Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii

Two new nitrate assimilation-related genes, Nrt2;3 and Nar5, have been identified in Chlamydomonas reinhardtii. The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters. Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway. The three Nrt2 genes of C. reinhardtii are differentially regulated by the nitrogen source. Expression of Nrt2;3 and of Nrt2;1, a nitrate/nitrite-bispecific transporter gene, was induced by nitrate and more efficiently by nitrite. Accumulation of mRNA of Nrt2;2, the nitrate-specific transporter gene, was only induced efficiently by nitrate. The Nar5 gene is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium. The Nrt2;3 and Nar5 genes are overexpressed in a deletion mutant that lacks nitrate assimilation loci. Received: 6 October 1997 / Accepted: 30 December 1997     

Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii

Two new nitrate assimilation-related genes, Nrt2;3 and Nar5, have been identified in Chlamydomonas reinhardtii. The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters. Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway. The three Nrt2 genes of C. reinhardtii are differentially regulated by the nitrogen source. Expression of Nrt2;3 and of Nrt2;1, a nitrate/nitrite-bispecific transporter gene, was induced by nitrate and more efficiently by nitrite. Accumulation of mRNA of Nrt2;2, the nitrate-specific transporter gene, was only induced efficiently by nitrate. The Nar5 gene is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium. The Nrt2;3 and Nar5 genes are overexpressed in a deletion mutant that lacks nitrate assimilation loci.     

The Chlamydomonas reinhardtii Nar1 gene encodes a chloroplast membrane protein involved in nitrite transport

A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO(2) conditions.     

Nitrite transport to the chloroplast in Chlamydomonas reinhardtii: molecular evidence for a regulated process

Nitrite transport to the chloroplast is not a well documented process in spite of being a central step in the nitrate assimilation pathway. The lack of molecular evidence, as well as the easy diffusion of nitrite through biological membranes, have made this physiological process difficult to understand in plant nutrition. The aim of this review is to illustrate that nitrite transport to the chloroplast is a regulated step, intimately related to the efficiency of nitrate utilization. In Chlamydomonas reinhardtii, the Nar1;1 gene has been shown to have this role in nitrate assimilation. NAR1;1 corresponds to a plastidic membrane transporter protein related to the bacterial formate/nitrite transporters. At least four Nar1 genes might exist in Chlamydomonas. The existence of orthologous Nar1 genes in plants is discussed.     

Regulation of assimilatory nitrate reduction at the level of nitrite in Chlorella fusca

Batch cultures of Chlorella fusca excreted nitrite into the medium if gassed with air (0.03% CO₂), but they did not if supplied with air containing 5% CO₂. After a change from high to low CO₂ concentration in the gas stream, nitrite excretion started immediately. After an increase in CO₂ concentration to 5%, nitrite uptake started within only 30 min. Changes of in-vitro activities of nitrate reductase, nitrite reductase and glutamine synthetase did not correspond to changes of nitrite concentration in the medium and therefore could not explain these observations. A nitrite-binding site, whose activity corresponded with both nitrite excretion and uptake, was detected at the chloroplast envelope. From these data an additional regulatory step in the assimilatory nitrate-reduction sequence is suggested. This includes an envelopeprotein fraction probably regulating the availability of nitrite within the chloroplast.Abbreviations FMN riboflavin 5-phosphate - GS glutamine synthetase - NIR nitrite reductase - NR nitrate reductase     

Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa

Although nitrate is a macronutrient and can serve as good nitrogen source for many species of phytoplankton, high nitrate concentrations do not benefit the growth of phytoplankton. We hypothesise that algae cultured under high nitrate concentrations can accumulate intracellular nitrite, which is produced by nitrate reductase (NR) and can inhibit the growth of algae. To assess the validity of this hypothesis, Microcystis aeruginosa was grown under different nitrate concentrations from 3.57 to 21.43 mM in low CO₂ and high CO₂ conditions for 15 days. We observed that, with increasing nitrate concentrations, the intracellular nitrite concentrations of the alga increased and the growth rates and photosynthesis declined. When grown under high CO₂ conditions, M. aeruginosa showed lower intracellular nitrite concentrations and higher growth rates and \textP_\textm^\textchla {\text{P}}_{\text{m}}^{{\text{chl}}a} , \textR_\textd^\textchla {\text{R}}_{\text{d}}^{{\text{chl}}a} , α^chla than under low CO₂ conditions. These results suggest that the accumulation of intracellular nitrite could be the cause of inhibition of algal growth under high nitrate concentrations.     

