NITRATE TRANSPORTER GENES FROM THE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE): mRNA LEVELS CONTROLLED BY NITROGEN SOURCE AND BY THE CELL CYCLE

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Abstract
The molecular characterization of components involved in nitrate uptake and assimilation in phytoplankton is likely to provide new insights into these processes, their regulation, and their effect on primary production. We report the cloning and initial characterization of the first nitrate transporter genes in a marine organism, from the diatom Cylindrotheca fusiformis Reimann et Lewin. A clone isolated from a silicon-responsive cDNA library was shown by sequence comparison to encode a homolog of high-affinity nitrate transporters. The C. fusiformis nitrate transporter cDNA was named NAT (NitrAte Transporter). The NAT cDNA was used to isolate a genomic clone that contained two additional nitrate transporter genes, NAT1 and NAT2, arranged in tandem. The cDNA and two genomic sequences were highly conserved, and only 18 of 1446 nucleotides in the coding region differed. At least four copies of NAT genes were present in C. fusiformis and as shown by hybridization, multiple copies were present in other diatom species. The transcript abundance of NAT genes in cultures with different nitrogen sources was monitored by RNase protection assays. NAT mRNA levels were high in the presence of nitrate, at nearly the same level during nitrogen starvation, and also high in urea-containing cultures. Lower mRNA levels occurred in nitrite-grown cultures. NAT transcript levels were highly repressed with NH4 Cl or NH4 NO3 as the nitrogen source, although very low amounts were detected. These results suggested that monitoring NAT mRNA levels could serve as a marker for (1) nitrate uptake in nitrate medium, (2) nitrogen starvation, and (3) ammonium use by virtue of absence of expression. NAT mRNA levels were not directly regulated by light or dark, but were apparently related to cellular growth and protein synthesis. Using light/dark synchronized cultures to monitor cell cycle responses, NAT mRNA levels were high in early G1 phase, decreased through the remainder of G1 , then increased during DNA synthesis in S phase and into G2 , and finally decreased after M phase. In silicon-starvation synchronized cultures, levels were high at the G1 /S phase boundary, high throughout S and G2 , and finally decreased after M phase. It was clear that NAT expression, and by inference nitrate uptake, did not occur at continuous levels throughout the cell cycle. The results of the RNase protection experiments suggested that transcriptional regulation is a major contributing factor in the control of diatom nitrate uptake. The cloning of the C. fusiformis nitrate transporter genes provides a new tool for investigating diatom nitrogen uptake and metabolism. In addition, the regulation of NAT expression by nitrogen source is likely to be useful in developing techniques to specifically control the expression of genes fused to NAT regulatory sequences in transgenic diatoms.