Chengwei Liang

'Green tides' are overwhelming the coastline of our blue planet: taking the world's largest example

A broad spectrum of events that come under the category of green tide are recognized world-wide as a response to elevated levels of seawater nutrients in coastal areas. Green tides involve a wide diversity of sites, macroalgal species, consequences, and possible causes. Here we review the effect of natural and man-induced environmental fluctuations on the frequency and apparent spread of green tides. This article highlights the need for interdisciplinary research aimed at shedding light on the basic mechanisms governing the occurrence and succession of green algae in coastal seas. This will result in more effective management and mitigation of the effects of green tides, thus safeguarding the intrinsic and commercial value of coastal marine ecosystems.

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.

De Novo Sequencing and Global Transcriptome Analysis of Nannochloropsis sp. (Eustigmatophyceae) Following Nitrogen Starvation

Nannochloropsis sp. is an economically and nutritionally important microalga. Recently it has been demonstrated that Nannochloropsis sp. has significant potential for biofuel production. To determine the mechanisms of lipid formation and accumulation during nitrogen starvation, a transcriptomic study was performed to compare gene expression during growth with and without nitrogen. Digital expression analysis identified 1,855 differentially expressed genes between cells grown under nitrogen-replete and nitrogen-deprived conditions; this provided novel insights into the molecular mechanisms of lipid formation by Nannochloropsis sp. under stress. As expected, nitrogen deprivation induced genes involved in nitrogen metabolism and lipid biosynthesis. Although the chlorophyll content decreased following nitrogen deprivation, a subset of genes putatively encoding light-harvesting complex (LHC) proteins were upregulated. These upregulated LHCs may play a role on photoprotection. The sequence data were confirmed using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The expressions of a number of genes involved in acetyl-CoA metabolism were also affected under nitrogen-deprived stress, which may change fatty acids indirectly. Overall, we found low gene expression levels for fatty acid synthesis, suggesting that the buildup of precursors for the acetyl-CoA carboxylases may play a more significant role in TAG synthesis compared with the actual enzyme levels of acetyl-CoA carboxylases per se. The changes in transcript abundance in Nannochloropsis sp. following nitrogen deprivation provided a potential source for exploration of molecular mechanisms of lipid formation and accumulation. Furthermore, a set of simple sequence repeat motifs were identified from the expressed sequence tags, which provide useful genetic markers for further genetic analysis.

Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR

Quantitative real-time reverse transcription PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. To date, few studies on reference genes have been undertaken for Nannochloropsis sp. In this study, 12 potential reference genes were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms by RT-qPCR. The results showed that the best reference genes differed depending on the treatments: different light intensities (DL), the diurnal cycle (DC), high light intensity (HL) and low temperature treatments (LT). A combination of ACT1, ACT2 and TUA would be appropriate as a reference panel for normalizing gene expression data across all the treatments. ACT2 showed the most stable expression across all tested samples but was not the most stable one for individual treatments. Though 18S showed the least stable expression considering all tested samples, it is the most stable one for LT using geNorm. The expression of Lhc confirmed that the appropriate reference genes are crucial. These results provide a foundation for more accurate use of RT-qPCR under different experimental conditions in Nannochloropsis sp. gene analysis.

Identification and expression analysis of the gene lhcSR associated with adaptation to light and low temperature stress in the green tide forming alga Ulva prolifera

Abstract As the dominant species of the green tide-forming algae in the Yellow Sea of China during the past four years, Ulva prolifera was proposed as having evolved a perfect response mechanism to deal with different light and temperature stress. The LHCSR (also known as LI818 in green algae and LHCX in diatoms) protein is a stress-related member of the LHC family found only in algae. The homologues in the green alga Chlamydomonas were up-regulated in response to a variety of stresses and required for energy quenching. In this study, we cloned a putative lhcSR full-length cDNA sequence from U. prolifera and analysed the expression in response to various temperature and illumination gradients. The results showed that expression of lhcSR was up-regulated by light and transiently accumulated in 1 h. Expression of lhcSR could also be induced by low-temperature stress and could sustain the up-regulation for up to 6 days. These data suggest that the lhcSR gene in U. prolifera is involved in the adaption to the light and low-temperature stress in both the short and long term.