Molecular Components of Nitrate and Nitrite Efflux in Yeast

Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.     

dr and spr/sr mutations of Chlamydomonas reinhardtii affecting D1 protein function and synthesis define two independent steps leading to chronic photoinhibition and confer differential fitness

The effects of introduced chloroplast gene mutations affecting D1 synthesis, turnover and function on photosynthesis, growth and competitive ability were examined in autotrophic cultures of Chlamydomonas reinhardtii (Chlorophyta) adapted to low or high irradiance. Few discernible effects were evident when the mutants were grown in low light (LL, 70 μmol m^?2 s^?1). The herbicide-resistant psbA mutation Ser²⁶⁴→ Ala (dr) slowed electron transfer and accelerated D1 degradation in cells grown under high light (HL, 600 μmol m^?2 s^?1). The maximum rate of light-and CO₂-saturated photosynthesis, cell growth rate and competitive ability in the dr mutant were reduced compared to wild type under HL. However, the wild-type rate of D1 synthesis in dr was adequate to compensate for accelerated D1 degradation. 16S rRNA mutations conferring resistance to streptomycin and spectinomycin (spr/sr) that altered chloroplast ribosome structure and assembly were used to inhibit chloroplast protein synthesis. In spr/sr cells grown under HL, D1 synthesis was reduced by 40–60% compared to wild type and D1 degradation was accelerated, leading to a 4-fold reduction in D1 pool size. The reduced D1 levels were accompanied by an elevation of F_o and a decline in F_v/F_m, quantum yield and maximum rate of CO₂-saturated photosynthesis. Chemostat experiments showed that the growth rate and competitive ability of spr/sr were reduced against both wild type and dr.     

Intracellular nitrite accumulation: The cause of growth inhibition of Microcystis aeruginosa exposure to high nitrite level

The aim of the present study is to test the role of intracellular nitrite in external nitrite suppressing algal growth. We examined the growth of Microcystis aeruginosa at different nitrite levels under high nitrate conditions and without nitrate conditions. There were higher intracellular nitrite and lower P_m^chla, R_d ^chla, α^chl, maximum cell density and specific growth rate in high nitrate group than nitrate absence group at 5 mg NO₂^?‐N L^?1. At 10 and 15 mg NO₂^?‐N L^?1, P_m^chla, R_d ^chla, α^chl, maximum cell densities and specific growth rates in the high nitrate group became higher than those of the nitrate absence group, while a lower intracellular nitrite in the high nitrate group than nitrate absence group was observed. In addition, the intracellular nitrite and the growth of M. aeruginosa in the high nitrate group did not change from 5 to 10 mg NO₂^?‐N L^?1. In the nitrite uptake experiment, with nitrite concentration increasing from 5 to 15 mg NO₂^?‐N L^?1, maximum nitrite uptake rate of alga increased, and half‐saturation constant of alga decreased. These results indicate that external nitrite inhibited algal growth through stimulating intracellular nitrite rise, which resulted from overexpression of nitrite transporter.     

