Molecular Cloning and Characterization of High-affinity Nitrate Transporters in Marine Phytoplankton

Nitrate transporter systems initiate nitrate uptake in photoautotrophic organisms including marine phytoplankton. The functional genes encoding highaffinity nitrate transporters (Nrt2) in marine phytoplankton were investigated in this study. Direct PCR amplification with newly developed primers was used to detect Nrt2 genes in cultured marine phytoplankton species: Dunaliella tertiolecta Butcher (Chlorophyceae); Emiliania huxleyi (Lohmann) W. H. Hay et H. Mohler (Haptophyceae); and four diatoms, including Thalassiosira weissflogii (Grunow) G. A. Fryxell et Hasle, Skeletonema costatum (Grev.) Cleve, Ditylum brightwellii (T. West) Grunow, and Chaetoceros muelleri Lemmerm. (Bacillariophyceae). Phylogenetic analysis of the detected Nrt2 genes identified three distinct clusters grouped by each class of phytoplankton species. Transcriptional abundance (mRNA) of Nrt2 genes in D. tertiolecta, E. huxleyi, T. weissflogii, and C. muelleri were measured using quantitative reverse transcriptase-PCR assays under various nitrogen conditions. The diatoms and E. huxleyi induced Nrt2 gene transcripts both in the presence of nitrate and under nitrogen (N) starvation, while the Nrt2 transcript in D. tertiolecta was induced only when nitrate was present. Chaetoceros muelleri exhibited the highest transcript induction of the Nrt2 gene. The transcriptional abundance of the Nrt2 genes in C. muelleri was further examined with short-term nitrate incubation of cells grown under N-sufficient or N-starved conditions. The maximum induction of Nrt2 gene transcripts was observed within 1 h after nitrate was added. Thus, we report differential induction and regulation of Nrt2 genes in the selected phytoplankton species to varying conditions of external N.