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.

Isolation and expression analyses of methyl-d-erythritol 4-phosphate (MEP) pathway genes from Haematococcus pluvialis

Haematococcus pluvialis is a green alga known to accumulate the keto-carotenoid astaxanthin under stress conditions. In H. pluvialis, carotenoids are derived from isopentenyl diphosphate (IPP), which is synthesized via the non-mevalonate methyl-d-erythritol 4-phosphate (MEP) pathway. The present study revealed that several treatments caused changes in pigment profiles and the expression levels of genes encoding MEP pathway enzymes. Additionally, photosynthesis fluorescence was monitored. Generally, under stress conditions, there was an increase in astaxanthin, along with a decrease in total chlorophyll and photo capacity. Six IPP biosynthetic genes were cloned from H. pluvialis. Expression analysis revealed that these transcripts were upregulated under stress culture conditions. However, the extent of MEP pathway gene expression varied with the stress conditions. 4-Diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) synthase (CMS) and CDP-ME kinase (CMK) exhibited significantly higher transcriptional expression under nitrogen starvation treatments. While 1-deoxy-d-xylulose 5-phosphate (DXP) synthase (DXS), CMS, CMK 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) synthase (HDS), and HMBPP reductoisomerase (HDR) showed significant upregulation on the second day under nitrogen starvation and high light (HL-N). The enhanced expression of these genes was also observed on the third day under high light. The high expression of MEP pathway genes was correlated with the accumulation of astaxanthin under HL-N stress. This is the first report of the isolation of IPP biosynthetic genes and their differential expression in H. pluvialis under stress conditions. The present study revealed the influence of stress conditions on the expression of MEP pathway genes and changes in pigment profiles.

Conserved and novel heat stress-responsive microRNAs identified by deep sequencing in Pyropia yezoensis

Pyropia yezoensis is commonly known as a valuable marine red alga. As a cold-temperate species, high temperature is a critical abiotic stress factor that can affect the growth and development of this seaweed. Exploring the regulatory mechanisms of P. yezoensis resistance to high temperatures has significance in breeding high temperature-resistant strains. To investigate the potential role of microRNA (miRNA) regulation in heat stress, we constructed and sequenced four libraries (one control and three heat stressed). A total of 1213 miRNAs, corresponding to 174 miRNA families and 10 miRNAs precursors producing 14 novel mature miRNAs were identified. Among them, 98 miRNAs were differentially expressed under heat stress. The quantitative PCR of six selected miRNAs verified the deep sequencing data. This study represents the first set of heat-responsive miRNAs from P. yezoensis, and provides valuable information for understanding the miRNA-mediated heat stress responses and resistance mechanisms in P. yezoensis. The results offer a foundation for future studies on the genetic improvement of Pyropia heat stress tolerance.

Characterization and production of tetraspore-derived seedlings of Grateloupia lanceolata (Halymeniales, Rhodophyta) in northeastern China

Ye N., Zhang X., Wang H., Lian W., Mao Y., Xu D., Li B., Liang C., Zou J., Zhu B., Jiang Z., Gao Z. and Li D. 2012. Characterization and production of tetraspore-derived seedlings of Grateloupia lanceolata (Halymeniales, Rhodophyta) in northeastern China. Phycologia 51: 330–339. DOI: 10.2216/11-03.1 In this study, Grateloupia lanceolata, newly recorded from northeastern China, was classified on the basis of morphology and rbcL sequence analysis and also raised in a laboratory to evaluate the feasibility of producing tetraspore-derived seedlings. Analysis of the rbcL sequence showed the samples to be monophyletic with G. lanceolata and different from any other species of the genus. Analysis of variance indicated that time has a significant (P < 0.01) effect on both tetraspore output and the percentage of total tetraspores released; those released in the first 2 d made up more than 72% of the total sporulation output; whereas, those released in the last 2 d contributed only 2.52%. In culture, tetraspores released from the unilocular sporangia developed into small discs in one week, and 87% ± 5.7% of the tetraspores that settled developed into sporelings after 9 wk of culture. Tetraspore culture of G. lanceolata in tanks and in the open sea proved to be a feasible method of generating large numbers of individual gametophytes economically.