Quantification of denitrification by strain T1 during anaerobic degradation of toluene

Summary Strain T1 is a denitrifying bacterium that is capable of toluene degradation under anaerobic conditions. During anaerobic growth on toluene, the specific growth rate of strain T1 was 0.14 h^–1. Nitrite accumulated in the medium stoichiometrically with the depletion of nitrate. When nitrate was nearly depleted from the medium nitrite reduction and dinitrogen formation began. A non-kinetic model was formulated that was based on a hypothesis of non-simultaneous nitrate and nitrite reduction, independent of the concentrations of nitrate and nitrite. The model was verified experimentally over a wide range of conditions that included nitrate and nitrite limitation, toluene limitation, and various ratios of nitrate to nitrite. The model and its experimental verification demonstrated that strain T1 reduces nitrate and nitrite non-simultaneously, even if nitrite is initially present in the medium in addition to nitrate. Offprint requests to: L. Y. Young     

Effects of growth and measurement light intensities on temperature dependence of CO2 assimilation rate in tobacco leaves

Effects of growth light intensity on the temperature dependence of CO₂ assimilation rate were studied in tobacco (Nicotiana tabacum) because growth light intensity alters nitrogen allocation between photosynthetic components. Leaf nitrogen, ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) contents increased with increasing growth light intensity, but the cyt f/Rubisco ratio was unaltered. Mesophyll conductance to CO₂ diffusion (g_m) measured with carbon isotope discrimination increased with growth light intensity but not with measuring light intensity. The responses of CO₂ assimilation rate to chloroplast CO₂ concentration (C_c) at different light intensities and temperatures were used to estimate the maximum carboxylation rate of Rubisco (V_cmax) and the chloroplast electron transport rate (J). Maximum electron transport rates were linearly related to cyt f content at any given temperature (e.g. 115 and 179 µmol electrons mol^?1 cyt f s^?1 at 25 and 40 °C, respectively). The chloroplast CO₂ concentration (C_trans) at which the transition from RuBP carboxylation to RuBP regeneration limitation occurred increased with leaf temperature and was independent of growth light intensity, consistent with the constant ratio of cyt f/Rubisco. In tobacco, CO₂ assimilation rate at 380 µmol mol^?1 CO₂ concentration and high light was limited by RuBP carboxylation above 32 °C and by RuBP regeneration below 32 °C.     

Effect of light and darkness on nitrate assimilation by Rhodopseudomonas capsulata E1F1

The photosynthetic nonsulfur purple bacterium Rhodopseudomonas capsulata strain E1F1 assimilated nitrate or nitrite only in illuminated cultures under anaerobic conditions. The bacterial cells grew aerobically in the dark only when ammonia or other forms of reduced nitrogen were present in the medium. However, nitrate reductase was detected either in light-anaerobic or in dark-aerobic conditions upon addition of nitrate to the media. Changes from light-anaerobic to dark-aerobic conditions and vice versa markedly influenced growth, nitrate uptake and the nitrate reductase levels. Growth on nitrate in the light and nitrate reductase activity were dependent on the presence of molybdenum in the medium whereas the addition of tungstate inhibited both growth and enzyme activity.     

Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol. The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O₂ pressure of 20 kPa. The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O₂ pressure. Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium. Crude extracts from R. capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate. A constitutive catechol 1,2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R. capsulatus. Further degradation of 4-nitrocatechol included both nitrite- and CO₂-releasing steps since: (1) a strain of R. capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R. capsulatus growing microaerobically produced low amounts of ¹⁴CO₂ from radiolabeled p-nitrophenol. The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled ¹⁴C-p-nitrophenol. From these results we concluded that the xenobiotic is used as a carbon source by R. capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source. Received: 30 December 1996 / Accepted: 3 September 1997     

Heterotrophic nitrogen removal by <Emphasis Type="Italic">Providencia rettgeri</Emphasis> strain YL

Providencia rettgeri strain YL was found to be efficient in heterotrophic nitrogen removal under aerobic conditions. Maximum removal of NH₄ ⁺–N occurred under the conditions of pH 7 and supplemented with glucose as the carbon source. Inorganic ions such as Mg²⁺, Mn²⁺, and Zn²⁺ largely influenced the growth and nitrogen removal efficiency. A quantitative detection of nitrogen gas by gas chromatography was conducted to evaluate the nitrogen removal by strain YL. From the nitrogen balance during heterotrophic growth with 180 mg/l of NH₄ ⁺–N, 44.5% of NH₄ ⁺–N was in the form of N₂ and 49.7% was found in biomass, with only a trace amount of either nitrite or nitrate. The utilization of nitrite and nitrate during the ammonium removal process demonstrated that the nitrogen removal pathway by strain YL was heterotrophic nitrification-aerobic denitrification. A further enzyme assay of nitrate reductase and nitrite reductase activity under the aerobic condition confirmed this nitrogen removal pathway.     

