Expression of nitrate assimilation related genes in Chlamydomonas reinhardtii

Abstract

The mRNA accumulation pattern of the Chlamydomonas reinhardtii nitrate assimilation-related gene cluster has been elucidated. In ammonium-grown wild-type cells, nit-1 (nitrate reductase, NR), nar-1, nar-2 and nar-3 (nitrate transporter) genes showed very similar kinetics of expression when transferred to nitrate medium. Transcripts of all these genes accumulated transiently in ammonium-grown wild-type cells after a one-hour incubation in nitrogen-free medium, and practically disappeared at about 2 hours. Mutant strains lacking functional nitrate reductase showed similar accumulation kinetics of these transcripts during both nitrate induction and derepression in nitrogen-free media. In contrast to the other nar transcripts, that nar-4, a gene sharing similar sequences with nar-3, accumulated in small amounts in wild-type cells, and only increased after a long nitrate induction period. Nitrate and light showed a strong positive effect on the accumulation of nit-1 gene transcripts. Acetate as a carbon source allowed accumulation of nit-1 mRNA in the dark, indicating the existence of interactions between light and carbon metabolism in nit-1 gene expression. Our data strongly suggest that NR negatively autoregulates its own expression and that of nar genes.

The Chlamydomonas reinhardtii Nar1 Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport

“…A protein band of ‫ف‬ 30 kD was immunodetected only from chloroplast membranes from cells that had been induced with nitrate; the size is compatible with that expected from the sequence data for the mature NAR1 protein. The expression pattern of the protein also matches the data for expression of Nar1 transcripts, which are induced in nitrate-grown and repressed in ammonium-grown cells (Quesada et al, 1993). Antibodies against NRT2;1 were used as a control in both membrane preparations.…”

“…The 14-mer oligopeptide used to raise the specific NAR1 antibody is underlined. nitrate assimilation appeared elusive, given that this strain grows in nitrite-containing media and efficiently assimilates nitrite (Quesada et al, 1993;Rexach et al, 1999). Accordingly, to gain information on the role of Nar1 in nitrate assimilation, we developed the following strategy.…”

“…Chlamydomonas strains having or not having functional genes for NR and the HANT ( Nar2, Nrt2;1 ) and either possessing or lacking Nar1 were isolated (see scheme illustrated in Figure 4A). The starting strain D2 is a mutant carrying a 15-kb deletion on the nitrate gene cluster, thus lacking Nar1, Nia1, Nar2, Nrt2;1 , and Nrt2;2 (Quesada et al, 1993). First, a functional NR gene was introduced by genetic cross in a D2 genetic background to obtain the strain PAD41.…”

“…In Chlamydomonas, six genes related to nitrate assimilation are clustered within a 45-kb genome region in linkage group IX, the same region in which the Nar1 gene is located at the 3 Ј end of the NR gene Nia1 ( Figure 1A) (Quesada et al, 1993(Quesada et al, , 1998b. Nar1 seems to have a role in nitrate assimilation, because it shows coordinated regulation with other genes for nitrate assimilation in Chlamydomonas: induction by nitrate, repression by ammonium, and control by the positive regulatory gene Nit2 (Quesada et al, 1993(Quesada et al, , 1998b.…”

“…Nar1 seems to have a role in nitrate assimilation, because it shows coordinated regulation with other genes for nitrate assimilation in Chlamydomonas: induction by nitrate, repression by ammonium, and control by the positive regulatory gene Nit2 (Quesada et al, 1993(Quesada et al, , 1998b.…”