A comparison of ammonium, nitrate and proton net fluxes along seedling roots of Douglas-fir and lodgepole pine grown and measured with different inorganic nitrogen sources

Significant spatial variability in NH4+, NO3- and H+ net fluxes was measured in roots of young seedlings of Douglas-fir (Pseudotsuga menziesii) and lodgepole pine (Pinus contorta) with ion-selective microelectrodes. Seedlings were grown with NH4+, NO3-, NH4NO3 or no nitrogen (N), and were measured in solutions containing one or both N ions, or no N in a full factorial design. Net NO3- and NH4+ uptake and H+ efflux were greater in Douglas-fir than lodgepole pine and in roots not exposed to N in pretreatment. In general, the rates of net NH4+ uptake were the same in the presence or absence of NO3-, and vice versa. The highest NO3- influx occurred 0-30 mm from the root apex in Douglas-fir and 0-10 mm from the apex in lodgepole pine. Net NH4+ flux was zero or negative (efflux) at Douglas-fir root tips, and the highest NH4+ influx occurred 5-20 mm from the root tip. Lodgepole pine had some NH4+ influx at the root tips, and the maximum net uptake 5 mm from the root tip. Net H+ efflux was greatest in the first 10 mm of roots of both species. This study demonstrates that nutrient uptake by conifer roots can vary significantly across different regions of the root, and indicates that ion flux profiles along the roots may be influenced by rates of root growth and maturation.     

不同氮源对小麦幼苗谷氨酰胺合成酶的影响

利用ＤＥＡＥ－纤维素柱层析、酶活性测定、Ｎｏｒｔｈｅｒｎ 分子杂交等技术,研究了小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．）幼苗的根、叶和离体叶在不同氮源培养条件下谷氨酰胺合成酶（ＧＳ）活性和同工酶变化, 以及不同氮源对ＧＳ基因转录－ＧＳ－ｍ ＲＮＡ 的影响． 同时与硝酸还原酶（ＮＲ）活性进行比较, 结果表明∶当以ＮＨ＋４ 作唯一氮源时,小麦幼苗根谷氨酰胺合成酶（ＧＳｒ）和叶细胞质谷氨酰胺合成酶（ＧＳ１）活性要比以ＮＯ－３ 作唯一氮源的高．当以ＮＯ－３ 为唯一氮源时, ＮＯ－３ 则促进完整叶片和离体叶片叶绿体谷氨酰胺合成酶（ＧＳ２）活性． 从转录水平上看,ＮＨ＋４ 促进根ＧＳ－ｍ ＲＮＡ 的合成,而ＮＯ－３ 促进叶ＧＳ－ｍ ＲＮＡ 的合成     

Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea (Camellia sinensis) plants

BACKGROUND AND AIMS: Tea (Camellia sinensis) is considered to be acid tolerant and prefers ammonium nutrition, but the interaction between root zone acidity and N form is not properly understood. The present study was performed to characterize their interaction with respect to growth and mineral nutrition. METHODS: Tea plants were hydroponically cultured with NH4+, NO3- and NH(4+) + NO3-, at pH 4.0, 5.0 and 6.0, which were maintained by pH stat systems. KEY RESULTS: Plants supplied with NO3- showed yellowish leaves resembling nitrogen deficiency and grew much slower than those receiving NH4+ or NH(4+) + NO3- irrespective of root-zone pH. Absorption of NH4+ was 2- to 3.4-fold faster than NO3- when supplied separately, and 6- to 16-fold faster when supplied simultaneously. Nitrate-grown plants had significantly reduced glutamine synthetase activity, and lower concentrations of total N, free amino acids and glucose in the roots, but higher concentrations of cations and carboxylates (mainly oxalate) than those grown with NH4+ or NH(4+) + NO3-. Biomass production was largest at pH 5.0 regardless of N form, and was drastically reduced by a combination of high root-zone pH and NO3-. Low root-zone pH reduced root growth only in NO(3-)-fed plants. Absorption of N followed a similar pattern as root-zone pH changed, showing highest uptake rates at pH 5.0. The concentrations of total N, free amino acids, sugars and the activity of GS were generally not influenced by pH, whereas the concentrations of cations and carboxylates were generally increased with increasing root-zone pH. CONCLUSIONS: Tea plants are well-adapted to NH(4+)-rich environments by exhibiting a high capacity for NH4+ assimilation in their roots, reflected in strongly increased key enzyme activities and improved carbohydrate status. The poor plant growth with NO3- was largely associated with inefficient absorption of this N source. Decreased growth caused by inappropriate external pH corresponded well with the declining absorption of nitrogen.     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.     

