Shanli Mou

De novo sequencing and analysis of the Ulva linza transcriptome to discover putative mechanisms associated with its successful colonization of coastal ecosystems.

BackgroundThe green algal genus Ulva Linnaeus (Ulvaceae, Ulvales, Chlorophyta) is well known for its wide distribution in marine, freshwater, and brackish environments throughout the world. The Ulva species are also highly tolerant of variations in salinity, temperature, and irradiance and are the main cause of green tides, which can have deleterious ecological effects. However, limited genomic information is currently available in this non-model and ecologically important species. Ulva linza is a species that inhabits bedrock in the mid to low intertidal zone, and it is a major contributor to biofouling. Here, we presented the global characterization of the U. linza transcriptome using the Roche GS FLX Titanium platform, with the aim of uncovering the genomic mechanisms underlying rapid and successful colonization of the coastal ecosystems.ResultsDe novo assembly of 382,884 reads generated 13,426 contigs with an average length of 1,000 bases. Contiguous sequences were further assembled into 10,784 isotigs with an average length of 1,515 bases. A total of 304,101 reads were nominally identified by BLAST; 4,368 isotigs were functionally annotated with 13,550 GO terms, and 2,404 isotigs having enzyme commission (EC) numbers were assigned to 262 KEGG pathways. When compared with four other full sequenced green algae, 3,457 unique isotigs were found in U. linza and 18 conserved in land plants. In addition, a specific photoprotective mechanism based on both Lhc SR and Psb S proteins and a C4-like carbon-concentrating mechanism were found, which may help U. linza survive stress conditions. At least 19 transporters for essential inorganic nutrients (i.e., nitrogen, phosphorus, and sulphur) were responsible for its ability to take up inorganic nutrients, and at least 25 eukaryotic cytochrome P450s, which is a higher number than that found in other algae, may be related to their strong allelopathy. Multi-origination of the stress related proteins, such as glutamate dehydrogenase, superoxide dismutases, ascorbate peroxidase, catalase and heat-shock proteins, may also contribute to colonization of U. linza under stress conditions.ConclusionsThe transcriptome of U. linza uncovers some potential genomic mechanisms that might explain its ability to rapidly and successfully colonize coastal ecosystems, including the land-specific genes; special photoprotective mechanism based on both Lhc SR and Psb S; development of C4-like carbon-concentrating mechanisms; muti-origin transporters for essential inorganic nutrients; multiple and complex P450s; and glutamate dehydrogenase, superoxide dismutases, ascorbate peroxidase, catalase, and heat-shock proteins that are related to stress resistance.

Detection and quantitation of lipid in the microalga Tetraselmis subcordiformis (Wille) Butcher with BODIPY 505/515 staining.

BODIPY 505/515, a lipophilic bright green fluorescent dye was tested for lipid detection in the microalga Tetraselmis subcordiformis. A concentration of 0.28 μg ml(-1) and staining for 6 min was optimal. Lipid bodies stained with BODIPY505/515 had a characteristic green fluorescence. Their volumes were determined using the sphere volume formula. Lipid accumulation under different nitrogen concentrations was analyzed. With an increase in NaNO(3) concentration from 0 to 240 mg L(-1), the maximum algal concentration increased from 8.23 ± 0.62 (× 10(5) cells ml(-1)) to 1.61 ± 0.13 (×10(6) cells ml(-1)), while the maximum volume of intracellular neutral lipid decreased from 9.78 ± 1.77 μm(3) cell(-1) to 6.00 ± 0.59 μm(3) cell(-1). A comparison of the lipid contents measured by BODIPY 505/515 staining and the gravimetric method showed a positive correlation coefficient of R(2) = 0.93. BODIPY 505/515 staining is a promising method in lipid quantitation in T. subcordiformis.

