Regulation by ammonium of nitrate and nitrite assimilation in Chlamydomonas reinhardtii

The inhibitor of mRNA synthesis, 6-methylpurine, inhibited nitrate reductase derepression in either ammonium-grown or methylammonium-treated wild-type cells of Chlamydomonas reinhardtii, but not in nitrogen-starved cells. In contrast, 6-methylpurine did not inhibit nitrate reductase synthesis in the methylammonium-resistant mutant 2170 (ma-1) either grown on ammonium, treated with methylammonium or nitrogen starved, but did inhibit the continuous synthesis of nitrate reductase, which required the presence of nitrate in the media. In both wild-type and mutant 2170 grown on ammonium and transferred to nitrate media, cycloheximide immediately prevented nitrate reductase derepression when added either at the beginning or at different times of induction treatment. Unlike wild-type cells, mutant 2170 was able to take up either nitrate or nitrite simultaneously with ammonium in whose presence nitrate and nitrite reductases were synthesized. However, synthesis of nitrate reductase was progressively inhibited in the mutant cells when the intracellular ammonium levels were raised as a result of an increase in either the external pH or the extracellular ammonium concentrations. The results rule out the existence of maturase-like proteins in Chlamydomonas and indicate that ammonium has a double effect on the regulation of nitrate reductase synthesis: (a) it prevents nitrate reductase mRNA production; and (b) it controls negatively the expression of this mRNA.     

Response of conifer seedlings to nitrate and ammonium sources of nitrogen

Summary Differences in growth responses of Douglas fir, western hemlock, Sitka spruce, and white spruce to nitrate and ammonium N sources were examined in sand culture and artificial soil culture. Effects of the two forms of N on growth, needle area, and N uptake of three Douglas fir halb-sib progenies were examined in a second sand culture. Response of Douglas fir to the two forms of N was followed over two years in nursery soil of different pH levels. In sand culture 1 mean seedling dry weight of all species, except hemlock, was greatest when ammonium N and nitrate N were provided in equal amounts. In all species, except Sitka spruce, ammonium alone resulted in greater growth than nitrate alone. Use of ammonium N resulted in greater growth of all species, than was obtained with nitrate N, at pH values in the region 5.4 and 7.5 in artificial soil culture. Only Douglas fir showed substantial differences due to N source below pH 5. Growth of all species was greater at pH 5.4 than at 7.5 in each N source treatment. Growth of Douglas fir seedlings was greatest with ammonium N and least with nitrate N in sand culture 2. Supply of nitrate and ammonium in equal proportions resulted in intermediate growth. Leaf area/plant weight ratio was unaffected by N source. Analysis of nutrient solutions showed appreciable nitrification of ammonium N during the 7 days between solution changes. In the three greenhouse experiments, with little exception, increase in proportion of ammonium in N supply resulted in increase of seedling tissue N concentration. This effect was more pronounced in roots than shoots. Total N uptake by ammonium fed seedlings was about double the N uptake of nitrate fed seedlings in sand culture 2. Nursery grown Douglas fir seedlings showed greater growth response to ammonium sulphate than to calcium nitrate, and this appeared due entirely to form of N supply in the first year. A similar response in the second year was partly due to greater soil acidification by ammonium sulphate. Compared with calcium nitrate, ammonium sulphate increased N concentration of one-year old shoots, but this difference was not detected by foliar analysis of two-year old seedlings.     

Spontaneous mutations in the ammonium transport gene AMT4 of Chlamydomonas reinhardtii

Evidence in several microorganisms indicates that Amt proteins are gas channels for NH(3) and CH(3)NH(2), and this has been confirmed structurally. Chlamydomonas reinhardtii has at least four AMT genes, the most reported for a microorganism. Under nitrogen-limiting conditions all AMT genes are transcribed and Chlamydomonas is sensitive to methylammonium toxicity. All 16 spontaneous methylammonium-resistant mutants that we analyzed had defects in accumulation of [(14)C]methylammonium. Genetic crosses indicated that 12 had lesions in a single locus, whereas two each had lesions in other loci. Lesions in different loci were correlated with different degrees of defect in [(14)C]methylammonium uptake. One mutant in the largest class had an insert in the AMT4 gene, and the insert cosegregated with methylammonium resistance in genetic crosses. The other 11 strains in this class also had amt4 lesions, which we characterized at the molecular level. Properties of the amt4 mutants were clearly different from those of rh1 RNAi lines. They indicated that the physiological substrates for Amt and Rh proteins, the only two members of their protein superfamily, are NH(3) and CO(2), respectively.     

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.     

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.     

Nitrate reductase activity and growth response of forest species to ammonium and nitrate sources of nitrogen

Summary Three tree species,Eucalyptus regnans (F. Muell.),E. obliqua (L'Herit.),Pinus radiata (D. Don) were grown in sand culture with different proportions of nitrate and ammonium. Nitrate Reductase Activity (NRA) was induced in root tissue of all species and in leaf tissue of the eucalypts. An increasing proportion of nitrate resulted in increasing NRA in all species and hence NRA alone is no indication of N-preference. The highest NRA was found withE. regnans, a result which has also been obtained in the mature forest. The growth ofE. regnans was least with NH₄ ⁺ alone, whereas that ofE. obliqua was least with NO₃ ^– alone. The soils of matureE. regnans forest have a high potential for nitrification while those ofE. obliqua forest show little nitrification. Thus the preference for particular N sources shown by seedlings in culture is supported by related properties of mature forests. It is postulated however, that the inducibility of a high level of RNA in seedlings is more likely a result of a preference for NO₃ ^– than a cause.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

The LPB1 gene is important for acclimation of Chlamydomonas reinhardtii to phosphorus and sulfur deprivation

