Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.     

Effect of growth conditions on accumulation of internal nitrate,ammonium, amino acids,and protein in three marine diatoms

The capacity of marine phytoplankton to change their cellular content of nitrate, ammonium, amino acids, and protein in response to different growth conditions was systematically investigated. Cellular concentrations of these compounds were measured in N-starved, N-deficient, and N-sufficient Skeletonema costatum (Grev.) Cleve and in N-deficient Chaetoceros debilis Cleve and Thalassiosira gravida Cleve, both before and after the addition of a pulse of nitrogen.N-sufficient Skeletonema costatum contains high concentrations of protein, large persistent pools of amino acids, and, if it is growing on nitrate, sizeable amounts of nitrate. As it becomes N-starved, the total cellular nitrogen decreases, the internal nitrate and amino acids become entirely depleted, and the protein content is drastically reduced. After nitrogen additions to N-deficient and N-starved cultures, transient pools of unassimilated nitrogen form which can account for a large fraction of newly taken up nitrogen. The size and kind of pool which accumulates is determined by the preconditioning of the cells, the nitrogen compound which is added, and the species identity. The pools which form in S. costatum indicate that nitrate reduction is the slowest step in nitrogen assimilation, the synthesis of protein from amino acids is the next slowest, and the incorporation of ammonium into amino acid is the fastest. However, the rate limiting steps may vary between diatom species.For the first time, measurements of the variation in cellular nitrogen compounds over a wide range of environmental conditions reveal the ability of some phytoplankton to buffer the effects of a changing, and sometimes growth-limiting, nitrogen supply. They accomplish this by utilizing stored internal nitrogen for growth when the external supply is low and by quickly storing unassimilated nitrogen when the external supply is suddenly increased beyond their ability to immediately assimilate it. The accumulation of large pools of unassimilated nitrogen compounds can explain the often observed difference between nitrogen uptake rates and growth rates.     

Resistance to acetohydroxamate acquired by slow adaptive increases in urease in cultured tobacco cells

Urease activity of tobacco XD cells (1U cells) had undergone a 4-fold increase (4U cells) during a year of growth on urea (Skokut and Filner 1980 Plant Phvsiol 65: 995-1003). A clone of 4U cells gave rise to 12U cells during another year of growth on urea. The doubling time of 12U cells on urea is 2.2 days, compared to about 4 days for 1U cells, while 1U and 12U cells double in 2 days on nitrate. Acetohydroxamic acid (AHA), a specific inhibitor/reversible inactivator of jack bean urease, affects tobacco cell urease similarly. Fifty per cent inhibition of growth by AHA occurred at 20 micromolar in 1U cells growing on urea and at 165 micromolar in 12U cells growing on urea, but at 600 micromolar for either 1U or 12U cells growing on nitrate. When 12U cells were grown on urea with 100 micromolar AHA, extractable urease activity decreased 80% within 2.5 hours and remained at this level for 2 weeks; the doubling time increased to 3.7 days, and intracellular urea rose 2-fold, compared to 12U cells grown on urea without AHA. Urease of 12U cells inactivated by AHA in vivo could be reactivated to its pre-AHA level by incubation at 30 C after extraction and separation from free AHA. AHA inhibited incorporation of ¹⁵N from [¹⁵N]urea into Kjeldahl nitrogen in the cells, in spite of the increased intracellular urea. These results indicate that AHA acts primarily by inhibiting urease action, rather than by inhibition of formation of urease protein or of uptake of urea. Because 12U cells are 8 times more tolerant of AHA than 1U cells, it is likely that growth on urea in the presence of AHA should select strongly for cells with high urease.     

Regulation of phenylalanine oxidase synthesis in Proteus mirabilis.

