Ammonium Treatment Research Articles

Molecular cloning, characterization and expression analysis of three aldehyde oxidase genes from Pisum sativum L.

Aldehyde oxidase (AO, EC 1.2.3.1) is a molybdenohydroxylase that is considered to catalyze the last step of abscisic acid (ABA) and indole-3-acetic acid (IAA) synthesis. Three cDNAs encoding aldehyde oxidase proteins in Pisum sativum (cv. Little Marvel) were obtained based on RT-PCR (reverse transcriptase-polymerase chain reaction) strategy. The cloned genes, designated as PsAO1, PsAO2 and PsAO3, are 4630, 4347, 4600 bp in length, respectively, and show high sequence identity to each other and to aldehyde oxidases from other plant species. The deduced PsAO1, PsAO2, and PsAO3 proteins are 1373, 1367, 1367 amino acids in length, respectively, and contain consensus sequences for two iron-sulfur centers, a FAD binding domain, and a molybdenum cofactor (Moco) binding domain. PsAO1 and PsAO2 were mainly expressed in leaves of seedlings and young leaves of adult plants, while the highest PsAO3 transcript level was observed in aging leaves and matured seeds. PsAO2 mRNA was not affected by salinity or ammonium treatment, whereas the transcript level of PsAO3 increased significantly under both stress conditions, with the most pronounced changes in aging leaves, fully expanded leaves and roots. The PsAO1 transcript level was enhanced only in the presence of ammonium in the nutrient medium, but not under salinity. Based on the molecular mass of the deduced proteins and on organ-specific gene expression, studied both under control and stress conditions, the contribution of each PsAO cDNA in the formation of the previously described three dimeric pea AO isoforms and the possible involvement of the PsAO3 in abscisic acid (ABA) synthesis is discussed.

Evaluation of nitrate and ammonium as sources of NO and N 2O emissions from black earth soils (Haplic Chernozem) based on 15N field experiments

Nitrous oxide (N 2O) and nitric oxide (NO) released from soil is a concern since it can act as a potential atmospheric pollutant and it represents a loss of N from the soil. These gases are present in the atmosphere in trace amounts and are important to atmospheric chemistry and earth's radiative balance. Nitric oxide (NO) does not directly contribute to the greenhouse effect, but it contributes to climate forcing through its role in photochemistry of hydroxyl radicals and ozone and plays a key role in air quality issues. Nitrification and denitrification have been identified as major controlling microbial processes in soils responsible for the formation of NO and N 2O. To elucidate the contribution of both processes to the release of NO and N 2O from loess-black earth soils under field conditions—i.e. to evaluate nitrate and ammonium as sources of NO and N 2O emission—two field experiments with either [ 15N] nitrate (NO 3 −) or [ 15N] ammonium (NH 4 +) labelling have been conducted at two sites differing in soil organic matter content (high and normal SOM). [ 15N] nitrate treatments revealed that denitrification of NO 3 − represents the main pathway of soil N 2O release. On average 76% and 54% of N 2O was emitted during denitrification from soils with high and normal SOM content, respectively. Contrarily, denitrification contributed on average only 17% and 12% of released NO from soil with high and normal SOM content, respectively. The [ 15N]ammonium treatments revealed that nitrification of NH 4 + is the major process responsible for soil NO emission. SOM content of the loess-black earth soil significantly influenced NO and N 2O emission. The soil with the higher SOM content showed lower NO emission but drastically increased N 2O emission after nitrate fertilisation. In particular the soil with high SOM content exhibited a high sorption capacity for ammonium ions which led to unexpected results after fertilisation with [ 15N]ammonium. To explain this results a revised concept containing three different interacting soil ammonium pools have been hypothesised.

Membrane Sequestration of PII Proteins and Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus

Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the R. capsulatus membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a glnK mutant, more GlnB was found to be membrane associated under these conditions. In a glnB mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in R. capsulatus.

