Nitrite reductase mutants as an approach to understanding nitrate assimilation in Chlamydomonas reinhardtii

Abstract

We constructed mutant strains lacking the nitrite reductase (NR) gene in Chlamydomonas reinhardtii. Two types of NR mutants were obtained, which either have or lack the high-affinity nitrate transporter (Nrt2;1, Nrt2;2, and Nar2) genes. None of these mutants overexpressed nitrate assimilation gene transcripts nor NR activity in nitrogen-free medium, in contrast to NR mutants. This finding confirms the previous role proposed for NR on its own regulation (autoregulation) and on the other genes for nitrate assimilation in C. reinhardtii. In addition, the NR mutants were used to study nitrate transporters from nitrite excretion. At high CO(2), only strains carrying the above high-affinity nitrate transporter genes excreted stoichiometric amounts of nitrite from 100 microM nitrate in the medium. A double mutant, deficient in both the high-affinity nitrate transporter genes and NR, excreted nitrite at high CO(2) only when nitrate was present at mM concentrations. This suggests that there exists a low-affinity nitrate transporter that might correspond to the nitrate/nitrite transport system III. Moreover, under low CO(2) conditions, the double mutant excreted nitrite from nitrate at micromolar concentrations by a transporter with the properties of the nitrate/nitrite transport system IV.

Figure 1

Strategy to obtain NiR mutants from C. reinhardtii by genetic cross between strains G1 and 04-1. A, Scheme of the genetic cross showing genotypes of parental strains and isolated segregants lacking NiR. Details on these strains are indicated in “Materials and Methods.” B, RNA transfer analysis of NiR mutant strains induced in 4 mm nitrate medium over 3 h using DNA-specific probes for the indicated transcripts. C, Immunodetection of NiR in crude extracts (50 μg of protein) from the indicated strains induced in 4 mm nitrate medium after SDS-PAGE and using a NiR-specific antibody.

Figure 2

Expression of Nia1, Nrt2;1, Nrt2;2, and Nar2 transcripts in the NiR mutants M1, M2, and M4, the NR mutant 305cw15, the HANT mutant S10, and the wild-type strain. Total RNA was extracted from the indicated strains after 1.5 h of incubation in 0.1 mm nitrate or nitrogen-free medium (−N). Total RNA (20 μg) was analyzed in RNA transfer hybridizations using the specific DNA probes indicated in “Materials and Methods” to detect Nia1 and Nrt2;2 transcripts (A) or these from Nrt2;1 and Nar2 (B).

Figure 3

Nitrate uptake and nitrite excretion activity in wild-type (WT) and NiR mutant strains due to the HANT systems I and II. Strains M1, M2, M3, and M4, and the wild-type 6145c were grown in ammonium and then transferred to medium containing 100 μm nitrate at a cell concentration of about 15 to 25 μg chlorophyll/mL. The media were bubbled with CO₂-enriched air and, at the indicated times, nitrate (●) and nitrite (○) were determined.

Figure 4

Nitrite excretion activity under high-CO₂ conditions by the NiR mutant strain M4. Cells from strain M4 were induced under 4% to 5% CO₂ in medium containing 4 mm nitrate for 4 h. Then cells were transferred to medium containing different nitrate concentrations from 0.1 to 40 mm (●, 0.1 mm; ○, 1.0 mm; ▪, 2.0 mm; □, 5 mm; ▴, 10 mm; ▵, 20 mm; and ⋄, 40 mm) and kept bubbling with 4% to 5% CO₂. A, The nitrite concentration in the medium was determined at the indicated times. B, The nitrite excretion rate activity was calculated and represented as a function of the initial nitrate concentration. Chl, Chlorophyll.

Figure 5

Nitrite excretion activity under limiting CO₂ conditions by the NiR mutant strain M4. Strain M4 was induced in media containing 4 mm nitrate for 4 h in cultures bubbled with air filtered through a CO₂ trap. Then cells were transferred to media containing different nitrate concentrations from 25 μm to 40 mm (▾, 25 μm; ▿, 50 μm; ●, 100 μm; ○, 1.0 mm; □, 5 mm; ▴, 10 mm; ⋄, 40 mm) and kept under limiting CO₂ conditions. A, The nitrite concentration excreted to the media was determined at the indicated times. B, The nitrite excretion rate activity was calculated and represented as a function of the initial nitrate concentration. Chl, Chlorophyll.

Figure 6

Effect of ammonium, chloride, and high CO₂ on the nitrite excretion activity induced under limiting CO₂ in the NiR mutant strain M4. Cells from strain M4 were induced as indicated in Figure 5, and transferred to medium containing 100 μm nitrate alone (○), plus 0.5 mm ammonium sulfate (●), plus 10 mm NaCl (▵), plus 10 mm KCl (▴), or bubbled with 4% to 5%-enriched air (□). Nitrite in the medium was determined at the indicated times.