The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.     

Expression of characteristics of ammonium nutrition as affected by pH of the root medium

To study the effect of root-zone pH on characteristic responses of NH4+ -fed plants, soybeans (Glycine max?L.? Merr. cv. Ransom) were grown in flowing solution culture for 21 d on four sources of N (1.0 mol m-3 NO3-, 0.67 mol m-3 NO3- plus 0.33 mol m-3 NH4+, 0.33 mol m-3 NO3- plus 0.67 mol m-3 NH4+, and 1.0 mol m-3 NH4+) with nutrient solutions maintained at pH 6.0, 5.5, 5.0, and 4.5. Amino acid concentration increased in plants grown with NH4+ as the sole source of N at all pH levels. Total amino acid concentration in the roots of NH4+ -fed plants was 8 to 10 times higher than in NO3(-)-fed plants, with asparagine accounting for more than 70% of the total in the roots of these plants. The concentration of soluble carbohydrates in the leaves of NH4+ -fed plants was greater than that of NO3(-)-fed plants, but was lower in roots of NH4+ -fed plants, regardless of pH. Starch concentration was only slightly affected by N source or root-zone pH. At all levels of pH tested, organic acid concentration in leaves was much lower when NH4+ was the sole N source than when all or part of the N was supplied as NO3-. Plants grown with mixed NO3- plus NH4+ N sources were generally intermediate between NO3(-)- and NH4+ -fed plants. Thus, changes in tissue composition characteristic of NH4+ nutrition when root-zone pH was maintained at 4.5 and growth was reduced, still occurred when pH was maintained at 5.0 or above, where growth was not affected. The changes were slightly greater at pH 4.5 than at higher pH levels.     

铵态氮和硝态氮营养与大豆幼苗的抗氰呼吸作用

在植物体内,NO云还原成NHI是一个耗能很多的生化过程,每还原一个NO。大约要消耗15个ATP分子（Salsac等1987）。植物直接吸收和利用NHi＋,可以减少能量消耗,所节约的能量可用于植物的生长。因此,从理论上讲,供NHI的植株要比供NOt的植株能够获得更高的生物产量。然而,对于大多数植物,供NOS的植株常具有更大的生长量和产量。对于这种现象,过去人们一直在矿物质和有机物积累的差异（Robin和Salsac1985）、根际的酸化程度（Ruftv等1983,Tolley－Henry和Rapen1986）、NHt有害浓度的积累（Haynes和Goh1978）、对光合作用的…     

Response of Anabaena species to different nitrogen sources

Nitrogenase activity, ammonia excretion and glutamine synthetase (GS) activity were examined in five strains of Anabaena (A. anomala ARM 314, A. fertilissima ARM 742, A. variabilis ARM 310, A. oryzae ARM 313 and A. oryzae ARM 570) in the presence of 2.5 mM NO3-N (KNO3), 2.5 mM NH-4-N [(NH4)2SO4] and diatomic nitrogen (N2). Ammonium-N was more inhibitory to nitrogenase activity as compared to NO3-N in all the strains. Maximum GS activity was exhibited in NO3-N medium, irrespective of the cyanobacterial strains studied. Uninduced release of ammonia was observed in all the species, with A. oryzae ARM 313 and Anabaena variabilis ARM 310 exhibiting maximum excretion of 0.25-0.31 and 0.27-1.23 mu moles NH4 mg Chl(-1) respectively on the 15th day of incubation. The glutamine synthetase activity of A. oryzae ARM 313 was relatively very high as compared to Anabaena variabilis ARM 310. There was no nitrate reductase activity in any of the Anabaena sp. grown on NH3-N or N2-N on the 15th day of incubation.     