Activation of crystalline cellulose to cellulose IIII results in efficient hydrolysis by cellobiohydrolase

The crystalline polymorphic form of cellulose (cellulose I(alpha)-rich) of the green alga, Cladophora, was converted into cellulose III(I) and I(beta) by supercritical ammonium and hydrothermal treatments, respectively, and the hydrolytic rate and the adsorption of Trichoderma viride cellobiohydrolase I (Cel7A) on these products were evaluated by a novel analysis based on the surface density of the enzyme. Cellobiose production from cellulose III(I) was more than 5 times higher than that from cellulose I. However, the amount of enzyme adsorbed on cellulose III(I) was less than twice that on cellulose I, and the specific activity of the adsorbed enzyme for cellulose III(I) was more than 3 times higher than that for cellulose I. When cellulose III(I) was converted into cellulose I(beta) by hydrothermal treatment, cellobiose production was dramatically decreased, although no significant change was observed in enzyme adsorption. This clearly indicates that the enhanced hydrolysis of cellulose III(I) is related to the structure of the crystalline polymorph. Thus, supercritical ammonium treatment activates crystalline cellulose for hydrolysis by cellobiohydrolase.

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and α-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the α-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the α-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U- 13C]glucose (2.5 mM) and isoleucine (1 mM) or [U- 13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U- 13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U- 13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U- 13C]glucose. Labeling in glutamate and aspartate from [U- 13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U- 13C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.

Differential Expression of Rice Genes Under Different Nitrogen Forms and Their Relationship with Sulfur Metabolism

Microarray analysis was initially performed to screen for differentially expressed genes between nitrate-and ammonium-fed rice (Oryza sativa L.) leaves. In total, 198 genes were shown to have a unique expression response to each treatment and most of the genes for which function is known were involved in signal transduction, plant stress resistance, transcriptional regulation, and basic metabolism. Northern blotting analysis confirmed that expression of the MT and PCS genes was highly upregulated in ammonium-fed leaves compared with expression in nitrate-fed leaves and it was further revealed that ammonium-fed leaves accumulated more cysteine and glutathione. The upregulated expressions of the MT and PCS genes and the higher levels of cysteine and glutathione in ammonium-fed leaves indicate that ammonium may be able to accelerate sulfur assimilation metabolism in rice leaves. Unexpectedly, Northern blotting analysis showed that the expression of the two key enzymes in the sulfur assimilation pathway, namely adenosine 5′-phosphosulfate reductase and O-acetylserine(thiol)lyase, was not upregulated by ammonium treatment. It was found that the total content of free amino acids was much higher in ammonium-fed leaves compared with nitrate-fed leaves, mainly resulting from an increase in several amino acids such as serine, asparagine, glutamine, and arginine. The increased amino acids, in particular serine (as a central substrate for the synthesis of the thiol metabolites), may have promoted sulfur assimilation metabolism under conditions of ammonium nutrition. (Managing editor: Ping He)

Nitrogenous preference of toxigenic Pseudo-nitzschia australis (Bacillariophyceae) from field and laboratory experiments

Field and laboratory experiments were designed to determine the differential growth and toxin response to inorganic and organic nitrogen additions in Pseudo-nitzschia spp. Nitrogen enrichments of 50 μM nitrate (KNO 3), 10 μM ammonium (NH 4Cl), 20 μM urea and a control (no addition) were carried out in separate carboys with seawater collected from the mouth of the San Francisco Bay (Bolinas Bay), an area characterized by high concentrations of macronutrients and iron. All treatments showed significant increases in biomass, with chlorophyll a peaking on days 4–5 for all treatments except urea, which maintained exponential growth through the termination of the experiment. Pseudo-nitzschia australis Frenguelli abundance was 10 3 cells l −1 at the start of the experiment and increased by an order of magnitude by day 2. Particulate domoic acid (pDA) was initially low but detectable (0.15 μg l −1), and increased throughout exponential and stationary phases across all treatments. At the termination of the experiment, the urea treatment produced more than double the amount of pDA (9.39 μg l −1) than that produced by the nitrate treatment (4.26 μg l −1) and triple that of the control and ammonium treatments (1.36 μg l −1 and 2.64 μg l −1, respectively). The mean specific growth rates, calculated from increases in chlorophyll a and from cellular abundance of P. australis, were statistically similar across all treatments. These field results confirmed laboratory experiments conducted with a P. australis strain isolated from Monterey Bay, CA (isolate AU221-a) grown in artificial seawater enriched with 50 μM nitrate, 50 μM ammonium or 25 μM of urea as the sole nitrogen source. The exponential growth rate of P. australis was significantly slower for cells grown on urea ( ca. 0.5 day −1) compared to the cells grown on either nitrate or ammonium ( ca. 0.9 day −1). However the urea-grown cells produced more particulate and dissolved domoic acid (DA) than the ammonium- or nitrate-grown cells. The field and laboratory experiments demonstrate that P. australis is able to grow effectively on urea as the primary source of nitrogen and produced more pDA when grown on urea in both natural assemblages and unialgal cultures. These results suggest that the influence of urea from coastal runoff may prove to be more important in the development or maintenance of toxic blooms than previously thought, and that the source of nitrogen may be a determining factor in the relative toxicity of west coast blooms of P. australis.

