Ammonium Transporter Expression Research Articles

Moderately Reducing Nitrogen Application Ameliorates Salt-Induced Growth and Physiological Damage on Forage Bermudagrass.

Nitrogen (N) application is one of the most effective methods to alleviate salt-induced damage on plants. Forage bermudagrass has higher utilization potential on saline soil, but whether its N requirement changed under high salt stress has not been studied. Through examining plant growth-related traits, salt-stress-responsive physiological traits, photosynthesis, N metabolism, and forage quality supplied with different N concentrations under high salt stress (200 mM NaCl), we noticed that the optimum N requirement of forage bermudagrass reduced. When supplied with 10 mM N under higher salt stress, plants had a similar biomass, turf color, and chlorophyll content with plants supplied with 15 mM N, accompanied by a lower firing rate and Na+ content of leaves. The N content, crude protein, crude fat content, the expression of AMTs (ammonium transporters), NR (nitrate reductase), GS (glutamine synthetase), and GOGAT (glutamate synthetase), the chlorophyll fluorescence curve, and parameters of leaves (e.g., PIABS; PICS; ABS/RC; TRo/RC; ETo/RC) all peaked under 10 mM N under high salt stress instead of 15 mM N. Through exploring the proper N application under higher salt stress and its alleviation mechanisms, our results indicated that moderate reduction in N application under high salt level had a maximum promotion effect on the salt tolerance of forage bermudagrass without growth or forage quality inhibition. These response mechanisms obtained can provide a useful reference for N application in moderation rather than in excess on forage bermudagrass, especially in higher salinity areas.

Nitrogen nutrition modifies the susceptibility of Arabidopsis thaliana to the necrotrophic fungus, Alternaria brassicicola.

The impact of the form of nitrogen (N) source (nitrate versus ammonium) on the susceptibility to Alternaria brassicicola, a necrotrophic fungus, has been examined in Arabidopsis thaliana at the rosette stage. Nitrate nutrition was found to increase fungal lesions considerably. There was a similar induction of defence gene expression following infection under both N nutritions, except for the phytoalexin deficient 3 gene, which was overexpressed with nitrate. Nitrate also led to a greater nitric oxide production occurring in planta during the saprophytic growth and lower nitrate reductase (NIA1) expression 7 days after inoculation. This suggests that nitrate reductase-dependent nitric oxide production had a dual role, whereby, despite its known role in the generic response to pathogens, it affected plant metabolism, and this facilitated fungal infection. In ammonium-grown plants, infection with A. brassicicola induced a stronger gene expression of ammonium transporters and significantly reduced the initially high ammonium content in the leaves. There was a significant interaction between N source and inoculation (presence versus absence of the fungus) on the total amino acid content, while N nutrition reconfigured the spectrum of major amino acids. Typically, a higher content of total amino acid, mainly due to a stronger increase in asparagine and glutamine, is observed under ammonium nutrition while, in nitrate-fed plants, glutamate was the only amino acid which content increased significantly after fungal inoculation. N nutrition thus appears to control fungal infection via a complex set of signalling and nutritional events, shedding light on how nitrate availability can modulate disease susceptibility.

Thymol ameliorates ammonium toxicity via repressing polyamine oxidase-derived hydrogen peroxide and modulating ammonium transporters in rice root

BackgroundAmmonium is an indispensable nutrient for crop growth, but anoxic conditions or inappropriate fertilizer usage result in the increase in ammonium content in soil. Excessive ammonium causes phytotoxicity. Thymol is a kind of natural phenolic compound with anti-microbial properties. However, little is known about the role of thymol in modulating plant physiology. Here we find the novel role of thymol in protecting rice from ammonium toxicity.ResultsThymol remarkably rescued rice seedlings growth from ammonium stress, which may resulted from the attenuation of reactive oxygen species (ROS) accumulation, oxidative injury, and cell death in both shoots and roots. Polyamine oxidase (PAO) metabolizes polyamines to produce ROS in plants in response to stress conditions. Thymol blocked ammonium-induced upregulation of a set of rice PAOs, which contributed to the decrease in ROS content. In rice seedlings upon ammonium stress, thymol downregulate the expression of ammonium transporters (AMT1;1 and AMT1;2); thymol upregulated the expression of calcineurin B-like interacting protein kinase 23 (CIPK23) and calcineurin B-like binding protein 1 (CBL1), two negative regulators of AMTs. This may help rice avoid ammonium overload in excessive ammonium environment. Correlation analysis indicated that PAOs, AMTs, and CBL1 were the targets of thymol in the detoxification of excessive ammonium.ConclusionThymol facilitates rice tolerance against ammonium toxicity by decreasing PAO-derived ROS and modulating ammonium transporters. Such findings may be applicable in the modulation of nutrient acquisition during crop production.Graphical abstract

Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility

The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH3/NH4+) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid-base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH4+ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.

