In Chlorella sorokiniana 13 nitrate reductase (NR) mutants have been found; all lack the ability to grow on nitrate (but retain the ability to grow on nitrite or ammonium), and all lack the ability to take up nitrate (Knobloch and Tischner, 1989). In Chlorella, some mutants are devoid of all NR activities and produce no NR protein as detected by Western blotting, and are putative regulatory mutants (Knobloch and Tischner, 1989).

Nitrite-reductase (NiR; EC 1.7.7.1)-deficient mutants of Chlorella sorokiniana absorb nitrate with rates close to those measured for the wild type, and excrete nitrite into the medium (Burhenne and Tischner, 2000).

Nichols et al (1978) suggested that the NR protein is responsible not only for nitrate reduction, but also for nitrate uptake in Chlamydomonas. Thus, NR-deficient mutants of Chlamydomonas are unable to take up nitrate (Nichols et al, 1978). However, three overlapping clones covering a Chlamydomonas reinhardtii genomic region of about 32 kb appear to contain five genes potentially involved in nitrate assimilation in addition to the nitrate reductase structural locus nit-1 (Quesada et al, 1993). These new loci produced transcripts of 2.8, 2.2, 1.8 and 1.7 kb in nitrate-induced wild-type cells that, like the 3.4 kb transcript of nit-1, were undetectable in cells grown in ammonium (Quesada et al, 1993). In the Chlamydomonas reinhardtii mutant defective at the regulatory locus, nit-2 for nitrate assimilation, which does not express nit-1, accumulation of the four other transcripts was also blocked. These four other loci have been named nar (nitrate assimilation related) genes. The nar-1 and nar-2 loci are transcribed in the same orientation as nit-1, whereas the nar-3 and nar-4 loci are transcribed divergently from nit-1 (Quesada et al, 1993). Mutants lacking nar transcripts and the regulatory mutant nit-2 are incapable of accumulating intracellular nitrate (Quesada et al, 1993). Nar-2 may encode a nitrate transporter (Navarro et al, 2000). The Chlamydomonas reinhardtii Nar1 gene encodes a chloroplast membrane protein involved in nitrite transport; the deduced amino acid sequence has substantial identity to putative formate and nitrite transporters in bacteria (Rexach et al, 2000).

Other high-affinity nitrate transporter (HANT) genes in Chlamydomonas reinhardtii include Nrt2;1 and Nrt2;2 (Navarro et al, 2000). The gene Nrt2;2 is closely linked to the nitrite reductase (NiR) structural gene Nii1 (Queseda et al, 1998). Only when the two mRNAs prepared from Nrt2;1 and Nar2 were expressed in Xenopus oocytes could a high affinity nitrate transport activity be measured. This suggest that two gene products are required to produce a functional high affinity nitrate transport system (Zhou et al, 2000). Three nitrate assimilation related high affinity transport systems operate in C. reinhardtii: one specific for nitrite, a second encoded by nar2/Nrt2;2 specific for nitrate, and another encoded by nar2/Nrt2;1, which is bispecific for these two anions (Galvan et al, 1996). The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters (Queseda et al, 1998). Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway (Queseda et al, 1998). Nar5 is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium (Queseda et al, 1998).

nit-2 (Nit2) is a positive regulatory gene in Chlamydomonas (Schnell and Lefebvre, 1993). Other putative regulatory genes include nit-8 and far-1 (free from ammonia repression), a putative negative regulatory gene required for the repression of the NR gene (nit-1) in Chlamydomonas (Chen and Silflow, 1996; Zhang and Lefebvre, 1997).

The nitA locus (encoding NR) in the muticellular green alga Volvox carteri has been used to identify a transposable element, named Jordan (Miller et al, 1993). Mutants defective in NR are more resistant to chlorate; therefore nitA can be used as a "transposon trap".

Burhenne N, Tischner R 2000 Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k). Planta 211: 440-445.

Chen Q, Silflow CD 1996 Isolation and characterization of glutamine synthetase genes in Chlamydomonas reinhardtii. Plant Physiol. 112: 987-996.

Galvan A, Quesada A, Fernandez E 1996 Nitrate and nitrate are transported by different specific transport systems and by a bispecific transporter in Chlamydomonas reinhardtii. J. Biol. Chem. 271: 2088-2092.

Knobloch O, Tischner R 1989 Characterization of nitrate reductase deficient mutants of Chlorella sorokiniana. Plant Physiol. 89: 786-791.

Miller SM, Schmitt R, Kirk DL 1993 Jordan, an active Volvox transposable element similar to higher plant transposons. Plant Cell 5: 1125-1138.

Navarro MT, Guerra E, Fernandez E, Galvan A 2000 Nitrite reductase mutants as an approach to understanding nitrate assimilation in Chlamydomonas reinhardtii. Plant Physiol. 122: 283-290.

Nichols GL, Shehata SAM, Syrett PJ 1978 Nitrate reductase deficient mutants of Chlamydomonas reinhardii. Biochemical characteristics. J. Gen. Microbiol. 108: 79-88.

Quesada A, Hidalgo J, Fernandez E 1998 Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii. Mol. Gen. Genet. 258: 373-377.

Rexach J, Fernandez E, Galvan A 2000 The Chlamydomonas reinhardtii Nar1 gene encodes a chloroplast membrane protein involved in nitrite transport. Plant Cell 12: 1441-1454.

Zhang D, Lefebvre PA 1997 FAR1, a negative regulatory locus required for the repression of the nitrate reductase gene in Chlamydomonas reinhardtii. Genetics 146: 121-133.

Zhou JJ, Fernandez E, Galvan A, Miller AJ 2000 A high affinity nitrate transport system from Chlamydomonas requires two gene products. FEBS Lett. 466: 225-227.