Differential effects of nitrate and ammonium supply on nodule initiation, development, and distribution on roots of pea (Pisum sativum)

Low, static concentrations of ammonium have less negative effects on nodulation of pea ( Pisum sativum L.) than nitrate and in some cases may actually stimulate nodulation. Two experiments were carried out to assess the effects of supplying both forms of mineral N, separately and in combination, on nodule initiation, nodule development, nodule distribution between primary and lateral (secondary) roots, tertiary root development, and N2 fixation in pea. Pea plants were grown for up to 24 d after inoculation in hydroponic culture with no mineral N (zero N), NO3– (0.5 mmol·L–1), NH4+ (0.5 mmol·L–1), or NO3– (0.25 mmol·L–1) plus NH4+ (0.25 mmol·L–1). Concentrations of nitrate and ammonium were monitored on a daily basis and held relatively constant by continuous, automatic additions of stock solutions. Pea plants accumulated the most total dry mass (DM) and total N when supplied with the combination of nitrate plus ammonium but had the lowest nodule DM and percentage of nitrogen derived from the atmosphere. Whole-plant nodulation (nodules per plant) and DM-specific nodulation (nodules·g–1 root DM) were 2.3- and 2.4-fold greater, respectively, in pea plants receiving NH4+ at 0.5 mmol·L–1 than in those supplied with NO3– at 0.5 mmol·L–1. The nodulation responses of plants receiving NO3– at 0.25 mmol·L–1 plus NH4+ at 0.25 mmol·L–1 were more similar to those of plants receiving only nitrate than only ammonium, indicating that when both forms of mineral N are available to plants, nitrate has a predominant effect on the nodulation response. Assessment of the stage of development of nodule primordia and nodules during the time course of the experiments indicated that nitrate not only decreased the degree of nodule initiation but also the rate at which those nodules developed. Microscopic observations indicated that the more negative effects of the nitrate treatment on DM-specific nodulation as compared with the ammonium treatment were consistent on both the primary and lateral roots. Quantification of nodulation and tertiary root development on lateral roots indicated that the stimulating effects of ammonium were specific to nodulation; the effects on tertiary root development were different. The study demonstrates for the first time that when both forms of mineral N are available at equal concentrations, the nodulation response in pea is influenced more by nitrate than by ammonium and that the effects of nitrate and ammonium on tertiary root initiation and development are unlike those on nodulation.