Combined Transcriptome and Proteome Analysis of Masson Pine (Pinus massoniana Lamb.) Seedling Root in Response to Nitrate and Ammonium Supplementations.

Nitrogen (N) is an essential nutrient for plant growth and development. Plant species respond to N fluctuations and N sources, i.e., ammonium or nitrate, differently. Masson pine (Pinus massoniana Lamb.) is one of the pioneer plants in the southern forests of China. It shows better growth when grown in medium containing ammonium as compared to nitrate. In this study, we had grown masson pine seedlings in medium containing ammonium, nitrate, and a mixture of both, and performed comparative transcriptome and proteome analyses to observe the differential signatures. Our transcriptome and proteome resulted in the identification of 1593 and 71 differentially expressed genes and proteins, respectively. Overall, the masson pine roots had better performance when fed with a mixture of ammonium and nitrate. The transcriptomic and proteomics results combined with the root morphological responses suggest that when ammonium is supplied as a sole N-source to masson pine seedlings, the expression of ammonium transporters and other non-specific NH4+-channels increased, resulting in higher NH4+ concentrations. This stimulates lateral roots branching as evidenced from increased number of root tips. We discussed the root performance in association with ethylene responsive transcription factors, WRKYs, and MADS-box transcription factors. The differential analysis data suggest that the adaptability of roots to ammonium is possibly through the promotion of TCA cycle, owing to the higher expression of malate synthase and malate dehydrogenase. Masson pine seedlings managed the increased NH4+ influx by rerouting N resources to asparagine production. Additionally, flavonoid biosynthesis and flavone and flavonol biosynthesis pathways were differentially regulated in response to increased ammonium influx. Finally, changes in the glutathione s-transferase genes suggested the role of glutathione cycle in scavenging the possible stress induced by excess NH4+. These results demonstrate that masson pine shows increased growth when grown under ammonium by increased N assimilation. Furthermore, it can tolerate high NH4+ content by involving asparagine biosynthesis and glutathione cycle.

Characterization and Expression of Ammonium Transporter in Peach (Prunus persica) and Regulation Analysis in Response to External Ammonium Supply

Characterization and Expression of Ammonium Transporter in Peach (Prunus persica) and Regulation Analysis in Response to External Ammonium Supply

Over-expression of OsPTR6 in rice increased plant growth at different nitrogen supplies but decreased nitrogen use efficiency at high ammonium supply

Nitrogen (N) plays a critical role in plant growth and productivity and PTR/NRT1 transporters are critical for rice growth. In this study, OsPTR6, a PTR/NRT1 transporter, was over-expressed in the Nipponbare rice cultivar by Agrobacterium tumefaciens transformation using the ubiquitin (Ubi) promoter. Three single-copy T2 generation transgenic lines, named OE1, OE5 and OE6, were produced and subjected to hydroponic growth experiments in different nitrogen treatments. The results showed the plant height and biomass of the over-expression lines were increased, and plant N accumulation and glutamine synthetase (GS) activities were enhanced at 5.0mmol/L NH4+ and 2.5mmol/L NH4NO3. The expression of OsATM1 genes in over-expression lines showed that the OsPTR6 over expression increased OsAMT1.1, OsATM1.2 and OsAMT1.3 expression at 0.2 and 5.0mmol/L NH4+ and 2.5mmol/L NH4NO3. However, nitrogen utilisation efficiency (NUE) was decreased at 5.0mmol/LNH4+. These data suggest that over-expression of the OsPTR6 gene could increase rice growth through increasing ammonium transporter expression and glutamine synthetase activity (GSA), but decreases nitrogen use efficiency under conditions of high ammonium supply.

