Abstract
Eukaryotic nitrate reductase (NR) catalyzes the first step in nitrate assimilation and is regulated transcriptionally in response to external cues and intracellular metabolic status. NRs are also regulated post-translationally in plants by phosphorylation and binding of 14-3-3 proteins at conserved serine residues. 14-3-3 binding motifs have not previously been identified in algal NRs. A novel NR (NR2-2/2HbN) with a 2/2 hemoglobin domain was recently described in the alga Chattonella subsalsa. Here, a second NR (NR3) in C. subsalsa is described with a 14-3-3 binding motif but lacking the Heme-Fe domain found in other NRs. Transcriptional regulation of both NRs was examined in C. subsalsa, revealing differential gene expression over a diel light cycle, but not under constant light. NR2 transcripts increased with a decrease in temperature, while NR3 remained unchanged. NR2 and NR3 transcript levels were not inhibited by growth on ammonium, suggesting constitutive expression of these genes. Results indicate that Chattonella responds to environmental conditions and intracellular metabolic status by differentially regulating NR transcription, with potential for post-translational regulation of NR3. A survey of algal NRs also revealed the presence of 14-3-3 binding motifs in other algal species, indicating the need for future research on regulation of algal NRs.
Highlights
Nitrate and ammonium are the most abundant nitrogen sources that phytoplankton utilize for their growth and metabolism: approximately 40% of global primary production is the result of assimilating these two nitrogenous nutrients by phytoplankton[1]
The deduced amino acid sequences of NR3 cDNA clones in plasmids CsNR3-P2 and CsNR3-P10 were identical to the NR3 sequence obtained from the Marine Microbial Eukaryote Transcriptome Sequencing Project[29] (MMETSP), except that the sequence obtained from cloned cDNA included 7 sites that were undetermined in the original sequence, 1 additional amino acid at site 166, and 15 additional amino acids (HASSQEVFNNESTTA) within the hinge 1 region at site number 418 to 432 (Supplementary Data S1)
A region of the sequence spanning from the dimer interface (DI) domain to the hinge 2 region showed that the deduced amino acid sequence of NR3 lacked both the 2/2 HbN domain present in NR2 as well as the cytb-5 (Heme-Fe) binding domain found in other nitrate reductase (NR) (Fig. 2)
Summary
Nitrate and ammonium are the most abundant nitrogen sources that phytoplankton utilize for their growth and metabolism: approximately 40% of global primary production is the result of assimilating these two nitrogenous nutrients by phytoplankton[1]. The tight regulation of NR plays an important role for organisms to optimize nitrogen assimilation and adjust different metabolic pathways, and prevents the accumulation of potentially toxic nitrite and formation of reactive oxygen and nitrogen species[10,11]. Both NR gene expression and activity respond to nitrogen source, temperature and light. Vergara et al demonstrated that NR gene expression in the marine diatom Thalassiosira weissflogii was regulated by the photosynthetic organic carbon accumulated in the light period, indicating coordination with photosynthetic activity and light- dark cycle regulation instead of endogenous control by a circadian clock[17]. Consistent with reports of NR sequences from other algae, a 14-3-3 binding site motif was not identified in the translated amino acid sequences of HsNR1 or the NR2 sequences for either H. akashiwo or C. subsalsa[2]
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