Insights into Symbiotic Nitrogen Fixation in Medicago truncatula

In silico analysis of the Medicago truncatula gene index release 8.0 at The Institute for Genomic Research identified approximately 530 tentative consensus sequences (TC) clustered from 2,700 expressed sequence tags (EST) derived solely from Sinorhizobium meliloti-inoculated root and nodule tissues. A great majority (76%) of these TC were derived exclusively from nitrogen-fixing and senescent nodules. A cDNA filter array was constructed using approximately 58% of the in silico-identified TC as well as cDNAs representing selected carbon and nitrogen metabolic pathways. The purpose of the array was to analyze transcript abundance in M. truncatula roots and nodules following inoculation by a wild-type S. meliloti strain, a mutant strain that forms ineffective nodules, an uninoculated root control, and roots following nitrate or ammonium treatments. In all, 81 cDNAs were upregulated in both effective and ineffective nodules, and 78% of these cDNAs represent in silico-identified TC. One group of in silico-identified TC encodes genes with similarity to putative plant disease resistance (R) genes of the nucleotide binding site-leucine-rich repeat type. Expression of R genes was enhanced in effective nodules, and transcripts also were detected in ineffective nodules at 14 days postinoculation (dpi). Homologous R gene sequences also have been identified in the Medicago genome. However, their functional importance in nodules remains to be established. Genes for enzymes involved in organic acid synthesis along with genes involved in nitrogen metabolism were shown to be coexpressed in nitrate-fed roots and effective nodules of M. truncatula.

Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation

A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.

Habitat of Origin and Changes in Water Chemistry Influence Development of Western Chorus Frogs

A variety of biotic and abiotic variables have been shown to affect length of larval period and size of juveniles at metamorphosis in amphibians. However, influence of water quality on phenotypic plasticity of growth and development of tadpoles generally has received less attention. We examined how abiotic factors in the larval environment change over time and how these changes affect the growth and development of larval amphibians. Western Chorus Frogs, Pseudacris triseriata, in tallgrass prairie breed in ephemeral aquatic habitats including intermittent streams and bison wallows. Our objectives were to determine whether abiotic factors in the larval environment of P. triseriata changed predictably as pools dried and to determine whether these changes affected growth and development of tadpoles when the environment was simulated in the laboratory. In our field studies, pH increased gradually in wallows, whereas ammonium increased in streams, as each habitat dried. In the laboratory, we examined the effects of increased levels of pH and ammonium on growth and development of tadpoles collected from both wallows and streams. Tadpoles collected from streams metamorphosed significantly faster in the high ammonium treatment than tadpoles from wallows. In contrast, tadpoles from wallows metamorphosed faster in the high pH treatment than tadpoles collected from streams. Growth rates of tadpoles from streams were not significantly affected by high pH, whereas those from wallows were not significantly affected by high ammonium treatments. We suggest that changes in abiotic factors over the course of the larval period may influence developmental rate and that natal habitat may determine how tadpoles respond to changes in abiotic factors.

Protein phosphatase PphA from Synechocystis sp. PCC 6803: the physiological framework of PII-P dephosphorylation

The phosphorylated signal transduction protein P(II) (P(II)-P) in the cyanobacterium Synechocystis sp. strain PCC 6803 is dephosphorylated by PphA, a protein phosphatase of the 2C family (PP2C). In this study, the physiological conditions of P(II)-P dephosphorylation were investigated with respect to the in vivo specificity of P(II)-P towards PphA and the cellular abundance of PphA in cells growing under different nitrogen regimes. Furthermore, the consequences of impaired P(II)-P dephosphorylation with respect to short-term inhibition of glutamine synthetase (GS) were studied. With a contribution of approximately 15 % of total Mn(2+)-dependent p-nitrophenyl phosphate hydrolysis activity, PphA has only a minor impact on the total PP2C activity in Synechocystis extracts. Nevertheless, residual P(II)-P dephosphorylation in PphA-deficient cells could only be observed after prolonged incubation in the presence of ammonium. The abundance of PphA correlates with the phosphorylation state of P(II) under nitrogen-replete conditions and is specifically enhanced by nitrite. Regulation of pphA expression operates at the post-transcriptional level. In the presence of nitrate/nitrite, PphA is present in molar excess over P(II)-P, enabling the cells to rapidly dephosphorylate P(II)-P in response to changing environmental conditions. A PphA-deficient mutant is not impaired in short-term inhibition of GS activity following ammonium treatment. Down-regulation of GS occurs by induction of gif genes (encoding GS inactivating factors 7 and 17), which is controlled by NtcA-mediated gene repression. Thus, impaired P(II)-P dephosphorylation does not affect ammonium-prompted inactivation of NtcA.

