AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots

Despite the fact that urea is a ubiquitous nitrogen source in soils and the most widespread form of nitrogen fertilizer used in agricultural plant production, membrane transporters that might contribute to the uptake of urea in plant roots have so far been characterized only in heterologous systems. Two T-DNA insertion lines, atdur3-1 and atdur3-3, that showed impaired growth on urea as a sole nitrogen source were used to investigate a role of the H+/urea co-transporter AtDUR3 in nitrogen nutrition in Arabidopsis. In transgenic lines expressing AtDUR3-promoter:GFP constructs, promoter activity was upregulated under nitrogen deficiency and localized to the rhizodermis, including root hairs, as well as to the cortex in more basal root zones. Protein gel blot analysis of two-phase partitioned root membrane fractions and whole-mount immunolocalization in root hairs revealed the plasma membrane to be enriched in AtDUR3 protein. Expression of the AtDUR3 gene in nitrogen-deficient roots was repressed by ammonium and nitrate but induced after supply of urea. Higher accumulation of urea in roots of wild-type plants relative to atdur3-1 and atdur3-3 confirmed that urea was the substrate transported by AtDUR3. Influx of 15N-labeled urea in atdur3-1 and atdur3-3 showed a linear concentration dependency up to 200 microM external urea, whereas influx in wild-type roots followed saturation kinetics with an apparent Km of 4 microM. The results indicate that AtDUR3 is the major transporter for high-affinity urea uptake in Arabidopsis roots and suggest that the high substrate affinity of AtDUR3 reflects an adaptation to the low urea levels usually found in unfertilized soils.     

Gene structure and expression of the high-affinity nitrate transport system in rice roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.     

Nitrogen acquisition in four co-existing species from an upland acidic grassland

The concentration of both nitrate and ammonium nitrogen was measured in soil taken from an upland acidic (pH 4.5) grassland habitat, containing four co-existing species, Deschampsia flexuosa (L.) Trin., Festuca ovina L., Juncus squarrosus L. and Nardus stricta L. Both nitrate and ammonium nitrogen were found to be present in the soil, in similarly small quantities. The effect of both sources of nitrogen on relative growth rate was studied, and an attempt was made to determine whether nitrate or ammonium nitrogen is the immediate source of nitrogen for these plants using assays of nitrate reductase (EC 1.6.6.2) and ammonium uptake. All four species showed larger growth rates on the same concentration of ammonium nitrogen compared to nitrate nitrogen. All species showed low activities of leaf nitrate reductase, even in plants grown on 18 mol nitrate m⁻³. Ammonium uptake activity appeared to be higher in species which showed the lowest nitrate reductase activity and least response to increasing nitrate concentration in the growth medium.     

Effects of NaCl salinity on 15N-nitrate fluxes and specific root length in the halophyte Plantago maritima L.

The effect of salinity on nitrate influx, efflux, nitrate net uptake rate and net nitrogen translocation to the shoot was assessed in a ¹⁵N steady state labelling experiment in the halophyte Plantago maritima L. raised for 14 days on solution supplied with 50, 100 and 200 mol m^–3 sodium chloride or without sodium chloride. Additionally, salinity induced changes in root morphology were determined. Specific root length increased upon exposure to elevated sodium chloride concentrations due to variations in biomass allocation and length growth of the tap root. Changes in root morphology, however, had a minor effect on nitrate fluxes when expressed on a root fresh weight basis. The decreased rate of nitrate net uptake in plants grown on elevated levels of sodium chloride was almost entirely due to a decrease in nitrate influx. Expressed as a proportion of influx, nitrate efflux remained unchanged and was even lower at the highest salinity level. At all sodium chloride concentrations applied the initial rate of nitrogen net translocation to the shoot decreased relative to the rate of nitrate net uptake. It is concluded that under steady state conditions the negative effect of sodium chloride on the rate of nitrate net uptake at non growth-limiting salinity levels was due to the interaction between sodium chloride and nitrate transporters in the root plasma membrane and/or processes mediating the translocation of nitrogen compounds, possibly nitrate, to the shoot.     

