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.     

Ammonia assimilation in Corynebacterium glutamicum and a glutamate dehydrogenase-deficient mutant

In the wild-type of Corynebacterium glutamicum, the specific activity of glutamate dehydrogenase (GDH) remained constant at 1.3 U (mg protein)–1 when raising the ammonia (NH4) concentration in the growth medium from 1 to 90 mM. In contrast, the glutamine synthetase (GS) and glutamate synthase (GOGAT) activities decreased from 1.1 U (mg protein)–1 and 42 mU (mg protein)–1, respectively, to less than 10 % of these values at NH4 concentrations > 10 mM suggesting that under these conditions the GDH reaction is the primary NH4 assimilation pathway. Consistent with this suggestion, a GDH-deficient C. glutamicum mutant showed slower growth at NH4 concentrations 10 mM and, in contrast to the wild-type, did not grow in the presence of the GS inhibitor methionine sulfoximine. © Rapid Science Ltd. 1998     

Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.)

A major source of inorganic nitrogen for rice plants grown in paddy soil is ammonium ions. The ammonium ions are actively taken up by the roots via ammonium transporters and subsequently assimilated into the amide residue of glutamine (Gln) by the reaction of glutamine synthetase (GS) in the roots. The Gln is converted into glutamate (Glu), which is a central amino acid for the synthesis of a number of amino acids, by the reaction of glutamate synthase (GOGAT). Although a small gene family for both GS and GOGAT is present in rice, ammonium-dependent and cell type-specific expression suggest that cytosolic GS1;2 and plastidic NADH-GOGAT1 are responsible for the primary assimilation of ammonium ions in the roots. In the plant top, approximately 80% of the total nitrogen in the panicle is remobilized through the phloem from senescing organs. Since the major form of nitrogen in the phloem sap is Gln, GS in the senescing organs and GOGAT in developing organs are important for nitrogen remobilization and reutilization, respectively. Recent work with a knock-out mutant of rice clearly showed that GS1;1 is responsible for this process. Overexpression studies together with age- and cell type-specific expression strongly suggest that NADH-GOGAT1 is important for the reutilization of transported Gln in developing organs. The overall process of nitrogen utilization within the plant is discussed.     

Effect of Immobilization on the Kinetic and Stability Properties of O-Acetyl-L-Serine Sulfhydrylase from: Chlamydomonas reinhardtii

The O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) from Chlamydomonas reinhardtii has been immobilized either by ionic binding to DEAE-cellulose or by covalent bonding to alkylamine silica and to vinyl acetate-divinylethylene urea copolymers. The immobilized enzyme had improved stability and showed sigmoidal kinetic behaviour with respect to O-acetyl-L-serine, but without major alterations in the corresponding apparent Km value or in the inhibitory effects of this substrate observed with the enzyme in solution. In addition, significant changes in optimum pH and reaction temperature for O-acetyl-L-serine sulfhydrylase activity were observed.     

Changes in the levels of enzymes involved in ammonia assimilation during the development of Phaseolus vulgaris seedlings.Effects of exogenous ammonia

Seeds of Phaseolus vulgaris L. cv. White Kidney were germinated and grown either in a nitrogen-free or in an ammonia-supplied medium. The changes in the soluble protein concentration and in the levels of glutamine synthetase (GS, EC 6.3.1.2), NADH–glutamate synthase (NADH-GOGAT, EC 1.4.1.14), ferredoxin-glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and glutamate dehydrogenase (GDH, EC 1.4.1.2), both NADH- and NAD⁺-dependent, were examined in cotyledons and roots during the first 10 days after sowing. Soluble protein declined rapidly in the cotyledons and increased slightly in the roots. GS activity was initially high both in cotyledons and roots but subsequently decreased during seedling growth. Exogenous ammonia hardly affected GS activity. High levels of NADH-GOGAT were present both in cotyledons and roots during the first days of germination. The activity then gradually declined in both organs. In contrast, Fd-GOGAT in cotyledons was initially low and progressively increased with seedling development. In roots, the levels of Fd-GOGAT were higher in young than in old seedlings. Supply of ammonia to the seedlings increased the levels of NADH-GOGAT and Fd-GOGAT both in cotyledons and roots. NADH-GDH (aminating) activity gradually increased during germination. In contrast, the levels of NAD⁺-GDH (deaminating) activity were highest during the first days of germination. Exogenous ammonia did not significantly affect the activities of GDH.     