Temperature regulates fatty acid desaturases at a transcriptional level and modulates the fatty acid profile in the Antarctic microalga Chlamydomonas sp. ICE-L

Chlamydomonas sp. ICE-L which can thrive in extreme environments of the Antarctic is a major biomass producer. The FAD genes in Chlamydomonas sp. ICE-L were obtained and sequence alignment showed that these genes are homologous to known FADs with conserved histidine motifs. In this study, we analyzed the transcription of five FADs and FA compositions at different temperatures. The results showed that the expressions of Δ9CiFAD, ω3CiFAD1 and ω3CiFAD2 were apparently up-regulated at 0°C, however, the up-regulation of Δ6CiFAD intensified with rising temperature. Meanwhile, analysis of the FA compositions showed that PUFAs were dominant compositions, accounting for more than 75% TFA in Chlamydomonas sp. ICE-L. Furthermore, PUFAs were significantly increased at 0 and 5°C, which may be attributed to higher proportions of C18:3 and C20:3. Moreover, PUFAs were significantly decreased at 15°C whereas SFAs were significantly increased.

Expression of fatty acid desaturase genes and fatty acid accumulation in Chlamydomonas sp. ICE-L under salt stress.

The Antarctic ice microalgae Chlamydomonas sp. ICE-L which is highly resistant to salt stress holds promise in providing an alternative species for the production of microalgal oil. We studied the effects of the alga in confrontation with NaCl stress on the growth, oil yield and expression of fatty acid desaturase genes. The growth rate of Chlamydomonas sp. ICE-L decreased with the gradual increase in NaCl concentration. Interestingly, we found that the highest lipid content was achieved at 16‰ NaCl, reaching 23% (w/w). Meanwhile, the expression of Δ9ACPCiFAD increased rapidly while Δ12CiFAD, ω3CiFAD2 and Δ6CiFAD showed a delayed elevation in response to altered salt stress. C18:3 was the dominant PUFA, which account for about 75% TFA in Chlamydomonas sp. ICE-L. Under 96‰ and 128‰ NaCl stress, the content of C20:5 almost approached that of C18:3. In contrast, low salinity enhanced the dominance of C18:3 at the expense of C20:3 and C20:5.

A transthylakoid proton gradient and inhibitors induce a non-photochemical fluorescence quenching in unicellular algae Nannochloropsis sp.

Non‐photochemical quenching (NPQ) of chlorophyll fluorescence is thought to be an indicator of an essential regulation and photoprotection mechanism against high‐light stress in photosynthetic organisms. In this report, special chemicals were used to perturb the kinetics of the ΔpH build‐up and the xanthophyll cycle (XC) in Nannochloropsis sp. We found that NPQ was stimulated rapidly on exposure to high light and relaxed rapidly in darkness. The ΔpH could be obligatory for NPQ and ΔpH alone was not sufficient to induce NPQ. The XC, being strictly mediated by ΔpH, was also essential for NPQ. The results demonstrate that the mechanism of NPQ in Nannochloropsis sp. resembled that of diatoms.

Activities of principal photosynthetic enzymes in green macroalga Ulva linza: functional implication of C4 pathway in CO2 assimilation

The green-tide-forming macroalga Ulva linza was profiled by transcriptome sequencing to ascertain whether the alga carries both C3 and C4 photosynthesis genes. The key enzymes involved in C4 metabolism including pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK) were found. When measured under normal and different stress conditions, expression of rbcL was higher under normal conditions and lower under the adverse conditions, whereas that of PPDK was higher under some adverse conditions, namely desiccation, high salinity, and low salinity. Both ribulose-1, 5-biphosphate carboxylase (RuBPCase) and PPDK were found to play a role in carbon fixation, with significantly higher PPDK activity across the stress conditions. These results suggest that elevated PPDK activity alters carbon metabolism in U. linza leading to partial operation of the C4 carbon metabolism, a pathway that, under stress conditions, probably contributes to the hardy character of U. linza and thus to its wide distribution.

Complete mitochondrial genome of the sea slug, Placida sp., contains unusual noncoding regions

The complete sequence (14,751 bp) of the mitochondrial DNA (mtDNA) of the opisthobranch gastropod Placida sp. was determined using long PCR and genome-walking techniques. The genome contains 13 protein-coding genes (PCG), 2 ribosomal RNA (rRNA), 22 transfer RNA (tRNA) genes that are typical of metazoan mtDNA, and 2 lengthy noncoding regions with a total length of 1441 bp. All the tRNA genes have general secondary structures without reduced T or D stems. The mitochondrial gene arrangement of Placida sp. is almost identical to that of sea slugs, whereas the tRNA gene arrangement is different from other animals outside Sacoglossa in the Euthyneura. A phylogenetic analysis based on the mitochondrial genome of Euthyneura indicated that Placida sp. and Placida dendritica are closely related to Elysia chlorotica as a sister taxon, which strongly supports their close phylogenetic affinity.