Cells of Proteus mirabilis could oxidize L-phenylalanine to phenylpyruvate only when grown in the presence of a number of amino acids, particularly, L-alanine, L-asparagine, L-glutamate, and L-glutamine. Production of phenylalanine oxidase was slowly lost upon growth in a minimal medium containing ammonium ions as a nitrogen source but was reversed by the addition of casein hydrolysate. Oxidase activity as well as a phenylalanine-dichlorophenolindophenol (DCIP) reductase activity increased in P. mirabilis only during cell multiplication. Both rifampin and nalidixic acid caused inhibition of oxidase synthesis. A phenylalanine-active transport was found to be operative when bacteria were grown in the absence of added amino acids. After anaerobic growth, cells of P. mirabilis had lost their ability to carry the phenylalanine oxidase reaction when assayed in the presence of air, and nitrate could not be used as an electron acceptor for the oxidation of phenylalanine. However, some phenylalanine-dichlorophenolindophenol reductase activity was still present in anaerobic bacteria at the early stage of cell multiplication.     

The effects of amino acids and ammonium on the growth of plant cells in suspension culture

A suspension culture of soybean (Glycine max L.) was grown on a defined medium in which the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The cells did not grow on nitrate unless the medium was supplemented with ammonium or glutamine. The l- and d-isomers of 12 amino acids tested singly could not replace ammonium. Most amino acids (4 mM) inhibited growth when the cells were cultured on nitrate and ammonium. Cells from five other plants (Reseda luteoli L.; Triticum monococcum L.; flax, Linum usitatissimum L.; horseradish, Amoracia lapathifolia Gilib; Haplopappus gracilis L.) grew on the defined medium with nitrate (25 mM) as the sole nitrogen source. Higher cell yields were obtained when ammonium (2 mM) or glutamine also was present. Supplementing the defined medium with high concentrations of ammonium (20 mM) inhibited growth of soybean, Haplopappus, and wheat cells. Addition of citrate (5 mM) relieved the inhibitory effects of ammonium in soybean and wheat cells but not in the Haplopappus cells.     

Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.     

Decreased Rubisco activity leads to dramatic changes of nitrate metabolism,amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants

Tobacco transformants that express an antisense RBCS construct were used to investigate the consequences of a lesion in photosynthetic carbon metabolism for nitrogen metabolism and secondary metabolism. The results show that an inhibition of photosynthesis and decrease in sugar levels leads to a general inhibition of nitrogen metabolism, and dramatic changes in the levels of secondary metabolites. The response was particularly clear in plants that received excess nitrogen. In these conditions, a decrease of Rubisco activity led to an inhibition of nitrate reductase activity, accumulation of nitrate, a decrease of amino acid levels that was larger than the decrease of sugars, and a large decrease of chlorogenic acid and of nicotine, which are the major carbon- and nitrogen-rich secondary metabolites in tobacco leaves, respectively. Similar changes were seen when nitrogen-replete wild-type tobacco was grown in low light. The inhibition of nitrogen metabolism was partly masked when wild-type plants and antisense RBCS transformants were compared in marginal or in limiting nitrogen, because the lower growth rate of the transformants alleviated the nitrogen deficiency, leading to an increase of amino acids. In these conditions, chlorogenic acid always decreased but the decrease of nicotine was ameliorated or reversed. When the changes in internal pools are compared across all the genotypes and growth conditions, two conclusions emerge. First, decreased levels of primary metabolites lead to a dramatic decrease in the levels of secondary metabolites. Second, changes of the amino acid : sugar ratio are accompanied by changes of the nicotine:chlorogenic acid ratio.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

The amino acid pool ofHansenula holstii: Characterisation,and changes mediated by environment

Amino acid pools extracted fromHansenula holstii grown in continuous culture with either ammonia or nitrate as sole source of nitrogen, under a variety of substrate limitations, were characterised and quantified. Pools from corresponding cultures were shown to be similar in size and composition, regardless of whether ammonia or nitrate was the nitrogen source. Large changes in pools (both quantitative and qualitative) occurred when cultures were grown under different substrate limitations. Such changes were particularly large in glutamate, glutamine, alanine, lysine and arginine; the possible significance of such environment-mediated changes is discussed.     

