Effect of Nitrogen Supply on the Kinetics and Regulation of Nitrate Assimilation in Chlamydomonas reinhardtii Dangeard

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Abstract
Concentration-dependent NO⊟3 uptake by C. reinhardtii was studied in cells cultured under three NO⊟3–growth regimes: 10 mM NO⊟3 (NO⊟3–grown), 0·25 mM NO⊟3 (N-limited) and an 18 h period of N-deprivation following growth on 10 mM NO⊟3 (N-starved). Two NO⊟3 uptake isotherms, with an apparent phase transition in uptake at 1·1 mM NO⊟3, were resolved in all three cell types over the concentration range tested (0·025 to 1·8 mM NO⊟3). The apparent Vmax for uptake by N-limited cells (0·72 x 10⊟8 μmol cell⊟1 h⊟1) exceeded that for both NO⊟3–grown (0–42 x 10⊟8 μmol cell⊟1 h⊟1) and N-starved (0·49 x 10⊟8 μmol cell⊟1 h⊟1) cells at NO⊟3 concentrations less than 1·1 mM (isotherm 0). When supplied with NO⊟3 concentrations in excess of 1·1 mM (isotherm 1) the apparent Bmax values for uptake by N-limited (0·64 x 10⊟8 μmol cell⊟1 h⊟1) and N-starved (0·41 x 10⊟8 μmol cell⊟1 h⊟1) cells were depressed relative to those for isotherm 0. In contrast, NO⊟3–grown cells exhibited a greater apparent Vmax for uptake (0·63 x 10⊟8 μmol cell⊟1 h⊟1) at the higher NO⊟3 concentrations. When N-limited and N-starved cells were supplied with 1·6 mM NO⊟3 (isotherm 1), the pattern of uptake showed a rapid linear increase after 60 min. This enhanced pattern of uptake did not occur in NO⊟3–grown cells supplied with either 0–6 or 1–6 mM NO⊟3 or in N-limited and N-starved cells supplied with 0·6 mM NO⊟3. Cycloheximide (10 μg cm⊟3), but not actinomycin D (5·0 μg cm⊟3), blocked the apparent ‘induction’ of NO⊟3 uptake in N-starved cells supplied with 1·6 mM NO⊟3. These results suggest that mechanisms controlling the observed ‘inducible’ aspect of NO⊟3 uptake in N-starved cells are dependent on a translational or post-translational event. It is possible that the NO⊟3-concentration dependent ‘inducible’ portion of uptake may provide the means by which C. reinhardtii cells may regulate uptake in accordance with their capacity for assimilation.