Expression of P-Type ATPases of Marine Green Microalga Dunaliella maritima under Hyperosmotic Salt Shock.

Partial sequences of P-type ATPases were cloned from the marine microalgae Dunaliella maritima, two putative H+-ATPases (DmHA1 and DmHA2) and two putative Ca2+-ATPases (DmCA1 and DmCA2). The probable functions of the cloned proteins were suggested on the basis of their primary structure similarity with the proteins whose functions have been already characterized. The transcriptional response of the cloned D. maritima ATPase genes to a sharp increase in the NaCl concentration in the culture medium (from 100 to 500 mM) was investigated by quantitative RT-PCR. Hyperosmotic salt shock led to a significant increase in the DmHA2 expression and to a slight increase in the DmCA2 expression, whereas the expression of the two other ATPases, DmHA1 and DmCA1, was decreased. These data indicate that the DmHA2 ATPase is involved in maintenance of ion homeostasis in D. maritima cells under hyperosmotic salt shock.

Molecular cloning and characterisation of SaCLCa1, a novel protein of the chloride channel (CLC) family from the halophyte Suaeda altissima (L.) Pall.

The SaCLCa1 gene, a putative orthologue of AtCLCa, the Arabidopsis thaliana gene encoding a NO3-/H+ antiporter, was cloned from the halophyte Suaeda altissima. It belonged to the CLC family, comprising anionic channels and anion/H+ antiporters. SaCLCa1 ion specificity was studied by heterologous expression of this gene in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encoded the only CLC family protein, the Cl- transporter Gef1p, in undisrupted strains of this organism. For comparison, the function of another recently identified S. altissima CLC family gene, SaCLCc1, was also characterised. Expression of SaCLCc1 in Δgef1 cells restored their ability to grow on selective media. This supported the chloride specificity of this transporter. By contrast, expression of SaCLCa1 did not complement the growth defect phenotype of Δgef1 cells. However, growth of the Δgef1 mutant on the selective media was partially restored when it was transformed with SaCLCa1(C562 T), encoding the modified protein SaCLCa1(P188S), in which proline responsible for NO3- selectivity in selective filter was replaced by serine providing chloride selectivity. Quantitative real-time polymerase chain reactions (qRT-PCR) showed that significant induction of SaCLCa1 occurred in the roots of S. altissima when plants were grown on nitrate-deficient medium, while SaCLCc1 activation was observed in S. altissima leaves of plants grown in increasing Cl- concentrations of nutrient solution. These results suggested that SaCLCa1 and SaCLCc1 function as anionic transporters with nitrate and chloride specificities, respectively.

[In silico Analyses of Transcriptomes of the Marine Green Microalga Dunaliella tertiolecta: Identification of Sequences Encoding P-type ATPases].

De novo assembled transcriptomes of the marine microalga Dunaliella tertiolecta (Chlorophyta) were analyzed. Transcriptome assemblies were performed using short-read RNA-seq data deposited in the SRA database (DNA and RNA Sequence Read Archive, NCBI). A merged transcriptome was assembled using a pooled RNA-seq data set. The goal of the study was in silico identification of nucleotide sequences encoding P-type ATPases in D. tertiolecta transcriptomes. P-type ATPases play a considerable role in the adaptation of an organism to a variable environment, and this problem is particularly significant for microalgae inhabiting an environment with an unstable ionic composition. Particular emphasis was given to searching for a sequence coding Na^(+)-ATPase. This enzyme is expected to function in the plasma membrane of D. tertiolecta like in some marine algae, in particular, in the closely related alga Dunaliella maritima. An ensemble of 12 P-type ATPases consisting of members belonging to the five main subfamilies of the P-type ATPase family was revealed in the assembled transcriptomes. The genes of the following P-type ATPases were found: (1) heavy metal ATPases (subfamily PIB); (2) Ca^(2+)-ATPases of SERCA type (subfamily P2A); (3) H^(+)-ATPases (subfamily P3); (4) phospholipid-transporting ATPases (flippases) (subfamily P4); (5) cation-transporting ATPases of uncertain specificities (subfamily P5). The presence of functional Na^(+)-ATPases in marine algae is presently undoubted. However, contrary to expectations, we failed to find a nucleotide sequence encoding a protein that could unequivocally be considered a Na^(+)-ATPase. Further study is necessary to elucidate the roles of in silico revealed D. tertiolecta ATPases in Na^(+) transport.