Detection of Nitrogen Sufficiency in Potato Plants Using Gene Expression Markers

In-season chemical or optical measures of crop N status can be effective tools in optimizing potato fertilizer N management. The feasibility of using a gene expression as an alternative approach for early detection of potato nitrate deficiency was examined using three potato cultivars (Shepody, Russet Norkotah, and Red Pontiac) with abundant (7.5 mM NO3), limited (0.75 mM NO3) or deficient (0 mM NO3) nitrate supply in nutrient culture over a 7 d period. RNA was extracted from the last fully expanded leaf and quantified using realtime RT-qPCR. Reduced nitrate supply had no measurable effect on shoot dry weight or leaf chlorophyll concentration, but decreased petiole nitrate concentration. Under deficit nitrate supply, down-regulation of nitrate reductase and nitrite reductase was measured within 3 days for all cultivars, and down-regulation of asparagine synthetase was measured in two cultivars. Nitrate supply had no effect on expression of ammonium transporter. In this experimental system, plant gene expression markers detected a reduction of nitrate supply prior to measureable reductions in plant growth or in N status measured using common chemical or optical methods.

Channel-like NH3 flux by ammonium transporter AtAMT2

Prokaryotes, plants and animals control ammonium fluxes by the regulated expression of ammonium transporters (AMTs) and/or the related Rhesus (Rh) proteins. Plant AMTs were previously reported to mediate electrogenic transport. Functional analysis of AtAMT2 from Arabidopsis in yeast and oocytes suggests that NH4+ is the recruited substrate, but the uncharged form NH3 is conducted. AtAMT2 partially co-localized with electrogenic AMTs and conducted methylamine with low affinity. This transport mechanism may apply to other plant ammonium transporters and explains the different capacities of AMTs to accumulate ammonium in the plant cell.

UNDERSTANDING NITROGEN LIMITATION IN AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE) THROUGH cDNA AND qRT-PCR ANALYSIS(1).

Brown tides of the marine pelagophyte Aureococcus anophagefferens Hargraves et Sieburth have been investigated extensively for the past two decades. Its growth is fueled by a variety of nitrogen (N) compounds, with dissolved organic nitrogen (DON) being particularly important during blooms. Characterization of a cDNA library suggests that A.anophagefferens can assimilate eight different forms of N. Expression of genes related to the sensing, uptake, and assimilation of inorganic and organic N, as well as the catabolic process of autophagy, was assayed in cells grown on different N sources and in N-limited cells. Growth on nitrate elicited an increase in the relative expression of nitrate and ammonium transporters, a nutrient stress-induced transporter, and a sensory kinase. Growth on urea increased the relative expression of a urea and a formate/nitrite transporter, while growth on ammonium resulted in an increase in the relative expression of an ammonium transporter, a novel ATP-binding cassette (ABC) transporter and a putative high-affinity phosphate transporter. N limitation resulted in a 30- to 110-fold increase in the relative expression of nitrate, ammonium, urea, amino acid/polyamine, and formate/nitrite transporters. A.anophagefferens demonstrated the highest relative accumulation of a transcript encoding a novel purine transporter, which was highly expressed across all N sources. This finding suggests that purines are an important source of N for the growth of this organism and could possibly contribute to the initiation and maintenance of blooms in the natural environment.

Responses of Rice Cultivars with Different Nitrogen Use Efficiency to Partial Nitrate Nutrition

There is increased evidence that partial nitrate (NO3-) nutrition (PNN) improves growth of rice (Oryza sativa), although the crop prefers ammonium (NH4+) to NO3- nutrition. It is not known whether the response to NO3- supply is related to nitrogen (N) use efficiency (NUE) in rice cultivars. Methods Solution culture experiments were carried out to study the response of two rice cultivars, Nanguang (High-NUE) and Elio (Low-NUE), to partial NO3- supply in terms of dry weight, N accumulation, grain yield, NH4+ uptake and ammonium transporter expression [real-time polymerase chain reaction (PCR)]. A ratio of 75/25 NH4+ -N/NO3- -N increased dry weight, N accumulation and grain yield of 'Nanguang' by 30, 36 and 21 %, respectively, but no effect was found in 'Elio' when compared with those of 100/0 NH4+ -N/NO3- -N. Uptake experiments with 15N-NH4+ showed that NO3- increased NH4+ uptake efficiency in 'Nanguang' by increasing Vmax (14 %), but there was no effect on Km. This indicated that partial replacement of NH4+ by NO3- could increase the number of the ammonium transporters but did not affect the affinity of the transporters for NH4+. Real-time PCR showed that expression of OsAMT1s in 'Nanguang' was improved by PNN, while that in 'Elio' did not change, which is in accordance with the differing responses of these two cultivars to PNN. Conclusions Increased NUE by PNN can be attributed to improved N uptake. The rice cultivar with a higher NUE has a more positive response to PNN than that with a low NUE, suggesting that there might be a relationship between PNN and NUE.

