Photorespiratory ammonium assimilation in chloroplasts of Chlamydomonas reinhardtii

The effect of CO₂ concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw-15 mutant (lacking cell wall) and in the high CO₂-requiring double mutant cia-3/cw-15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw-15 cells, both the extracellular (CA_ext) and chloroplastic (CA_chl) CA activities increased after transferring cells from media bubbled with 5% CO₂ in air (v/v, high-C_i cells) to 0.03% CO₂ (low-C_i cells), whereas in cia-3/cw-15 cells only the CA_ext was induced after adaptation to low-C_i conditions and the CA_chl activity was negligible. During adaptation to low-C_i conditions in the presence of 1 mM of l-methionine-D,L-sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw-15 in the presence of MSX was lower in cells grown and kept at 5% CO₂ than in high-C_i cells adapted to 0.03% CO₂. The double mutant cia-3/cw-15 excreted photorespiratory ammonium at a higher rate than did cw-15. Total GS activity (GS-1 plus GS-2) increased during adaptation to 0.03% CO₂ in both strains of C. reinhardtii. However, only the activity GS-2, which is located in the chloroplast, increased during the adaptation to low CO₂, whereas the cytosolic GS-1 levels remained similar in high and low-C_i cells. We conclude that: (1) cia-3/cw-15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS-2/GOGAT cycle; and (3) chloroplastic GS-2 concentration changes in response to the variation of environmental CO₂ concentration.     

Cooperative regulation by ammonium and ammonium derivatives of nitrite uptake in Chlamydomonas reinhardtii

The role of ammonium in the regulation of nitrite uptake in Chlamydomonas reinhardtii has been investigated under conditions that prevented ammonium assimilation. Prolonged carbon-starvation or inhibition of glutamine synthesis with l-methionine-dl-sulfoximine partially relieved ammonium inhibition of nitrite uptake. However, nitrite uptake was inhibited in both methionine sulfoximine-treated and carbon-starved cells preincubated with ammonium, the inhibition extent in the two cases being directly dependent on the ammonium concentration in the preincubation media. Methionine sulfoximine treatment caused an increase of intracellular ammonium levels. When methionine sulfoximine-treated cells were transferred to ammonium media there existed a linear correlation between intracellular and extracellular ammonium concentration. Addition of methionine sulfoximine to cells with their nitrite uptake system inhibited by ammonium counteracted the effect of ammonium and restored nitrite uptake rate. These results strongly suggest that ammonium itself and a (some) product(s) of its metabolism must act together to block completely nitrite uptake by C. reinhardtii cells. Partial inhibition of nitrite uptake by methylammonium, a structural analogue of ammonium incapable of being used for cell nutrition, supports the above conclusion.     

Effect of nitrogen starvation on ammonium assimilation by Chlamydomonas reinhardtii

In nitrogen-starved Chlamydomonas reinhardtii , wild type, strain 21 gr cells, consumption of nitrate, nitrite and ammonium may occur in the dark in the absence of an added carbon source. Consumption of ammonium in the dark was about 25% higher than in the light, while consumption of nitrate or nitrite in the dark was lower than in the light.
N starvation produced a linear increase with time in the intracellular level of glutamine synthetase (GS, EC 6.3.2.1) and glutamate synthase (NADH-GOGAT, EC 1.4.1.14 and ferredoxin-GOGAT, EC 1.4.7.1) activities in C. reinhardtii . The effect on GS₁ (3-fold) and NADH-GOGAT (4.5-fold) was higher than that on GS₂ (1.5-fold) and ferredoxin-GOGAT (1.5-fold).
Experiments with methylammonium, L-methionine-D, L-sulfoximine (MSX) and azaserine suggest that: 1) Ammonium itself decreases the intracellular levels of glutamine synthetase and ferredoxin-glutamate synthase activities; and 2) a metabolite resulting from ammonium assimilation by the alga may be a negative modulator of NADH-glutamate synthase activity.     

Utilization of nitrate,nitrite and ammonium by Chlamydomonas reinhardii

In phototrophically grown Chlamydomonas cells, ammonium strongly inhibited the utilization of nitrate or nitrite. Under darkness, or in the presence of an uncoupler or inhibitor of the non-cyclic photosynthetic electron flow, the utilization of nitrate, nitrite or ammonium was suppressed. l-Methionine-d,l-sulfoximine (MSX) or azaserine, which blocks the assimilation of ammonium, inhibited the consumption of nitrate, but not nitrite, by the cells. Ammonium produced an immediate inhibition of the permease for nitrate in Chlamydomonas growing with nitrate, while ammonium-grown cells lacked this permease. The synthesis of nitrate-reductase activity was dependent on an active permease. In N-starved Chlamydomonas cells, previously treated with MSX, the permease for nitrate was insensitive to inhibition by ammonium, and a significant amount of nitrate reductase was synthetized. These cells photoproduce ammonium by reducing nitrate. Nitrogen-repleted cells, treated with MSX, actively photoproduced ammonium by reducing nitrite, but not nitrate.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-di-methyl-urea - PCCP Carbonylcyanid-p-trifluoromethoxy-phenylhydrazone - Mops 2-(N-morpholino)propanesulfonic acid - MSX l-Methionine-d,l-sulfoximine     

Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

Inhibition of nitrate consumption by nitrite in entrapped Chlamydomonas reinhardtii cells.