Cyclobutane pyrimidine dimers photolyase from extremophilic microalga: remarkable UVB resistance and efficient DNA damage repair.

Bacteria living in the Antarctic region have developed several adaptive features for growth and survival under extreme conditions. Chlamydomonas sp. ICE-Lis well adapted to high levels of solar UV radiation. A putative photolyase was identified in the Chlamydomonas sp. ICE-L transcriptome. The complete cDNA sequence was obtained by RACE-PCR. This PHR encoding includes a polypeptide of 579 amino acids with clear photolyase signatures belonging to class II CPD-photolyases, sharing a high degree of homology with Chlamydomonas reinhardtii (68%). Real-time PCR was performed to investigate the potential DNA damage and responses following UVB exposure. CPD photolyase mRNA expression level increased over 50-fold in response to UVB radiation for 6h. Using photolyase complementation assay, we demonstrated that DNA photolyase increased photo-repair more than 116-fold in Escherichia coli strain SY2 under 100μw/cm(2) UVB radiation. To determine whether photolyase is active in vitro, CPD photolyase was over-expressed. It was shown that pyrimidine dimers were split by the action of PHR2. This study reports the unique structure and high activity of the enzyme. These findings are relevant for further understanding of molecular mechanisms of photo-reactivation, and will accelerate the utilization of photolyase in the medical field.

Reference genes for gene expression normalization in Chlamydomonas sp. ICE-L by quantitative real-time RT-PCR

The determination of a robust reference gene has become increasingly important since RT-qPCR used as a prominent technique for quantification of transcript in connection with their molecular and biological mechanisms. Only a few studies on reference genes have been conducted using Antarctic ice algae. In this work, 10 candidate reference genes of Chlamydomonas sp. ICE-L were evaluated for their stabilities. The results showed that the best references genes differed across the experimental samples. Based on NormFinder Analysis, EF-1α was the most suitable reference gene under the diurnal cycle, high light, high salinity and UV-B irradiation conditions, and GAPDH was the most stable gene under different light intensities. For all tested samples H2B was the best gene and 18S was the least. Pair-wise variation analysis revealed that H2B and EF-1α were the best gene combination for diurnal cycle and high light conditions. For different light intensities and high salinity samples, the best combinations were GAPDH + ACT and L32 + H2B, respectively. For UV-B irradiated samples, a minimum of three genes (EF-1α, L32 and 18S) were necessary for accurate normalization. Selecting appropriate reference gene was very important to achieve an accurate and reliable normalization of genes’ expression. These results provided guidelines for reference genes selection under different experimental conditions and also established a foundation for more accurate and widespread use of RT-qPCR in Chlamydomonas sp. ICE-L.

Phylogeny and characterisation of Nannochloropsis oceanica var. sinensis var. nov. (Eustigmatophyceae), a new oleaginous alga from China

Cao S., Zhang X., Fan X., Qiao H., Liang C., Xu D., Mou S., Wang W. and Ye N. 2013. Phylogeny and characterisation of Nannochloropsis oceanica var. sinensis var. nov. (Eustigmatophyceae), a new oleaginous alga from China. Phycologia 52: 573–577. DOI: 10.2216/13-164.1 Strain QD001, previously identified as Chlorella, was examined using molecular phylogenetic analysis (nuclear-encoded 18S rRNA, ITS, rbcL), ITS2 secondary structure, light and electron microscopy, high-performance liquid chromatography pigment analysis, and fatty acid analysis. The strain was shown to be phylogenetically distinct from all described taxa, and the strain was named Nannochloropsis oceanica var. sinensis var. nov. DNA sequences from the new variety were very similar to GenBank sequences for N. maritima nomen nudum.