Quantitative characterization of nitrogen regulation of OsAMT1;1, OsAMT1;2, and OsAMT2;2 expression in rice seedlings

The effects of nitrogen forms and supply levels on rice ammonium transporter expression have previously been studied by Northern blot or semiquantitative PCR in a model rice Oryza sativa L., cv. Nipponbare. However, most ammonium transporters (AMT) have low abundance in rice, and it is difficult to accurately analyze their transcript levels. In this study, an analysis of the transcript levels of the OsAMT1;1, OsAMT1;2, and OsAMT2;2 in rice seedlings, cv. Guidan 4 has been performed under various nitrogen conditions, using the technique of real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). The OsAMT1;1 and OsAMT2;2 mRNA levels in nitrogen prestarved plants were induced by supply of NH 4 + , NH4NO3, and NO 3 − , while OsAMT1;2 mRNA in roots was repressed by the same treatment. Expression of OsAMT1;2 in nitrogen-starved rice shoots was induced steeply when rice plants were exposed to 0.5 mM NH4NO3 or 2 mM NH 4 + -N whereas both OsAMT1;1 and OsAMT2;2 exhibited only modest changes under same conditions. OsAMT1;1 and OsAMT1;2 were expressed preferentially in roots while OsAMT2;2 was expressed evenly in roots and shoots, suggesting that OsAMT2;2 may play a different physiological role in ammonium uptake. The upregulation effect of NH4NO3 on OsAMT1;1 mRNA in roots is between that of NO 3 − and NH 4 + , and its induction effect on OsAMT2;2 mRNA level is higher than that of NO 3 − or NH 4 + in both roots and shoots. These three OsAMTs genes in the Guidan 4 rice cultivar show dissimilar transcriptional regulation by various levels of different nitrogen species, and the nitrogen-regulated expression patterns are different from those of the model rice cv. Nipponbare.

NITROGEN STRESS RESPONSE OF PROCHLOROCOCCUS STRAIN PCC 9511 (OXYPHOTOBACTERIA) INVOLVES CONTRASTING REGULATION OF ntcA AND amt11

The ntcA and amt1 genes (encoding a transcriptional activator of genes involved in nitrogen metabolism and a high affinity ammonium transporter respectively) from the axenic Prochlorococcus strain PCC 9511 were amplified, cloned, and sequenced. Cultures of this strain were exposed to nitrogen deprivation, and the expression of ntcA and amt1 was monitored along with cell density, photochemical quantum yield (as determined from Fv/Fm measurements), and cellular C:N ratios. Nitrogen deprivation led to arrested cell growth, reduced photochemical quantum yields, and increased cellular C:N ratios. Surprisingly, transcript accumulation patterns of ntcA and amt1 were not correlated. High levels of amt1 transcript were observed in both nitrogen‐replete and ‐deplete conditions. Expression of amt1 declined under severe deprivation of either phosphorus or nitrogen and showed a positive correlation with photochemical quantum yield. In contrast, ntcA expression was specifically enhanced in the absence of ammonium. High ntcA transcript levels were maintained over time and did not decline with low Fv/Fm values under severe N deprivation. Although ntcA transcript levels correlated somewhat with cellular C:N ratios, at the onset of nitrogen deprivation increases in ntcA mRNA levels occurred before increases in cellular C:N ratios. Constitutively high ammonium transporter expression together with low cellular ammonium requirements are likely to have important implications for the success of Prochlorococcus in the nitrogen‐poor oligotrophic waters where they are most abundant. The function of NtcA in Prochlorococcus strain PCC 9511 remains unclear.