Cell Wall Composition of Neurospora crassa Under Conditions of Copper Toxicity

The mycelia of Neurospora crassa grown in the presence of high concentrations of copper were blue in color, but only on a medium containing inorganic nitrate and phosphate as the nitrogen and phosphate sources, respectively. The cell wall isolate of the blue mycelia contained large amounts (12%) of copper and higher amounts of chitosan, phosphate, and amino groups, with a 42% decrease in the chitin content. Although all the glucosamine of the cell wall of control cultures could be released within 6 h of hydrolysis with acid, that of the blue mycelium required prolonged hydrolysis for 24 h. On removal of copper, the cell wall of the blue mycelium could quantitatively bind again to copper as well as to zinc. Although zinc binding was fivefold greater, copper alone was preferentially bound from a mixture of the two metal ions. Supplementation of iron along with copper in the culture medium resulted in the disappearance of the blue color of the mycelium and restoration of normal growth and composition of the cell wall, probably by limiting the uptake of copper from the medium. The possibility of the cell wall being a specific site of lesion in copper toxicity in the mold is discussed.     

Changes in fatty acids,amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grownIsochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.     

Glycolate metabolism is under nitrogen control in chlorella

The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR).

During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate.

     

Identification,Source, and Metabolism of N-Ethylglutamate in Escherichia coli

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-²H₄]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.During work on developing methods for the analysis of the amino acids generated by recombinant archaeal mutases, I developed procedures for the recovery and analysis of the free amino acids present in cell extracts of Escherichia coli. When these methods were applied to analysis of E. coli grown on LB broth, I always found a large amount of an unknown amino acid. Here I report on the identification of this amino acid as N-ethylglutamate (NEG). NEG has never been reported as a natural product. I demonstrate that NEG is readily taken up by E. coli and can serve as the sole source of nitrogen when the cells are grown on M9 glucose medium.     

Regulation of exocellular protease in Neurospora crassa: induction and repression under conditions of nitrogen starvation.

Exponential-phase cells of Neurospora crassa require the continued presence of a protein inducer and nitrogen starvation to induce exocellular protease under conditions where protein is the sole nitrogen source. The nature of the protein inducer appears relatively unimportant, since both soluble proteins (e.g., myoglobin) and insoluble proteins (e.g., corn zein) will effect induction. Nonstarved cells of N. crassa appear to have small nitrogen pools, since nitrogen starvation of exponential cells prior to transfer into a medium where protein is the sole nitrogen source effects starvation-time-dependent decreases in protease biosynthesis. Ammonium ion represses protease synthesis, with apparent specificity at low concentrations. The amino acids arginine, tryptophan, and threonine effect repression of protease biosynthesis under conditions of nitrogen starvation. Under conditions of sulfur starvation, the amino acids cysteine, methionine, and cystine repress protease biosynthesis. In carbon-starved cells, all of the above amino acids, plus histidine, isoleucine, leucine, lysine, phenylalanine, and valine, effect repression. Examination of amino acid pools formed when cells are grown on protein as the sole nitrogen source demonstrated that the amino acids which repress protease biosynthesis under conditions where protein is the sole carbon source accumulate in significant amounts during the course of protease induction, with kinetics consonant with the induction process.     

The development of nitrate reductase in Chlorella and its repression by ammonium

Summary Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.     

COMPARISON OF GROWTH AND SINKING RATES OF NON-COCCOLITH- AND COCCOLITH-FORMING STRAINS OF EMILIANIA HUXLEYI(PRYMNESIOPHYCEAE) GROWN UNDER DIFFERENT IRRADIANCES AND NITROGEN SOURCES1