Light acclimation in leaves of the juvenile and adult life phases of ivy (Hedera helix)

Hoflacher, H. and Bauer, H. 1982. Light acclimation in leaves of the juvenile and adult life phases of ivy (Hedera helix). – Physiol. Plant. 56: 177–182. Light acclimation was investigated during the juvenile and adult life phases of the whole-plant-development in Hedera helix L. For this purpose, cuttings of the juvenile and adult parts of one single parent plant were grown under low-light (PAR 30–50 μmol photons m^?2 s^?1) and high-light (PAR 300–500 μmol m^?2 s^?1) conditions: CO₂ exchange, chloroplast functions, and specific anatomy of fully developed leaves differentiated under these conditions were determined. In juvenile plants the leaves formed under low and high light had light-saturated rates of net photosynthesis of 6.5 and 11.1 mg CO₂ (dm leaf area)^?2 h^?1, respectively. In adult plants the rates were 9.4 and 22.2 mg dm^?2 h^?1, indicating a more pronounced capacity for acclimation to strong light in the adult life phase. Higher photosynthetic capacities were accompanied by higher conductances for the CO₂ transfer through the stomata, leading to almost the same CO₂ concentration in the intercellular spaces. Thus, stomatal conductances were not primarily responsible for the different photo-synthetic capacities. The higher rates in adult and high-light grown leaves were mainly the result of formation of thicker leaves with more chloroplasts per unit leaf area. Expressed per chloroplast, the photosynthetic capacity, the Hill reaction, and the activity of ribulose bisphosphate carboxylase were almost identical in plants grown in low-light and high-light. Measurements of photosynthetic capacity and thickness of leaves of Hedera sampled from field habitats with contrasting light regimes confirm the results of growth chamber studies. It is, therefore, concluded that both life phases of Hedera are capable of acclimating to strong light, but that during the juvenile phase this capacity is not fully developed.     

Factors involved in the coordinate appearance of nitrite reductase and glutamine synthetase in the mustard (Sinapis alba L.) seedling

The extent to which the appearances of nitrite reductase (NIR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) are coordinated was studied in mustard (Sinapis alba L.) seedlings. It was established by immunotitration that the increased activities of NIR and GS in the presence of light and nitrate can be attributed to the de-novo synthesis of enzyme protein. The bulk of the NIR and GS was found in the developing cotyledons. In the absence of nitrate in the growth medium there was no coordinate appearance of NIR and GS. While light strongly stimulated the appearance of GS, the level of NIR was hardly affected and remained low. On the other hand, in the presence of nitrate in the medium the appearances of NIR and GS were strictly coordinated, the GS level being considerably above that of NIR. It is argued that phytochrome-controlled synthesis of GS in the absence of nitrate is part of the mechanism to reassimilate ammonium liberated during proteolysis of storage protein and metabolism of the resulting amino acids, whereas the strictly coordinated synthesis in the presence of light and nitrate indicates the dominance of nitrate assimilation under these circumstances. The fact that the level of GS was always considerably above that of NIR appears to be a safety measure to prevent ammonium accumulation.Abbreviations FR standardized far-red light (3.5 W·m^–2), to drive the high-irradiance reaction of phytochrome - GS glutamine synthetase, EC 6.3.1.2 - NIR nitrite reductase, EC 1.7.7.1 This work was supported by Heidelberger Akademie der Wissenschaften (Forschungsstelle Nitratassimilation).