Organisms exhibit a diverse set of responses when exposed to low-phosphate conditions. Some of these responses are specific for phosphorus limitation, including responses that enable cells to efficiently scavenge phosphate from internal and external stores via the production of high-affinity phosphate transporters and the synthesis of intracellular and extracellular phosphatases. Other responses are general and occur under a number of different environmental stresses, helping coordinate cellular metabolism and cell division with the growth potential of the cell. In this article, we describe the isolation and characterization of a mutant of Chlamydomonas reinhardtii, low-phosphate bleaching (lpb1), which dies more rapidly than wild-type cells during phosphorus limitation. The responses of this mutant to nitrogen limitation appear normal, although the strain is also somewhat more sensitive than wild-type cells to sulfur deprivation. Interestingly, depriving the cells of both nutrients simultaneously allows for sustained survival that is similar to that observed with wild-type cells. Furthermore, upon phosphorus deprivation, the lpb1 mutant, like wild-type cells, exhibits increased levels of mRNA encoding the PHOX alkaline phosphatase, the PTB2 phosphate transporter, and the regulatory element PSR1. The mutant strain is also able to synthesize the extracellular alkaline phosphatase activity upon phosphorus deprivation and the arylsulfatase upon sulfur deprivation, suggesting that the specific responses to phosphorus and sulfur deprivation are normal. The LPB1 gene was tagged by insertion of the ARG7 gene, which facilitated its isolation and characterization. This gene encodes a protein with strong similarity to expressed proteins in Arabidopsis (Arabidopsis thaliana) and predicted proteins in Oryza sativa and Parachlamydia. A domain in the protein contains some similarity to the superfamily of nucleotide-diphospho-sugar transferases, and it is likely to be localized to the chloroplast or mitochondrion based on programs that predict subcellular localization. While the precise catalytic role and physiological function of the putative protein is not known, it may function in some aspect of polysaccharide metabolism and/or influence phosphorus metabolism (either structural or regulatory) in a way that is critical for allowing the cells to acclimate to nutrient limitation conditions.     

The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii

The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynthesis in the dark in Chlamydomonas reinhardtii, was precisely replaced by either the Klebsiella pneumoniae nifH (encoding the structural component of nitrogenase Fe protein) or the Escherichia coli uidA reporter gene encoding beta-glucuronidase. Homoplasmic nifH or uidA transformants were identified by Southern blots after selection on minimal medium plates for several generations. All the uidA transformants had the "yellow-in-the-dark" phenotype characteristic of chlL mutants, whereas homoplasmic nifH transformants exhibited a partial "green-in-the-dark" phenotype. NifH protein was detected in the nifH transformants but not in the wild-type strain by Western blotting. Fluorescence emission measurements also showed the existence of chlorophyll in the dark-grown nifH transformants, but not in the dark-grown uidA transformants. The nifH transplastomic form of C. reinhardtii that lacks the chlL gene can still produce chlorophyll in the dark, suggesting that the nifH product can at least partially substitute for the function of the putative "chlorophyll iron protein" encoded by chlL. Thus, introducing nitrogen fixation gene directly into a chloroplast genome is likely to be feasible and providing a possible way of engineering chloroplasts with functional nitrogenase. Notably, to introduce foreign genes without also introducing selective marker genes, a novel two-step chloroplast transformation strategy has been developed.     

In vivo localization of centrin in the green alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii has been used as a model system to study flagellar assembly, centriole assembly, and cell cycle events. These processes are dynamic. Therefore, protein targeting and protein-protein interactions should be evaluated in vivo. To be able to study dynamic processes in C. reinhardtii in vivo, we have explored the use of the green fluorescent protein (GFP). A construct containing a fusion of centrin and GFP was incorporated into the genome as a single copy. The selected clone shows expression in 25-50% of the cells. Centrin-GFP was targeted in vivo to the nuclear basal body connectors and the distal connecting fibers. At the electron microscopic level, it was also localized to the flagellar transitional regions. EM data of transformants indicate that there are some abnormalities in the centrin-containing structures. The transitional region consists of only the transverse septum or has lesions in the H-piece. The distal connecting fibers are thinner and their characteristic crossbands seem to be incomplete. Deflagellation is not affected since more than 95% of the cells deflagellate. Also basal body segregation is not affected since cells with an abnormal flagellar number were not detected. Functional studies of the centrin-GFP fusion show the characteristic calcium-induced mobility shift in SDS-PAGE. Immunofluorescence revealed that during cell division, centrin-GFP remains associated with the basal bodies. In vivo localization of the fusion protein during cell division shows that in metaphase centrin-GFP appears as two opposing spots located close to the spindle poles. The distance between the spots increases as the cells progress through anaphase and then decreases during telophase. GFP is a useful tool to study dynamic processes in the cytoskeleton of C. reinhardtii.     

The effect of ammonium and nitrate nitrogen sources on copper uptake and amino acid status of cereals

Summary The effects of different nitrogen sources (NH₄, NO₃, and NH₄ NO₃) on the uptake of copper by wheat and barley growing in solution culture were compared in three experiments. Both the copper concentration and weight gain of shoots and roots were found to decrease in the order NO₃>NH₄ NO₃>NH₄ irrespective of the solution copper concentration. Ammonium nitrogen was also found to decrease the copper concentration of wheat grown on a copper deficient soil compared with a nitrate source of nitrogen. Increasing concentrations of ammonium ions in solution culture caused ammonium toxicity and reduced both plant copper concentrations and vegetative yield. Biochemical investigations using paper chromatography revealed that the amino acid asparagine was the major detoxification product of ammonia in wheat. Copper deficient plants were found to have elevated levels of amino acids compared with controls, irrespective of the nitrogen source.