Cadmium uptake by tobacco as affected by liming, N form, and year of cultivation

Tobacco is able to accumulate cadmium and reduction of cadmium content can reduce health hazards to smokers. Soil pH and form of N fertilizers are among the factors affecting Cd uptake by tobacco. This hypothesis was tested in an acid soil in northern Greece by a four year field experiment. The variability of Cd uptake by tobacco was attributed to the variation of soil Cd availability as affected by soil pH. Liming with 3000 kg Ca(OH) 2 ha −1 increased soil pH by 0.8 units and decreased extractable with DTPA soil and leaf Cd by 40% and 35%, respectively. The ammonium fertilizer caused the opposite, but weaker, effects. Liming reduced soil Cd more in the ammonium treatment than in nitrate or combined N treatments. The year of cultivation strongly affected soil and leaf Cd. Four years after tobacco cultivation, soil pH was reduced by 0.5 units, whereas soil and leaf Cd reduction was more than 60% in the limed treatments. Liming affected Cd uptake only in the first three years of cultivation.

Biochemical Background and Compartmentalized Functions of Cytosolic Glutamine Synthetase for Active Ammonium Assimilation in Rice Roots

Rice plants in paddy fields prefer to utilize ammonium as a major nitrogen source. Glutamine synthetase (GS) serves for assimilation of ammonium in rice root, and ameliorates the toxic effect of ammonium excess. Among the three isoenzymes of the cytosolic GS1 gene family in rice, OsGLN1;1 and OsGLN1;2 were abundantly expressed in roots. Analysis of the purified enzymes showed that OsGLN1;1 and OsGLN1;2 can be classified into high-affinity subtypes with relatively high V(max) values, as compared with the major high-affinity isoenzyme, GLN1;1, in Arabidopsis. Low-affinity forms of GS1 comparable to those in Arabidopsis (GLN1;2 and GLN1;3) were absent in rice roots. The OsGLN1;1 and OsGLN1;2 transcripts showed reciprocal responses to ammonium supply in the surface cell layers of roots. OsGLN1;1 accumulated in dermatogen, epidermis and exodermis under nitrogen-limited condition. By contrast, OsGLN1;2 was abundantly expressed in the same cell layers under nitrogen-sufficient conditions, replenishing the loss of OsGLN1;1 following ammonium treatment. Within the central cylinder of elongating zone, OsGLN1;1 and OsGLN1;2 were both induced by ammonium, which was distinguishable from the response observed in the surface cell layers. The high-capacity Gln synthetic activities of OsGLN1;1 and OsGLN1;2 facilitate active ammonium assimilation in specific cell types in rice roots.

Further investigation of the roles of auxin and cytokinin in the NH4+-induced stimulation of nodulation using white clover transformed with the auxin-sensitive reporter GH3:gusA

Although mineral N (nitrate and ammonium) is believed to have generally negative effects on nodulation in legume-rhizobia symbioses, previous studies have shown that low, static concentrations of ammonium stimulate nodulation in pea, and that this enhancement may be due to an elevation in cytokinin to auxin levels in roots. Here, the effects of ammonium (0.0, 0.1, 0.5 and 2.5mM) on nodulation and auxin levels were investigated in wild-type (WT) white clover (Trifolium repens cv. Haifa) and its transformants (lines 38 and 41) which contain the auxin-sensitive reporter gene (GH3:gusA). The effects of exogenous application (10 -10 , 10 -9 and 10 -8 M) of the cytokinin 6-benzylaminopurine (BAP) were also assessed. Whole-plant nodulation (nodules plant -1 ) and dry weight (DW)-specific nodulation (nodules g -1 root DW) were stimulated (up to 49%) in all white clover lines by 0.1mM NH 4 +. This represents the first confirmation of an NH 4 +-induced stimulation of DW-specific nodulation in a species other than pea. At 2.5mM NH 4 +, the effect was lost on whole-plant nodulation and was inhibitory on DW-specific nodulation. Rhizobial inoculation resulted in a decline in the expression of GH3:gusA in root tips as expected; however, ammonium treatment did not affect GH3 expression in any root zones. Exogenous application of BAP at 10 -9 and 10 -8 M stimulated whole-plant and DW-specific nodulation in wild-type white clover to a similar degree as treatment with 0.1 mM NH 4 +. These results support our previous hypothesis that the stimulation of nodulation by low concentrations of ammonium involves the alteration of the ratio of cytokinin to auxin, specifically by increasing cytokinin.

