in Antarctic aquatic mats. Our results highlight the importance of using artificial communities to validate the parameters of the bioinformatics procedure used to analyze natural communities, since pipeline‐dependent biases had a strong effect on the observed community structures. Analysis of microbial mats from five Antarctic lakes and an aquatic biofilm from the Sub‐Antarctic showed that HTS is a valuable tool for the assessment of cyanobacterial diversity. The majority of the operational taxonomic units retrieved were related to filamentous taxa such as Leptolyngbya and Phormidium, which are common genera in Antarctic lacustrine microbial mats. However, other phylotypes related to different taxa such as Geitlerinema, Pseudanabaena, Synechococcus, Chamaesiphon, Calothrix, and Coleodesmium were also found. Results revealed a much higher diversity than what had been reported using traditional methods and also highlighted remarkable differences between the cyanobacterial communities of the studied lakes. The aquatic biofilm from the Sub‐Antarctic had a distinct cyanobacterial community from the Antarctic lakes, which in turn displayed a salinity‐dependent community structure at the phylotype level.     

Molecular characterization of a glutathione peroxidase gene and its expression in the selected Vibrio-resistant population of the clam Meretrix meretrix

Glutathione peroxidase (GPx) is an important member of cellular enzymatic antioxidant system, which may be involved in pathogen defense of host. In the present study, a selenium-dependent glutathione peroxidase (MmeGPx) gene from clam Meretrix meretrix was cloned and analyzed. The MmeGPx gene was composed of two introns of 723 bp and 238 bp and an open reading frame (ORF) of 711 bp. The ORF encodes a protein of 237 amino acids with a putative selenocysteine residue encoded by an unusual stop codon. MmeGPx shares a higher level of similarity with human GPx 3 than with other human GPx isozymes. The level of MmeGPx mRNA roughly paralleled GPx enzyme activity in different tissues except in gills, with the highest mRNA expression and enzyme activity occurring in hepatopancreas. MmeGPx mRNA expressions were detected in different larval stages and the results showed that MmeGPx mRNA increased significantly in pediveliger stage, which may be a response to oxidative stress. After challenge of clam with a Vibrio parahaemolyticus-related bacterium (MM21), the expression of MmeGPx was significantly up-regulated at 6 h and 12 h in hepatopancreas, which suggested that MmeGPx may be involved in the immune response to MM21 infection. To better understand its role in the immunity of clam, the expression of MmeGPx in hepatopancreas was compared between a selected Vibrio-resistant population and a control population after immersion challenge with MM21. Early up-regulation of MmeGPx was observed in the resistant population. These results suggested that MmeGPx might be involved in maintaining the redox state of immune system, and the early immune response to pathogen infection may help the clam against pathogen infection.     

The constitutive expression of Chrysanthemum dichrum ICE1 in Chrysanthemum grandiflorum improves the level of low temperature, salinity and drought tolerance

The quality and productivity of chrysanthemum are severely compromised by various abiotic stresses. Here, we describe the isolation of CdICE1 from Chrysanthemum dichrum using RACE PCR, which shared identical nucleotide of ICE1 ORF from Chrysanthemum grandiflorum variety 'Jinba'. CdICE1 contains a conserved bHLH domain, a nuclear localization domain, a S-rich motif and a ACT domain. The constitutive expression of CdICE1 in C. grandiflorum improved the tolerance of C. grandiflorum to low temperature/freezing, drought and salinity. When the transgene was inserted in the antisense direction, the expression of the endogenous ICE1 gene was down-regulated, and the level of the plant's sensitivity to abiotic stress increased. The level of expression of CgDREBa and CgDREBb, activities of superoxide dismutase and peroxidase and the proline content were enhanced in the sense transgenic lines, and lowered in the antisense ones under stresses. In conclusion, CdICE1 represents a promising candidate for a biotechnological approach to improve the level of crop abiotic stress tolerance. Key message Overexpression of CdICE1 in C. grandiflorum confers the stress tolerance via its regulation of CgDREB involved in the oxidative and osmotic homeostasis pathways.     

Purification and characterization of a psychrophilic glutathione reductase from Antarctic ice microalgae <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. Strain ICE-L

A psychrophilic glutathione reductase from Antarctic ice microalgae Chlamydomonas sp. Strain ICE-L was purified by ammonium sulfate fractionation and three steps of chromatography. The yield was up to 25.1% of total glutathione reductase in the crude enzyme extract. The glutathione reductase activity was characterized by the spectrophotometric method under different conditions. Purified glutathione reductase was separated by SDS-PAGE, which furnished a homogeneous band. The native molecular mass of the enzyme was 115 kDa. Apparent Km values for NADPH and NADH (both at 0.5 mmol L⁻¹ oxidized glutathione) were 22.3 and 83.8 μmol L⁻¹, respectively. It was optimally active at pH 7.5, and it was stable from pH 5 to 9. Its optimum temperature was 25°C, with activity at 0°C 23.5% of the maximum. Its optimum ion strength and optimum Mg²⁺ were 50–90 and 7.5 mmol L⁻¹, respectively. Ca²⁺, Mg²⁺, and cysteine substantially increased the activity of the enzyme but chelating agents, heavy metals (Cd²⁺, Pb²⁺, Cu²⁺, Zn²⁺, etc.), NADPH, and ADP had significant inhibitory effects. This glutathione reductase can be used to study the adaptation and mechanism of catalysis of psychrophilic enzymes, and it has a high potential as an environmental biochemical indicator under extreme conditions.