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Nitrate transporter genes (Nrt2) encode high-affinity nitrate transporters in marine phytoplankton, and their transcript levels are potential markers of nitrogen deficiency in eukaryotic phytoplankton. For the proper interpretation of measured Nrt2 transcript abundances, a relative expression assay was proposed and tested in Isochrysis galbana Parke (Prymnesiophyceae) and Thalassiosira pseudonana (Hust.) Hasle et Heimdal (Bacillariophyceae). The minimal transcript levels of Nrt2 genes were achieved by the addition of 100 μM ammonium, which led to a rapid decline in Nrt2 transcripts in 10-30 min. Experiments using a concentration series revealed that the effective dosage of ammonium to create a minimal transcript level of ∼1 μmol · mol(-1) 18S rRNA was ≥25 μM in both species. On the other hand, the addition of l-methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, enhanced the Nrt2 transcript level in I. galbana but did not affect that in T. pseudonana. Nitrogen deprivation was used as an alternative means to create maximal Nrt2 transcript levels. By transferring cells into N-free medium for 24 h, Nrt2 transcript levels increased to ∼90 μmol · mol(-1) 18S rRNA in I. galbana, and to ∼800 μmol · mol(-1) 18S rRNA in T. pseudonana. The degree of nitrogen deficiency thus can be determined by comparing original Nrt2 transcript levels with the minimal and maximal levels.

Three DNA fragments belonging to genes involved in nitrogen uptake and assimilation, including a nitrate transporter gene (IgNrt2), an ammonium transporter gene (IgAmt), and a glutamine synthetase gene (IgglnII, were obtained from the marine haptophyte, Isochrysis galbana. Based on alignments of the deduced amino acid sequences, the IgNRT2 fragment shared 47%,, identity with the Cylindrotheca fusiformis (Bacillariophyceae) nitrate transporter, the IgAMT fragment shared 48%, identity with the C. fusiformis ammonium transporter, and the IgGSII fragment shared 61% identity with Skeletonema costatum (Bacillariophyceae) glutamine synthetase. Southern hybridization indicated that both IgNrt2 and IgAmt may have more than one copy in the genome of L galbana, but only a single copy was detected for IgglnII. The transcript abundances of these three genes under various nitrogen-supply conditions were monitored simultaneously by quantitative real-time polymerase chain reaction (PCR). In the presence of ammonium, both IgNrt2 and IgAmt were severely repressed with mean mRNA concentrations of 0.67 and 3.45 mu mol (mol 18S rRNA)(-1). IgNrt2 and IgAmt mRNA transcripts increased 41-fold and 8.3-fold, respectively, in the presence of nitrate, and increased 160-fold and 15-fold, respectively, in the ni trogen-depleted condition. The mRNA level of IgglnII was low in cells grown in the presence of ammonium [3.98 mu mol (mol 18S rRNA)(-1)], but mild increases in the range of 2- to 4-fold were observed in cells grown in both nitrate-containing and nitrogen-depleted cultures. Our results demonstrated that the expression patterns of IgNrt2, IgAmt, and IgglnII shared a common trend with their homologous genes in diatoms and green algae under various nitrogen-supply conditions. This group of genes could be used as indicators in natural environments to estimate the degree of nitrogen deficiency and chemical forms of nitrogenous nutrients in use on a species-specific basis