Relatively large nitrate efflux can account for the high specific respiratory costs for nitrate transport in slow-growing grass species

In this paper we address the question why slow-growing grass species appear to take up nitrate with greater respiratory costs than do fast-growing grasses when all plants are grown with free access to nutrients. Specific costs for nitrate transport, expressed as moles of ATP per net mole of nitrate taken up, were 1.5 to 4 times higher in slow-growing grasses than in fast-growing ones (Scheurwater et al., 1998, Plant, Cell & Environ. 21, 995–1005). The net rate of nitrate uptake is determined by two opposing nitrate fluxes across the plasma membrane: influx and efflux. To test whether differences in specific costs for nitrate transport are due to differences in the ratio of nitrate influx to net rate of nitrate uptake, nitrate influx and the net rate of nitrate uptake were measured in the roots of two fast-growing ( Dactylis glomerata L. and Holcus lanatus L.) and two slow-growing (Deschampsia flexuosa L. and Festuca ovina L.) grass species at four points during the diurnal cycle, using ¹⁵NO₃ ^-. Efflux was calculated by subtraction of net uptake from influx; it was assumed that efflux of nitrogen represents the flux of nitrate. Transfer of the plants to the solution containing the labelled nitrate did not significantly affect nitrate uptake in the present grass species. The net rate of nitrate uptake was highest during the middle of the light period in all species. Diurnal variation in the net rate of nitrate uptake was mostly due to variation in nitrate influx. Variation in nitrate efflux did not occur in all species, but efflux per net mole of nitrate taken up was higher during darkness than in the light in the slow-growing grasses. The two fast-growing species, however, did not show diurnal variation in the ratio of efflux to net nitrate uptake. Integrated over 24 hours, the slow-growing grasses clearly exhibited higher ratios of influx to net uptake than the fast-growing grass species. Our results indicate that the higher ratio of nitrate influx to net nitrate uptake can account for higher specific costs for nitrate transport in slow-growing grass species compared with those in their fast-growing counterparts, possibly in combination with greater activity of the non-phosphorylating alternative respiratory path. Therefore, under our experimental conditions with plants grown at a non-limiting nitrate supply, nitrate uptake is less efficient (from the point of ATP consumption) in slow-growing grasses than in fast-growing grass species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influx and efflux of K(+) in sunflower roots after transfer between solutions with different K(+) concentrations

It was investigated whether K(+) efflux, like K(+) influx, is affected when roots are transferred between solutions with different K(+) concentrations. Sunflower plants (Hehanthus annuus L. cv. Uniflorus) were grown on complete nutrient solutions with 0.1, 1.0, 10 or 25 mM K(+) . This produced plants with K(+) concentrations in the roots varying between 9 and 110 μmol (g fresh weight)(-1) . At the beginning of the experiments the plants were transferred to an (86) Rb-labelled experimental solution initially containing 0.1 mM K(+) . At intervals during 6.5 h samples were removed from the solution and analyzed for K(+) and radioactivity. Based on the analyses K(+) ((86) Rb) influx, K(+) net uptake and K(+) efflux could be computed. In'low K(+) 'roots, K(+) ((86) Rb) influx and K(+) net uptake agreed, suggesting a very low K(+) efflux. This was contrary to'high K(+) 'roots, where K(+) efflux was initially higher than K(+) ((86) Rb) influx. After about 4 h, K(+) efflux declined to a low value also in these roots. When 2-4-dinitrophenol was included in the experimental solution, K(+) ((86) Rb) influx was generally depressed, whereas K(+) efflux was high throughout the experiment and directly proportional to the K(+) status of the roots. Our hypothesis is that after transfer of'high K(+) 'roots to a solution with low K(+) concentration, the K(+) efflux from the vacuoles of root cells transiently increases, until a new electrochemical equilibrium is attained.     

Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots

In Arabidopsis four root-expressed AMT genes encode functional ammonium transporters, which raises the question of their role in primary ammonium uptake. After pre-culturing under nitrogen-deficiency conditions, we quantified the influx of (15)N-labeled ammonium in T-DNA insertion lines and observed that the loss of either AMT1;1 or AMT1;3 led to a decrease in the high-affinity ammonium influx of approximately 30%. Under nitrogen-sufficient conditions the ammonium influx was lower in Columbia glabra compared with Wassilewskija (WS), and AMT1;1 did not contribute significantly to the ammonium influx in Col-gl. Ectopic expression of AMT1;3 under the control of a 35S promoter in either of the insertion lines amt1;3-1 or amt1;1-1 increased the ammonium influx above the level of their corresponding wild types. In transgenic lines carrying AMT-promoter-GFP constructs, the promoter activities of AMT1;1 and AMT1;3 were both upregulated under nitrogen-deficiency conditions and were localized to the rhizodermis, including root hairs. AMT gene-GFP fusions that were stably expressed under the control of their own promoters were localized to the plasma membrane. The double insertion line amt1;1-1amt1;3-1 showed a decreased sensitivity to the toxic ammonium analog methylammonium and a decrease in the ammonium influx of up to 70% relative to wild-type plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake capacity in Arabidopsis roots under nitrogen-deficiency conditions.     

Uptake of ammonium and nitrate by carob (Ceratonia siliqua) as affected by root temperature and inhibitors

Seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were grown in nutrient solution culture for 5 weeks, with or without nitrogen at different root temperatures (10, 16, 22, 30, 35 or 40deg;C) and with the air temperature kept between 20 and 24°C. The nitrogen was given as either ammonium or nitrate. At all root temperatures studied, nitrogen-depleted plants developed higher net uptake rates for nitrogen than plants grown in the presence of nitrogen. Temperature affected the kinetic parameters of nitrate uptake more than those of ammonium uptake. With increasing root temperature, the K_m of ammonium uptake decreased, but to a lesser extent than the K_m for nitrate. The increase in V_max of ammonium uptake with temperature was also less noticeable than that for nitrate uptake. Ammonium and nitrate uptakes were inhibited in a similar way by respiratory or protein synthesis inhibitors. It may be noted that ammonium uptake in the presence of inhibitors at 40°C was higher than uptake at 10°C without inhibitors. Some similarities between the transport mechanisms for nitrate and ammonium are underlined in the present work. Components of both transport systems displayed saturation kinetics and depended on protein synthesis and energy. The following components of nitrate uptake were distinguished: (a) a passive net influx into the apparent free space; (b) a constitutive active uptake and (c) active uptake dependent on protein synthesis. We may similarly define three ammonium uptake systems: (a) a passive influx into the apparent free space; (b) passive diffusion uptake at high temperature and (c) active uptake dependent on protein synthesis. The possible role of the ratio between mechanism (c) and mechanism (b) as determinant of ammonium sensitivity is discussed.     

The molecular physiology of ammonium uptake and retrieval

Plants are able to take up ammonium from the soil, or through symbiotic interactions with microorganisms, via the root system. Using functional complementation of yeast mutants, it has been possible to identify a new class of membrane proteins, the ammonium transporter/methylammonium permease (AMT/MEP) family, that mediate secondary active ammonium uptake in eukaryotic and prokaryotic organisms. In plants, the AMT gene family can be subdivided according to their amino-acid sequences into three subfamilies: a large subfamily of AMT1 genes and two additional subfamilies each with single members (LeAMT1;3 from tomato and AtAMT2;1 from Arabidopsis thaliana). These transporters vary especially in their kinetic properties and regulatory mechanism. High-affinity transporters are induced in nitrogen-starved roots, whereas other transporters may be considered as the 'work horses' that are active when conditions are conducive to ammonium assimilation. The expression of several AMTs in root hairs further supports a role in nutrient acquisition. These studies provide basic information that will be needed for the dissection of nitrogen uptake by plants at the molecular level and for determining the role of individual AMTs in nutrient uptake and potentially in nutrient efficiency.     