Isolation and characterization of a new transposable element in Chlamydomonas reinhardtii

A new transposable element, Tcr3, was identified in the unicellular green alga Chlamydomonas reinhardtii. The Tcr3 element contained imperfect terminal inverted repeat sequences of 56 bp and created a 2 bp target site duplication upon insertion. Insertion of Tcr3 into the 3-untranslated region of the NIT8 gene, which is essential for nitrate assimilation, prevented expression of the gene. Excision of the Tcr3 element correlated with reversion of the mutant phenotype and left behind a 3 bp footprint. Tcr3 was found in all Chlamydomonas isolates tested and should prove to be useful for transposon-tagging experiments in Chlamydomonas.     

Pathway of ammonia assimilation in illuminated Lemna minor

Plants of duckweed (Lemna minor) were grown under constant illumination and with a controlled supply of ammonium-N so as to maintain a constant low concentration. In two kinetic experiments (differing in illumination and N level) with ¹⁵N-ammonia, plants were periodically harvested and their free amino acids analysed for ¹⁵N abundance. Attempts were then made to fit the data by computer simulation models. Only models which had at least two or more intracellular compartments gave adequate fits. Two two-compartment models were tested fully. Both had in compartment 1 the glutamine synthetase-glutamate synthase cycle and in compartment 2 a second site of glutamine synthesis. In one model the glutamate for compartment 2 was derived by transport from compartment 1; in the second model it was synthesized from ammonia by glutamate dehydrogenase at a rate equivalent to 10% of the total N uptake. This second model was rejected after it was found that plants previously treated with methionine sulphoximine and aza-serine (inhibitors of the glutamate synthase cycle) were unable to incorporate ¹⁵N. In spite of wide differences in labelling pattern between the two experiments the first model gave acceptable fits to both when different pool sizes were allowed for. Operation of the glutamate synthase cycle was confirmed by the correspondence between model and data for labelling of glutamine amide, glutamine amino and glutamic acid. Consideration of enzyme distributions suggested that compartment 1 (the glutamate synthase system) is the chloroplasts and compartment 2 the cytosol. Analysis of asparagine and neutral amino acids made it possible to construct balance sheets for N uptake in the two experiments. They suggest that all glutamine synthesized in the chloroplast is used for glutamate and asparagine synthesis and that the cytosol enzyme meets the need of the cell for glutamine per se. The high turnover rates for asparagine indicate that this compound is an important intermediate even under steady state conditions, and carries between 20 and 50% of the products of N assimilation.     

