CONSTITUTIVE AND INDUCIBLE ASPECTS OF NITRATE-NITROGEN UPTAKE BY CHLAMYDOMONAS-REINHARDTII

Similarly to higher plant root systems, Chlamydomonas reinhardtii Dangeard (UTEX 90) cells exhibited biphasic NO3- uptake kinetics. The uptake pattern was similar in cells cultured in 10 mM NO3- (NO3--grown), 0.25 mM NO3- (N-limited) or 10 mM NO3- followed by an 18-h period of N-deprivation (N-starved). In all cell types there was an apparent phase transition in uptake at 1.1 mM NO3- although there were variations in the uptake V-max of both isotherms. The rate of uptake via isotherm 0 ([NO3-] <1.1 mM) in N-limited cells was higher than that of either NO3--grown or N-starved cells. In contrast, NO3--grown and N-limited cells exhibited comparable V-max values when supplied with 1.1 to 1.8 mM NO3(-) (isotherm 1). When supplied with 1.6 mM NO3-, both N-Iimited and N-starved cells exhibited enhanced linear uptake after 60 min of incubation. We ascribed this to an induction phenomenon. This trend was not observed when NO3--grown cells were supplied with 1.6 mM NO3-, or when N-limited and N-starved cells were supplied with 0.6 mM NOT. The 'inducible' aspect of uptake by N-limited cells was blocked by cycloheximide (10 mg 1(-1)), but not by actinomycin D (5 mg 1(-1)) thus indicating the involvement of a translational or post-translational event. To investigate this phenomenon further, we analysed the cell proteins of N-limited cells supplied with either 0.6 or 1.6 mM NO3- for 90 min, using two-dimensional gel electrophoresis. Comparison of protein profiles enabled the identification of a single cell membrane-associated polypeptide (21 kDa, pI ca 5.5) and ten soluble fraction polypeptides (17-73 kDa, pI ca 5.0 to 7.1) unique to the high NO3- treatment. We propose that the 'inducible' portion of NO3- uptake may provide the means by which C. reinhardtii cells regulate uptake in accordance with assimilatory capacity.