Cloning and Characterization of Two Putative P-Type ATPases from the Marine Microalga Dunaliella maritima Similar to Plant H+-ATPases and Their Gene Expression Analysis under Conditions of Hyperosmotic Salt Shock

The green microalga genus Dunaliella is mostly comprised of species that exhibit a wide range of salinity tolerance, including inhabitants of hyperhaline reservoirs. Na+ content in Dunaliella cells inhabiting saline environments is maintained at a fairly low level, comparable to that in the cells of freshwater organisms. However, despite a long history of studying the physiological and molecular mechanisms that ensure the ability of halotolerant Dunaliella species to survive at high concentrations of NaCl, the question of how Dunaliella cells remove excess Na+ ions entering from the environment is still debatable. For thermodynamic reasons it should be a primary active mechanism; for example, via a Na+-transporting ATPase, but the molecular identification of Na+-transporting mechanism in Dunaliella has not yet been carried out. Formerly, in the euryhaline alga D. maritima, we functionally identified Na+-transporting P-type ATPase in experiments with plasma membrane (PM) vesicles which were isolated from this alga. Here we describe the cloning of two putative P-type ATPases from D. maritima, DmHA1 and DmHA2. Phylogenetic analysis showed that both ATPases belong to the clade of proton P-type ATPases, but the similarity between DmHA1 and DmHA2 is not high. The expression of DmHA1 and DmHA2 in D. maritima cells under hyperosmotic salt shock was studied by qRT-PCR. Expression of DmHA1 gene decreases and remains at a relatively low level during the response of D. maritima cells to hyperosmotic salt shock. In contrast, expression of DmHA2 increases under hyperosmotic salt shock. This indicates that DmHA2 is important for overcoming hyperosmotic salt stress by the algal cells and as an ATPase it is likely directly involved in transport of Na+ ions. We assume that it is the DmHA2 ATPase that represents the Na+-transporting ATPase.

Transcriptomic survey reveals multiple adaptation mechanisms in response to nitrogen deprivation in marine Porphyridium cruentum

Single-cell red microalga Porphyridium cruentum is potentially considered to be the bioresource for biofuel and pharmaceutical production. Nitrogen is a kind of nutrient component for photosynthetic P. cruentum. Meanwhile, nitrogen stress could induce to accumulate some substances such as lipid and phycoerythrin and affect its growth and physiology. However, how marine microalga Porphyridium cruentum respond and adapt to nitrogen starvation remains elusive. Here, acclimation of the metabolic reprogramming to changes in the nutrient environment was studied by high-throughput mRNA sequencing in the unicellular red alga P. cruentum. Firstly, to reveal transcriptional regulation, de novo transcriptome was assembled and 8,244 unigenes were annotated based on different database. Secondly, under nitrogen deprivation, 2100 unigenes displayed differential expression (1134 upregulation and 966 downregulation, respectively) and some pathways including carbon/nitrogen metabolism, photosynthesis, and lipid metabolism would be reprogrammed in P. cruentum. The result demonstrated that nitrate assimilation (with related unigenes of 8–493 fold upregulation) would be strengthen and photosynthesis (with related unigenes of 6–35 fold downregulation) be impaired under nitrogen deprivation. Importantly, compared to other green algae, red microalga P. cruentum presented a different expression pattern of lipid metabolism in response to nitrogen stress. These observations will also provide novel insight for understanding adaption mechanisms and potential targets for metabolic engineering and synthetic biology in P. cruentum.

Toxicity Mechanisms of ZnO Nanoparticles in Nanochloropsis Oculata with a Comparative Approach with CuO and Ag Nanoparticles

The purpose of present work was the investigation of different concentrations of zinc oxide nanoparticles on the marine microalga Nannochloropsis oculata and compare the results of this study with previous studies. Dissolution of ZnO NPs in nanopure water was 0.378-3.12 mg/L and the rate solubility decreased with increasing the concentrations of ZnO NPs. ZnO NPs were toxic to this microalga with EC50 of 153/72 mg/L. The toxicity of 200 mg/L ZnO NPs was 59.36% for the cell number, 61.27% for MTT test, and 57.34% for the chlorophyll content. Increase the content of malondialdehyde and hydrogen peroxide in response to increasing the concentration of ZnO NPs was indicated the induction of oxidative stress in N. oculata. The activity of catalase and lactate dehydrogenase increased in the treated cells, while the activity of ascorbate peroxidase was decreased. Concurrently, an increase in the content of carotenoids and phenolic compounds was observed in the treated cells. SEM and TEM analyses confirmed the aggregation of algal cells, damages in cell membrane and atypical changes in morphology of cell wall after NPs treatments. The FTIR results cofirmed the interaction of ZnO NPs with C-H, C-O and C=O groups on the cell surface. All of these changes were indicated the significant toxic impacts of ZnO NPs on the N. oculata cells. Comparison between the results obtained in previous studies with our results showed that the defensive mechanisms of N. oculata probably was not effective against the oxidative stress by >10 mg/L of ZnO NPs, > 5 mg/L of CuO NPs and > 1 mg/L of Ag NPs. Therefore, N. oculata is sensitive to such concentrations of these NPs.