MOLECULAR CHARACTERIZATION OF THE ASSIMILATORY NITRATE REDUCTASE GENE AND ITS EXPRESSION IN THE MARINE GREEN ALGA DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)¹

Abstract

Nitrate is used as a nutrient for growth by many organisms through the nitrate assimilation pathway. The key enzyme involved in nitrate assimilation is nitrate reductase (NR). Transcriptional regulation of NR genes has been studied in various phototrophic eukaryotes from freshwater or terrestrial habitats. Here, we describe the first NR gene from a marine phytoplankton, the green alga Dunaliella tertiolecta Butcher. Its sequence is very similar to that of the other green algae, but its intron structure and transcriptional regulation differ significantly. A partial genomic NR gene fragment was cloned using direct PCR amplification with degenerate primers, and rapid amplification of cDNA ends was performed to obtain a complete NR gene from a cDNA of D. tertiolecta. The complete NR gene of D. tertiolecta is 2.7 kb in size and is similar at the amino acid level to the NRs from freshwater green algae Chlorella vulgaris (50.6%), Chlamydomonas reinhardtii (58.4%), and Volvox carteri (59.4%). Transcriptional regulation of D. tertiolecta NR was examined in response to different nitrogen sources and during the diel cycle. Dunaliella tertiolecta NR transcript was induced by nitrate and repressed by ammonium. In addition, transcription was not induced under nitrogen depletion. Induction of NR transcript responded to the diel cycle, and transcript levels were maximized within an hour after the beginning of the light period. The induction and repression of NR transcript were much slower than reported in freshwater green algae, which may be related to the size of internal nitrogen pools.

Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta)

“…They have substantial similarity, particularly within functional domains, but present large variation in GC content at the third codon position and in the number of introns. NR genes from green algae have 18 introns in Chlorella vulgaris (Dawson et al 1996), 15 in Chlamydomonas reinhardtii (Merchant et al 2007), ten in Volvox carteri (Gruber et al 1992), and also Dunaliella tertiolecta showed two introns in a partial region of the gene cloned (Song and Ward 2004). The intron positions are different between fungi and plants, but conserved within these groups (Zhou and Kleinhofs 1996).…”

A microarray for assessing transcription from pelagic marine microbial taxa

“…Although transcription of NR can be induced by the presence of nitrate alone in eukaryotes (Song and Ward, 2004;Poulsen and Krö ger, 2005), the lower Fv/Fm ratio was possibly due to insufficient N (Tolonen et al, 2006) for the dominant phytoplankton in chlorophyll a measurements, which at that time were eukaryotic phytoplankton. Similar to Prochlorococcus, eukaryotic phytoplankton downregulated rbcL genes in the Fe treatment, especially two Chrysophytes, Epipyxis pulchra and Ochromonas aestuarti, and two Prymnesiophytes, Chrysochromulina alifera and Chrysochromulina flava (Figure 5d).…”

Cloning and heterologous expression of nitrate reductase genes from Dunaliella salina

“…Analysis of NR genes showed that gene expression in higher plants and algae was regulated primarily at the transcriptional level. Song and Ward (2004) found a different transcriptional response of NR in the marine alga D. tertiolecta compared to that found in studies with freshwater green algae such as Chlamydomonas reinhardtii, V. carteri, and Chlorella vulgaris (Quesada and Fernandez 1994;Cannons and Pendleton 1994). In the freshwater green algae, NR transcription is induced by the removal of repressors such as ammonia and metabolites of ammonia and under N-depleted conditions.…”

“…During the last two decades, genes encoding NR have been cloned from several algae such as Chlamydomonas reinhardtii , Volvox carteri (Gruber et al 1992), Chlorella vulgaris (Dawson et al 1996), Dunaliella tertiolecta (Song and Ward 2004) and Phaeodactylum triconutum (Allen et al 2005). Moreover, Kindle et al (1989) developed a nuclear transformation system for C. reinhardtii, using micro-projectile bombardment to introduce the C. reinhardtii NR gene ) into a nit1 mutant strain that lacks NR activity.…”