Shaona Cao

Evidence of Coexistence of C3 and C4 Photosynthetic Pathways in a Green-Tide-Forming Alga, Ulva prolifera

Ulva prolifera, a typical green-tide-forming alga, can accumulate a large biomass in a relatively short time period, suggesting that photosynthesis in this organism, particularly its carbon fixation pathway, must be very efficient. Green algae are known to generally perform C3 photosynthesis, but recent metabolic labeling and genome sequencing data suggest that they may also perform C4 photosynthesis, so C4 photosynthesis might be more wide-spread than previously anticipated. Both C3 and C4 photosynthesis genes were found in U. prolifera by transcriptome sequencing. We also discovered the key enzymes of C4 metabolism based on functional analysis, such as pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK). To investigate whether the alga operates a C4-like pathway, the expression of rbcL and PPDK and their enzyme activities were measured under various forms and intensities of stress (differing levels of salinity, light intensity, and temperature). The expression of rbcL and PPDK and their enzyme activities were higher under adverse circumstances. However, under conditions of desiccation, the expression of rbcL and ribulose-1, 5-biphosphate carboxylase (RuBPCase) activity was lower, whereas that of PPDK was higher. These results suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C4-type carbon metabolism in U. prolifera, probably contributing to its wide distribution and massive, repeated blooms in the Yellow Sea.

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.

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.

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.

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.

Artificial leaf aids analysis of chlorophyll fluorescence and P700 absorbance in studies involving microalgae

Measuring chlorophyll fluorescence and P700 absorbance has been widely used to study photosynthesis in both terrestrial plants and algae. However, in order to apply these measurement techniques to study microalgae, a concentrated suspension of algae, which is usually prepared by centrifugation, is required. In this study, instead of using centrifugation, we concentrated microalgae on a nitrocellulose membrane using filtration to create an ‘artificial leaf’ before analysis. Overall, we were able to generate values of the appropriate photosynthetic parameters that were comparable to those obtained when chlorophyll fluorescence and P700 absorbance were measured following centrifugation. There were no statistically significant differences (P > 0.05) between the artificial leaf method and the traditional cuvette method for determining chlorophyll fluorescence or P700 absorbance at appropriate chlorophyll concentrations. We were also able to reduce background noise by using a filter membrane as a carrier. Therefore, an artificial leaf has the potential to be a valuable tool for phycologists interested in studying microalgal photosynthesis by enabling them to eliminate tedious centrifugation steps. In addition, fluorometers commonly used for studying the leaves of higher plants will also be suitable for studying microalgae.

Short-term retention of kleptoplasty from a green alga (Bryopsis) in the sea slug Placida sp. YS001

The capacity of sea slugs (sacoglossans) for retaining chloroplasts from food algae provides important insights into endosymbiotic relationships and kleptoplasty. A sea slug species was captured accidentally in the Yellow Sea and identified as Placida sp. YS001 based on phylogenetic analyses of the COX1 and 16S gene sequence. Its life cycle was recorded using microscope. Photosynthetic analysis by pulse amplitude modulated fluorometry during starvation revealed shortterm functional kleptoplasty. An ultrastructural comparison of the slug and alga showed that a change in the chloroplast structure and the phagosome might correspond to short-term endosymbiosis. The horizontally transferred genes, psbO and lectin, were not cloned in the adults or eggs. This study demonstrates the morphological adaptation that occurs during short-term endosymbiotic relationships and provides fresh insights.