Activity and protein level of AO isoforms in pea plants (Pisum sativum L.) during vegetative development and in response to stress conditions.

Among three AO isoforms detected in pea plants, the activity of PAO-1 was dominant in leaves of seedlings and young leaves of mature plants, while PAO-3 revealed the highest band intensity in old leaves and roots. PAO-1 and PAO-3 are homodimers consisting of 145 kDa and 140 kDa subunits, respectively, while PAO-2 is a heterodimer of one 145 kDa and one 140 kDa subunit. In leaves, the activity of PAO-1 disappeared gradually with leaf ageing, while in roots it was present only in seedlings but not in mature pea plants. PAO-3 could oxidize abscisic aldehyde, a precursor of abscisic acid, indicating the possible involvement of this isoform in ABA synthesis in pea. The ability of PAO-3 to oxidize abscisic aldehyde was higher in old leaves than in young ones and increased significantly both in roots and leaves of plants exposed to salinity and ammonium treatments. A marked increase of the AO protein level was observed after ammonium application but not under salinity. Interestingly, the activity of PAO isoforms may be transcriptionally and post-transcriptionally regulated during vegetative growth and in response to stress conditions, and such a regulation might be particularly important to adjust ABA levels to the recent requirements of the plant. The observations suggest that the AO isoforms have different metabolic roles and that the activity and protein level of each isoform is regulated not only by environmental conditions but also through plant developmental stages.

AMMONIUM AND UV RADIATION STIMULATE THE ACCUMULATION OF MYCOSPORINE‐LIKE AMINO ACIDS IN PORPHYRA COLUMBINA (RHODOPHYTA) FROM PATAGONIA, ARGENTINA1

The combined effects of ammonium concentration and UV radiation on the red alga Porphyra columbina (Montagne) from the Patagonian coast (Chubut, Argentina) was determined using short‐term (less than a week) experimentation. Discs of P. columbina were incubated with three ammonium concentrations (0, 50, and 300 μM NH4Cl) in anilluminated chamber (PAR=300 μmol photons·m−2·s−1, UVA=15 W·m−2, UVB=0.7 W·m−2) at 15°C. Algae incubated at 300 μM ammonium showed a significant increase (P<0.05) in the concentration of mycosporine‐like amino acids (MAAs) compared with the initial value or with the other ammonium treatments. The increase of MAAs was, however, a function of the quality of irradiance received, with a higher increase in samples exposed to UVA compared with UVB (29% and 5% increase, respectively). However, UVB radiation was more effective in inducing MAA synthesis per unit energy received by the algae. Samples exposed to PAR only had an intermediate increase in MAA concentration of 16%. Chl a concentration decreased through the incubation with the greatest decrease at high ammonium concentration. Phycobiliprotein (BP) decreased through time with the smallest decrease occurring at high ammonium concentration. Photoinhibition (as a decrease of optimal quantum yield) was significantly greater under nitrogen‐deprived conditions than that under replete ammonium levels. Maximal gross photosynthesis (GPmax), as oxygen evolution, and maximal electron transport rate (ETRmax), as chl fluorescence, increased with the ammonium concentration. Positive relationships between maximal GP or ETR and pigment ratios (BP/chl a and MAAs/chl a) and negative relationships with chl a concentration were found.

Is xanthine dehydrogenase involved in response of pea plants (Pisum sativum L.) to salinity or ammonium treatment?