Depression of Nitrate and Ammonium Transport in Barley Plants with Diminished Sulphate Status. Evidence of Co-regulation of Nitrogen and Sulphate Intake

When barley plants were grown in a solution with nitrate asthe sole N-source but deprived of sulphate (–Splants)for 1 to 5 d, the capacity for sulphate transport by the rootsincreased very markedly; subsequent measurement of influx using³⁵S-labelled showed increases of > 10-fold compared to plants continuously supplied with sulphate (+S plants).There were only small effects on plant growth over a 5 d periodand yet the influx of , labelled with the short-lived tracer ¹³N, was diminished by approximately 30%.By contrast, the influx of phosphate was little affected bysulphate-deprivation. When a sulphate supply was restored to– S plants, the sulphate influx was quickly repressedover the subsequent 24 h and the nitrate influx was restoredto >90% of the value in +S plants. When plants were grown in a solution with a mixed nitrate andammonium supply and deprived of sulphate for 1 d or 5 d thedepression of nitrate influx was more strongly marked (up to55% depression). The influx of ammonium was also depressed after5 d of sulphate-deprivation, but not at 1 d, nor where the concentrationof ammonium in the uptake solution was lowered to 20 mmol m^–3or less. Additional measurements with ¹⁵N-labelled nitrate and ammoniumover longer periods were used to determine net uptake. Net uptakeof nitrate was depressed to a similar extent to efflux, butnet ammonium uptake was depressed only in unbuffered uptakesolution where the pH decreased to pH 4.9 during the uptakeperiod. The ¹⁵N-tracer experiments showed that the translocationof label to the shoot, from both nitrate and ammonium, was depressedto a greater extent than net uptake in –S plants. Thedepression of nitrate influx, caused by 5 d of sulphate deprivation,could be relieved almost completely by providing plants with1.0 mol m^–3 L-methionine during the day prior to influxmeasurement. This treatment substantially decreased sulphateand potassium (⁸⁶Rb-labelled) influx in both +S and –Splants, but greatly increased total S-status of the plants.This methionine treatment had no effect on ammonium influx ornet uptake in – S plants but increased influx significantlyin +S ones. When plants were grown with sulphate but deprived of nitratefor 4 d there was a marked depression of the sulphate influx(by 48–65%) but a smaller effect on phosphate influx (21–37%of +N). The results are discussed in relation to the effects of sulphate-deprivationon growth rate and the root: shoot weight ratio. It is concludedthat the effects on influx and net uptake of nitrogen are moresevere than could be accounted for by these factors. The decreasedtranslocation of either nitrate, or the products of nitrateand ammonium assimilation from the roots, is suggested as areason for the depression of influx. The restoration of nitrateinflux and net uptake by methionine suggests that, for thision at least, a shortage of S-amino acids within the plant maylead to the accumulation of inhibitory concentrations of non-Samino acids in the transport pool. Key words: ¹³N, sulphate, nitrate, ammonium, ion-uptake, barley     

Inhibition of nitrification by waste tea (‘Tea Fluff’)

Summary Ammonium fertilizer applied to tea soils is readily converted to nitrate by the nitrifying bacteria in soil. Excess nitrate in soil could undergo rapid leaching losses under high rainfall conditions. Data is presented in this paper to show that waste tea could be effectively used to retard and delay nitrate production and thereby prevent loss of nitrogen as nitrate by leaching. Evidence is also presented to show that waste tea readily liberates ammonium nitrogen in soil.     