The effect of Cu2+ on uptake and assimilation of ammonium by cucumber seedlings

The ammonium uptake by cucumber seedlings was estimated from ammonium ions depletion in an uptake solution. The uptake of NH ₄ ⁺ was decreased by about 60 % after one hour and by about 90 % after two hours of 100 μM Cu²⁺ treatment. On the contrary the accumulation of ammonium in roots of Cu²⁺-treated seedlings at the same time was higher than in the control. Cu²⁺ in the concentration inhibiting NH ₄ ⁺ absorption during one hour inhibited also glutamine synthetase (GS) (EC 6.3.1.2) and NADH-glutamate dehydrogenase (NADH-GDH) (EC 1.4.1.2) activities both localized in the roots of seedlings. After one hour and at least up to the 4th hour Cu²⁺ accumulated mainly in roots (95 %). It was probably the reason of the GS activity in cotyledons of seedling treated with Cu²⁺ that it was at the same level as in the control. NADH-GDH activity in cotylcdons after one hour of the Cu²⁺ treatment was lower than in the control but the influence of Cu²⁺ action on the activity of this enzyme in roots was by far stronger. 100 μM Cu²⁺ did not affect the activities of both enzymes in in vitro experiments. Copper added into the incubation medium in 1000 μM concentration decreased GS activity, but still did not change NADH-GDH activity. These results suggested the indirect Cu²⁺ action on the investigated enzymes in in vivo experiments. However, no substantial effect on enzyme activities extracted from control plants was observed after the addition of the extract from Cu²⁺-treated plants into the incubation medium. The data suggest that the influence of Cu²⁺ on uptake and assimilation of ammonium may be connected not only with changes of plasma membrane properties in the root cells of Cu²⁺ treated seedlings but also with Cu²⁺ action on two major enzymes involved in NH ₄ ⁺ assimilation: glutamate synthetase and NADH-glutamate dehydrogenase.     

莱茵衣藻Nfr-4突变株中类胡萝卜素含量的变化及其对藻生长的影响

文章对相同条件下培养的莱茵衣藻野生型CC-137和八氢番茄红素脱氢酶(phytoene desaturase,PDS)基因突变株Nfr-4的生长进行分析;并用反相高效液相色谱分析总有色类胡萝卜素以及叶绿素含量变化,结果表明两者生长的差异明显;Nfr-4突变株的单细胞叶绿素和总有色类胡萝卜素含量高于野生型CC-137的。     

Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO₂, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.     

Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH₃ assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH₃ is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.     

Biochemical and genetic analysis of a Chlamydomonas reinhardtii mutant devoid of chloroplastic glutamine synthetase activity

A spontaneous double mutant of Chlamydomonas reinhardtii, designated ARF3, was resistant to L-methionine-S-sulfoximine (MSX), lacked chloroplastic glutamine synthetase (GS2) activity, and grew very poorly in all media tested. In segregants obtained after genetic crosses, the poor-growth phenotype was always linked to the lack of GS2 and to a diminished rate of consumption of ammonium, even under conditions where photorespiration was minimized. The ammonium permeases in mutant ARF3, however, were not altered. This indicates that, unlike in higher plants, GS2 contributes substantially to the primary assimilation of ammonia in this alga, and that its function cannot be replaced by the cytosolic glutamine synthetase. In genetic crosses, the MSX resistance and the lack of GS2 segregated independently, indicating that resistance was not due to an altered form of GS2. Received: 5 June 1998 / Accepted: 10 September 1998     

Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtü Dangeard, adenine or guanine can be used as the sole nitrogen source for growth by means of an inducible system which is repressed by ammonia. Cells grown on either adenine or guanine were able to take up both purines, although the adenine uptake rate was always about 40% of the guanine uptake rate. Both adenine and guanine were taken up by an inducible system(s) exhibiting hyperbolic kinetics with identical apparent A, values of 3-2 mmol m^?3 for adenine and 3-2mmol m^?3 for guanine. Adenine and guanine utilization depended on pH, with similar optimal pH values of 7·3 and 7·4, respectively. Adenine and guanine each acted as a competitive inhibitor of the other's uptake, and their utilization was also inhibited by hypoxanthine, xanthine and urate. Inhibition of adenine uptake by guanine and hypoxanthine was competitive, with A′, values of 5·5 and 1. 6 mmol m^?3 respectively. Guanine uptake was also inhibited competitively by adenine (K₁= 1·3mmol m^?3) and hypoxanthine (K₁= 3. 3 mmol m^?3). Utilization of both adenine and guanine was inhibited by cyanide, azide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone, and was also sensitive to p-hydroxymercuribenzoate and N-ethyl-maleimide. On the basis of these results, taken together, the possibility that adenine and guanine are translocated into Chlamydomonas by a common system is discussed.