The effect of salinity and different nitrogen sources on the level of xanthine dehydrogenase (XDH) activity in roots and leaves of pea plants was investigated. Two bands of xanthine dehydrogenase activity (XDH-R2, XDH-R3) were detected in roots after native PAGE and staining with hypoxanthine as substrate. Only one band of XDH activity (XDH-L1) was detected in leaf extracts. Within leaves of three different ages the highest XDH activity was detected in young leaves both under control as well as stress conditions. Salinity did not affect significantly the activity of XDH in pea roots, however, depressed XDH activity in leaves. A significant increase of XDH activity both in roots and leaves was observed only when ammonium was applied as the sole N source. Increased concentration of ureides in the xylem sap of pea plants was observed for both ammonium and high salt treatments, although the higher content of ureides in the xylem sap of 100 mM NaCl treated plants may be rather a result of lower rate of exudation from roots than of increased root ureide biosynthesis. Thus, the changes of root and leaf XDH activity in pea plants seem to be tightly correlated with ureide synthesis that is induced by NH 4 + , the product of N fixation, and rather than by salinity. A contribution of pea XDH in increased oxygen species or uric acid production under saline conditions seems to be less than likely.

The influence of nitrogen fertilisation on bacterial activity in the rhizosphere of barley

The effect of nitrogen addition on the activity of rhizosphere bacteria was studied using barley seedlings. Three different nitrogen sources were added to the soil (nitrate, ammonium and ammonium+nitrate) at four different concentrations (0, 100, 300 and 500 mg N kg −1 soil) and the plants were allowed to grow for 6 weeks. The bacterial activity was estimated by measuring thymidine and leucine incorporation into bacteria extracted using homogenisation–centrifugation. Bulk soil bacterial activity was low compared with that of rhizosphere bacteria. Nitrogen addition did not affect the activity of the bulk soil bacteria, indicating that the activity was not nitrogen limited. The thymidine and leucine incorporation rates of rhizosphere bacteria decreased when ammonium or ammonium+nitrate was applied compared with the non-amended controls. No effect on bacterial activity was found following nitrate addition. There was a significant positive correlation between rhizosphere bacterial activity and rhizosphere pH. Shoot length following ammonium treatment was significant lower than in the non-amended control, while nitrate and ammonium+nitrate addition had no effect. This indicates that the varying effects due to nitrogen sources on rhizosphere bacterial activity were not due to effects on plant growth.

Soil ammonium accumulation after sod cutting hampers the restoration of degraded wet heathlands

Summary Restoration of formerly species‐rich wet heaths and matgrass swards has not always been successful. The constraints on this restoration process are not yet fully understood and need further investigation, particularly the accumulation of ammonium in the soil after sod cutting, i.e. the removal of the vegetation and topsoil layer. This accumulation is known from sod cutting experiments in dry heaths, but had not previously been studied in wet heaths and matgrass ecosystems. In 2000, sods were cut from two degraded Dutch wet heaths. Soil chemistry and germination in the sod‐cut plots were measured at irregular intervals between April 2000 and August 2001. To test the influence of ammonium on germination and survival, a glasshouse dose–response experiment was conducted with two endangered wet heath plant species. In both wet heaths, an accumulation of KCl‐extractable ammonium up to 600 µmol kg−1 dry soil was found in the upper 10 cm of the soil within the first year after sod cutting. These high ammonium concentrations lasted for about 10 months. Germination was very low in the sod‐cut plots in 2000 and 2001, and few target species were found, although they were present in the vicinity. The dose–response experiment indicated a significant, negative correlation of both germination and survival with increasing ammonium addition for both plant species. Mean soil ammonium concentrations of the control, 100 and 250 µm ammonium treatments were significantly lower than those of the 500 and 1000 µm ammonium treatments (47, 45, 70, 144 and 252 µmol kg−1 dry soil, respectively). Maximum concentrations of KCl‐extractable ammonium in the field corresponded to water‐extractable concentrations that were higher than those found to be limiting germination and growth in the glasshouse experiments. The low germination in the field is likely to have been adversely affected by high concentrations of ammonium as a result of sod cutting. Synthesis and applications. High ammonium concentrations occur in wet heaths following sod cutting. Low rates of germination of restoration target plant species occur under such conditions. To increase the success of wet heath restoration, the accumulation of ammonium after sod cutting should be prevented by additional measures, such as liming. Because sod cutting is also applied as a restoration measure in the restoration of other ecosystems, such as fens, the effects on increased soil ammonium concentrations need further attention.