Multiple GS isoenzymes are expressed in most photosynthetic eukaryotes examined to date and are compartmentalized within the chloroplast and cytoplasm. Although the exact physiological function of the different isoenzymes is equivocal, a paradigm has emerged from studies of vascular plants. The model predicts that cytoplasmic isoenzymes assimilate ammonium from sources external to the cell and are involved in nitrogen mobilization, while chloroplastic isoenzymes assimilate ammonium produced from nitrate reduction. Despite its terminal role in both nitrate and ammonium assimilation, our understanding of GS expression and regulation in marine algae is poor compared to that in vascular plants. The purpose of this research was to determine the number and to examine the physiological function of GS isoenzymes expressed by the marine diatom Skeletonema costatum (Greville).

Two peaks of GS activity were resolved by anion-exchange chromatography from the S. costatum. The second peak of activity accounted for greater than 93% of total enzyme activity and this isoenzyme was purified over 200-fold. Results from denaturing gel electrophoresis and gel filtration suggest that six, 70 kD subunits comprise the 400 kD native enzyme. The structure of the diatom GS, therefore, appears more similar to a type of bacterial GS (GSIII) than to the type common among other eukaryotes. This form of GS does not appear unique to S. costatum as the antiserum raised against this protein recognized a similar sized protein in cell lysates of other chromophyte algae.

A cDNA encoding GS was isolated by PCR amplification. The predicted size of the encoded protein (45 kD), was smaller than the GS polypeptide (70 kD) identified by biochemical purification. The nucleic acid and deduced amino acid sequences of the diatom GS were greater than 50% identical to GS from green algae and vascular plants. The presence of a N-terminus signal sequence, identified based on sequence similarity with other chloroplast-localized proteins from diatoms, suggests that the encoded GS isoenzyme is localized to the chloroplast. Phylogenetic analyses indicate that genes encoding GS from the diatom and two species of green algae diverged prior to the gene duplication giving rise to the isoenzymes in vascular plants, supporting the hypothesis that GS isoenzymes in diatoms, green algae and vascular plants arose through independent evolutionary events.

GS activity and isoenzyme abundance were examined during a transition in nitrogen supply. GS expression was examined using antisera raised against GS from S. costatum and two heterospecific GS antisera. Expression of the putative GS polypeptides, detected with the different antisera, varied in response to the nitrogen treatment, suggesting that the isoenzymes have distinct physiological roles. The results indicate that antisera developed for specific GS isoenzymes will be useful in further refining our understanding of the physiological status of phytoplankton cells in situ.