Nitrogen balance studies for different Egyptian soils cropped with corn

Summary The nitrogen balance for four different Egyptian soils cropped with corn and fertilized with increasing amounts of ammonium sulphate was studied in pot experiments. The tested soils were clay loam, calcareous sandy loam, sandy loam and sand. Nitrogen added as seeds, fertilizers, irrigation water and insecticides, nitrogen removed by the plants including thinned plants and fallen leaves and the nitrogen content of the soils before sowing and after the harvest were determined and used to draw the balance sheet.The obtained results showed that nitrogen loss ranged between 6.8 and 51.5 per cent depending mainly on soil type and to some extent on the rate of applied ammonium sulphate. Nitrogen uptake by the corn plants was lowest from the clay loam soil and highest from the sandy soil. In all cases it increased slightly with the addition of nitrogen fertilizer. re]19720918     

Distribution and recovery of15N after fertilization of Douglas-fir saplings with different nitrogen sources

Summary Four-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) saplings planted in pots with a sand and peat mix (11) were fertilized at the rate of 200 kg N/ha of (¹⁵NH₂)₂CO (U-15),¹⁵NH₄NO₃ (A-15) and NH₄ ¹⁵NO₃(An-15). They were placed in a shadehouse and watered regularly to maintain soil moisture at field capacity over periods of one and two years. Quantity of¹⁵N in foliage generally increased from old to current growth, irrespective of the nitrogen source. Utilization of¹⁵N fertilizers by saplings after the first and second growing seasons following fertilization was greatest with nitrate labelled ammonium nitrate AN-15, and nearly equal for urea U-15 and ammonium labelled ammonium nitrate A-15. The soil immobilized more fertilizer nitrogen-15 from U-15 and A-15 than from AN-15. Data from the present study, in which leaching losses of fertilizer were minimized, demonstrated that in terms of nitrogen uptake by the saplings the nitrate fertilizer was superior to ammonium fertilizer.     

THE EFFECTS OF NITROGEN SUPPLY UPON THE VERTICILLIUM WILT OF ANTIRRHINUM

Isolates of Verticillium albo-atrum, V. dahliae and V. nigrescens grown on media adequately supplied with sodium nitrate induced wilt rather more rapidly in antirrhinum plants growing in soils with a normal and an excessive amount of nitrogen than in plants in nitrogen-deficient soil, though plants became diseased in all soils. Similarly treated isolates of V. nubilum and V. tricorpus induced a greater incidence of wilt in plants in soil supplied with heavy dressings of organic nitrogenous fertilizer than in plants in soil deficient in nitrogen, although V. tricorpus from a medium containing much sodium nitrate, in contrast to V. nubilum , was pathogenic to plants in such deficient soil. The ability of the nitrogen-starved isolates to penetrate the host plant was significantly diminished, and even when wound-inoculated into stems their effect upon the host was much reduced.     

Measurement of short-term nutrient uptake rates in cranberry by aeroponics

Aeroponics, a soil-less plant culture system in which fresh nutrient solutions are intermittently or continuously misted on to plant roots, is capable of sustaining plant growth for extended periods of time while maintaining a constantly refreshed nutrient solution. Although used relatively extensively in commercial installations and in root physiology research, use of aeroponics in nutrient studies is rare. The object of this study was to examine whether nutrient uptake rates could be calculated for aeroponic systems by difference using measurements of concentrations and volumes of input and efflux solutions. Data were collected from an experiment with cranberry plants (Vaccinium macrocarpon Ait. cv. Stevens) cultured aeroponically with nutrient solutions containing various concentrations of ammonium-N and isotopically labelled nitrate-N. Validation of the calculated uptake rates was sought by: (1) evaluating charge balance of the solutions and total ion uptake (including proton efflux) and (2) comparison with N-isotope measurements. Charge balance and proton efflux calculations required use of chemical modelling of the solutions to determine speciation of dissolved phosphate and acid-neutralizing capacity (ANC). The results show that charge balance requirements were acceptably satisfied for individual solution analyses and for total ion uptake when proton efflux was included. Relative rates of nitrate/ammonium uptake determined by difference were in agreement with those determined by isotopic techniques. Additional information was easily obtained from this experimental technique, including evidence of diurnal variation in nutrient uptake, correlation between ammonium uptake and proton efflux, and the relationship between ion concentration and uptake. Use of aeroponic systems for non-destructive measurement of water and ion uptake rates for numerous other species and nutrients appears promising.