Whereas in freely suspended cell cultures growing photoautotrophically under non-limiting carbon conditions nitrite and nitrate were simultaneously consumed after ammonium consumption was complete, in alginate-entrapped cell cultures a sequential consumption of nitrite (first) and nitrate was observed after ammonium had almost been fully removed. In this paper results are reported that show inhibition of nitrate consumption by nitrite in immobilized cells. However no inhibition of nitrate active transport was observed. The sequential consumption of ammonium, nitrite and nitrate by Ca-alginate immobilized cells is explained on the basis of local ammonium accumulation due to its photoproduction by photorespiration, that could be caused by the increase of the O2/CO2 ratio around the entrapped cells. Measurements of light-dependent oxygen production (LDOP) and activity levels of nitrogen assimilation enzymes, including nitrite reductase (NiR) and glutamine synthetase (GS) in immobilized cells, determined under photorespiration stimulating conditions, are shown that support this explanation.     

Metal toxicity in Chlamydomonas reinhardtii. Effect on sulfate and nitrate assimilation

Cadmium (Cd(2+)) or copper (Cu(2+)) ions are toxic for Chlamydomonas reinhardtii growth, at 300 microM, and the alga may accumulate about 0.90+/-0.02 and 0.64+/-0.02% of its dry weight, respectively. Metal contamination changes the elemental composition of dried alga biomass, which indicates the possibility to use C. reinhardtii as biosensor and bioremediator of the aquatic contamination by heavy metals. Either, Cd(2+) or Cu(2+), inhibits about 20% of the nitrate consumption rate by the cells, while only Cd(2+) increases about 40% the sulfate consumption rate. The presence of 1 mM calcium (Ca(2+)) in the culture medium increases the C. reinhardtii productivity (about 50%), the nitrate uptake rate (about 20%) and the sulfate uptake rate (about 30%). In addition, Ca(2+) overcomes the Cd(2+) (300 microM) toxicity by decreasing (about 35%) the intracellular accumulation of metal. Sulfur-starvation induces in C. reinhardtii the expression of serine acetyltransferase and O-acetylserine(thiol)lyase activities, but decreases 50% the consumption rate of nitrate by the cells. Sulfate is also required for the full expression of the nitrate reductase (NR), nitrite reductase (NiR) and glutamate synthase activities.     

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Post-translational modification of protein cysteine residues is emerging as an important regulatory and signaling mechanism. We have identified numerous putative targets of redox regulation in the unicellular green alga Chlamydomonas reinhardtii. One enzyme, isocitrate lyase (ICL), was identified both as a putative thioredoxin target and as an S-thiolated protein in vivo. ICL is a key enzyme of the glyoxylate cycle that allows growth on acetate as a sole source of carbon. The aim of the present study was to clarify the molecular mechanism of the redox regulation of Chlamydomonas ICL using a combination of biochemical and biophysical methods. The results clearly show that purified C. reinhardtii ICL can be inactivated by glutathionylation and reactivated by glutaredoxin, whereas thioredoxin does not appear to regulate ICL activity, and no inter- or intramolecular disulfide bond could be formed under any of the conditions tested. Glutathionylation of the protein was investigated by mass spectrometry analysis, Western blotting, and site-directed mutagenesis. The enzyme was found to be protected from irreversible oxidative inactivation by glutathionylation of its catalytic Cys¹⁷⁸, whereas a second residue, Cys²⁴⁷, becomes artifactually glutathionylated after prolonged incubation with GSSG. The possible functional significance of this post-translational modification of ICL in Chlamydomonas and other organisms is discussed.     

Nonthermal effect of microwave irradiation on nitrite uptake in Chlamydomonas reinhardtii

When cells of the unicellular green alga Chlamydomonas reinhardtii were subjected to microwave irradiation at 2.45 GHz, nitrite uptake kinetics still obeyed the Michaelis-Menten equation, the Km of the process remaining constant, whereas V _max increased, which indicates an enhanced nonthermal permeability in irradiated cells.     

Two different carriers transport both ammonium and methylammonium in Chlamydomonas reinhardtii

A new methylammonium-resistant mutant strain from Chlamydomonas reinhardtii, henceforth termed 2172 (ma-2), has been isolated. This mutant is affected in a single mendelian gene different from and linked to the ma-1 locus which is defective in the methylammonium-resistant mutant 2170. Both mutations in ma-1 (2170) and ma-2 (2172) are linked to the nit-1 gene coding for the nitrate reductase apoenzyme. Mutant 2172 is affected in methylammonium but not in ammonium uptake capacity and shows derepressed nitrate and nitrite reductase activities in media containing nitrate plus methylammonium but not in nitrate plus ammonium media. The following two enzymatic components for the transport of both ammonium and methylammonium in wild-type cells have been identified: component 1, with high Vmax and K values, which is constitutive, and component 2, with low Vmax and K values, which is ammonium-repressible. Mutant 2170 lacks component 1 whereas mutant 2172 lacks component 2 for both methylammonium and ammonium transport. From genetic and kinetic evidences we conclude that in C. reinhardtii two different carriers are responsible for the transport of both ammonium and methylammonium and that methylammonium (ammonium) transport is a reversible process probably inhibited by the intracellular ammonium which, in turn, regulates nitrate and nitrite reductase levels.