ESTABLISHMENT OF MINIMAL AND MAXIMAL TRANSCRIPT LEVELS FOR NITRATE TRANSPORTER GENES FOR DETECTING NITROGEN DEFICIENCY IN THE MARINE PHYTOPLANKTON ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE) AND THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)

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 mu M 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 similar to 1 mu mol center dot mol(-1) 18S rRNA was >= 25 mu M 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 similar to 90 mu mol center dot mol(-1) 18S rRNA in I. galbana, and to similar to 800 mu mol center dot 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.