We examined the effect of the presence or absence of coccoliths on the growth and sinking rates of an oceanic isolate of the coccolithophore Emiliania huxleyi (Lohmann) Hay et Mohler isolated from the northeastern subarctic Pacific. Coccolith-forming and non-coccolith-forming (i.e. naked, nonmotile) strains were obtained from the same isolate and grown under both saturating and limiting irradiance levels with either nitrate or ammonium as the primary nitrogen source. Sinking rate, growth rate, and cell volume (excluding coccoliths) were measured for each culture. Under saturating irradiance, coccolith-forming cells grew significantly slower than naked cells, had significantly higher sinking rates, and had larger cell volumes than naked cells. Under limiting irradiance levels, growth rates of the two strains were identical, sinking rates were higher for coccolith-forming cells in stationary-phase cultures only, and cell volumes remained greater for coccolith-forming cells. The sinking rates achieved for this ubiquitous coccolithophore ranged from <0.1 to 0.5 m · d^?1. Sinking rates were not statistically different between coccolith-forming and naked strains of E. huxleyi under limiting irradiance conditions for log-phase cultures, but sinking rates were greater for coccolith-forming cells under some of the other conditions tested. However, the average sinking rate was never more than twice as great as for coccolith-forming cells, with the exception of nitrate-grown, senescent cells under limiting irradiance (3.4 times greater). Cell volumes (excluding coccoliths) were consistently ca. 1.5 times greater for coccolith-forming cells than for naked cells. Nitrogen source had an effect on growth rate and cell volume, with ammonium-grown cultures growing faster and having larger cell volumes than nitrate-grown cultures of both strains. However, despite the difference in growth rate and cell volume, nitrogen source had little if any effect on sinking rate.     

Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH⁺₄ or NO-₃.     

Influence of abscisic acid on nitrogen partitioning, sucrose metabolism and nitrate reductase activity of chicory suspension cells

Batch suspension cultures of chicory cells (Cichorium intybusL. var. Witloof) possess a NADH-specific nitrate reductase activitythat peaks on day 3 of a 10 d growth cycle. When both nitrateand ammonium are used as nitrogen sources, chicory cells absorbnitrate irst. Ammonium uptake becomes predominant at day 3,even though NO₃^– was still present in the medium. Althoughabscisic acid impairs growth as well as ¹⁵NO₃^– uptakeand reduction, it promotes nitrate reductase activity as measuredboth in vivo and in vitro. Specific activity is 50% higher inABA-treated cells than in controls. These conflicting data maybe explained either in erms of nitrate reductase levels or bythe availability of reducing power and energy. Since NRA isgenerally controlled by the availability of the reducing power,the energy status of the cell, the adenylate nucleotide pools,were measured simultaneously with the carbohydrate levels withinthe cell and the growth medium. The energy charge was not modifiedduring the growth cycle, regardless of the rowth conditions.Yet ABA modified the intracellular carbohydrate metabolism andinhibited the acidic invertase, the sucrose synthase and thesucrose phosphate synthase activities. Modified assimilationrates of nitrate in chicory cells grown in the presence of ABA,were probably correlated to modified carbohydrate metabolismpathways leading to increased availability of reducing power,energy and C-skeletons. Key words: Abscisic acid, Cichorium intybus L, nitrate reductase, reductase, invertase, sucrose synthase, sucrose phosphate synthase     

NITROGEN METABOLISM OF AQUATIC ORGANISMS. II. THE ASSIMILATION OF NITRATE,NITRITE, AND AMMONIA BY BIDDULPHIA AURITA1

Biddulphia aurita, a centric diatom, can grow on either nitrate, nitrite, or ammonia as its sole nitrogen, source. Cells remove ammonium nitrogen from the medium 2.3–2.4 times faster than either nitrate or nitrite nitrogen and, when grown for 24 hr in the ammonium medium, contain higher levels of non-protein nitrogen than cells grown in the nitrate or nitrite medium for the same period of time. The nitrogenous compounds in the nonprotein nitrogen fraction from cells grown in the nitrate, nitrite, or ammonium medium contain the same level of soluble-free amino nitrogen, combined amino nitrogen, and ammonium nitrogen. The high level of soluble nonprotein nitrogen in the medium of the cells grown in the ammonium medium is due to soluble amide nitrogen which represents 18% of the total soluble nitrogen present in these cells, whereas it represents only 2% in cells from the nitrite medium, and its level is negligible in cells from the nitrate medium. Cells grown in the nitrate medium have both nitrate- and nitrite-reductase activity. Cells grown in the nitrite medium have only nitrite-reductase activity in significant levels, while cells grown in the ammonium medium lack both enzymes.