Constitutive expression of nitrate reductase changes the regulation of nitrate and nitrite transporters in Chlamydomonas reinhardtii

Abstract

Summary The effect of constitutive expression of nitrate reductase (NR) on the regulation pattern of nitrite and nitrate transporters (NT) has been analysed in Chlamydomonas reinhardtii. Strain Tx11‐8, bearing the chimeric Nia1 gene for NR under the control of the cabll‐1 gene promoter, showed a constitutive NR which was inactive in ammonium‐grown cells. In contrast to the wild‐type, incubation of Tx11‐8 in ammonium media containing nitrate at micromolar concentrations resulted in reactivation of inactive NR and excretion of nitrite. In addition, negligible amounts of NT gene transcripts were induced by nitrate in this strain. However, NT transcripts from Tx11‐8 cells were induced in amounts similar to those in the wild‐type, by preventing constitutive NR expression from the cabll‐1 gene promoter in the dark. Strains lacking high‐affinity NT and nitrite reductase activity and having the chimeric Nia1 gene have been constructed by genetic crosses. All these strains were sensitive to chlorate in ammonium‐containing media and excreted nitrite when nitrate was present at millimolar concentrations. Spontaneous chlorate‐resistant mutants isolated from these strains lacked constitutive NR activity though they constitutively expressed NR mRNA. The data strongly suggest that nitrate plays an important post‐transcriptional regulatory role and that NR is involved in the regulation of the nitrate/ nitrite transport activities in C. reinhardtii.

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

“…In Chlamydomonas, apparently four nitrate/nitrite transporters operate at the plasma membrane, each showing different substrate affinities and regulation patterns (Galván et al, 1996;Rexach et al, 1999). Systems I (NRT2;1/NAR2, a bispecific HANT/HANiT), II (NRT2;2/NAR2, a specific HANT), and III (NRT2;3, a specific HANiT) operate optimally at high CO 2 ; their activity is blocked by ammonium (Galván et al, 1996;Rexach et al, 1999;Navarro et al, 2000). System IV (NRT2;4, a bispecific HANT/HANiT) operates optimally under limiting CO 2 , and its activity is not blocked by ammonium but rather is inhibited by chloride (Rexach et al, 1999;Navarro et al, 2000).…”

“…In Chlamydomonas, the NR enzyme plays an important role in regulating nitrate/nitrite transporters (Galván et al, 1992;Navarro et al, 1996). In addition, a physical link between Nia1 and Nar1 genes exists in Chlamydomonas so that the Nar1 promoter region (1.5 kb of BamHI-EcoRI) is also the terminating region of the NR gene.…”

“…The selection of these NR ϩ strains, which are unable to grow in nitrate-containing media, was performed by measuring NR activity in the segregants. Second, strain PAD41 was transformed with plasmid pT2CO-5, which carried functional genes for HANT ( Nrt2;1 and Nar2 ); the transformants were selected from nitrate growth tests Galván et al, 1996), which allowed the isolation of strain JA4. Third, the Nar1 gene was transferred to strain JA4 by transformation with a plasmid (pBleNar1) carrying the Nar1 gene and the bleomycin resistance marker (Stevens et al, 1996).…”

“…a K s was determined from the progress curves (Galván et al, 1996). Statistical values represent the mean ϮSD from three independent experiments.…”

“…System I corresponds to a bispecific HANT/HANiT encoded by Nrt2;1/Nar2 ; system II corresponds to a monospecific HANT encoded by Nrt2;2/Nar2 Galván et al, 1996); system III is a bispecific HANiT and LANT, probably encoded by Nrt2;3 (Quesada et al, 1998a;Rexach et al, 1999;Navarro et al, 2000); and system IV is a bispecific HANT/HANiT, probably encoded by Nrt2;4 (Rexach et al, 1999;Navarro et al, 2000). These four systems are involved in the entry of nitrate/nitrite into the cell and thus help regulate the pathway by providing nitrate/nitrite to the cells.…”