不同浓度的NO^—3和NH^＋4对小麦根谷氨酰胺合成酶及其相关酶的影响

利用酶活性测定和 Northern分子杂交等技术 ,研究了小麦幼苗根在不同浓度的 Na NO3 和(NH4) 2 SO4的供应下 ,其谷氨酰胺合成酶 (GS)、天冬酰胺合成酶 (AS)、谷氨酸脱氢酶 (GDH)、硝酸还原酶 (NR)以及 GS- m RNA的变化。结果表明 :NH 4 处理的小麦 ,其根部 GS活性比 NO-3 处理的高 ;高浓度处理的比低浓度处理的高 ;Northern杂交结果说明 GS- m RNA转录量与 GS活性一致 ;3mmol/ L NO-3促进了 AS的活性。AS酶活性变化与 GS酶活性变化无明显依赖关系。在实验的条件下 ,没能测出 GDH的活性 ,不同浓度的 NO-3 和 NH 4 处理对 NR活性没有明显的规律。     

Initiation of nitric oxide (NO) synthesis in roots of etiolated seedlings of pea (Pisum sativum L.) under the influence of nitrogen-containing compounds

The level of nitric oxide (NO) in roots of 2-day-old etiolated pea (Pisum sativum L.) seedlings was investigated by fluorescence microscopy using the fluorescent probe 4,5-diaminofluorescein diacetate. Segments representing transversal (cross) cuts of the roots having thickness of 100 to 150 μm (a segment of the root located 10 to 15 mm from the apex) were analyzed. A substantial concentration of NO in the roots was registered when the seedlings were grown in water (control). Addition of 4 mM sodium nitroprusside, 20 mM KNO₃, 2 mM NaNO₂, 2 mM L-arginine into the growth medium increased NO concentration with respect to the control by 1.7- to 2.3-fold. Inhibitors of animal NO-synthase — 1 mM Nω-nitro-L-arginine methyl ester hydrochloride and 1 mM aminoguanidine hydrochloride — reduced the intensity of fluorescence in the root segments in the presence of all the studied compounds. In medium with KNO₃, the inhibitor of nitrate reductase ?150 μM sodium tungstate -lowered the fluorescence intensity by 60%. Scavengers of nitric oxide — 100 μM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and 4 μM hemoglobin — lowered NO concentration in all the studied variants. Potassium ferrocyanide (4 mM) as the inactive analog of sodium nitroprusside inhibited generation of NO. These results are discussed regarding possible pathways of NO synthesis in plants.     

氮素形态对黄檗幼苗生长及氮代谢相关酶类的影响

通过改变水培溶液中NH4+-N和NO3--N的比例, 研究了不同氮素形态对黄檗(Phellodendron amurense)幼苗生长及氮代谢相关酶类的影响。结果表明, 硝态氮比例较高的营养供给比铵态氮比例较高的营养供给有利于黄檗幼苗的生长, 叶片叶绿素含量和可溶性蛋白含量也高。在NH4+-N/NO3--N为25/75 时黄檗幼苗具有最大生物量。在铵态氮比例大的营养供给下, 黄檗幼苗的谷氨酰胺合成酶(GS)活性增强,而在硝态氮比例大的营养供给下幼苗的硝酸还原酶(NR)活性则较高, 叶片中的硝态氮较低。营养液的氮素形态及其组成通过影响GS与NR的活性而调控黄檗幼苗的氮素代谢。     

氮素形态对黄檗幼苗生长及氮代谢相关酶类的影响

通过改变水培溶液中NH4^＋-N和NO3^--N的比例,研究了不同氮素形态对黄檗（Phellodendron amurense）幼苗生长及氮代谢相关酶类的影响。结果表明,硝态氮比例较高的营养供给比铵态氮比例较高的营养供给有利于黄檗幼苗的生长,叶片叶绿素含量和可溶性蛋白含量也高。在NH4^＋-N／NO3^--N为25／75时黄檗幼苗具有最大生物量。在铵态氮比例大的营养供给下,黄檗幼苗的谷氨酰胺合成酶（GS）活性增强,而在硝态氮比例大的营养供给下幼苗的硝酸还原酶（NR）活性则较高,叶片中的硝态氮较低。营养液的氮素形态及其组成通过影响GS与NR的活性而调控黄檗幼苗的氮素代谢。     

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.     

Partial substitution of NO(3)(-) by NH(4)(+) fertilization increases ammonium assimilating enzyme activities and reduces the deleterious effects of salinity on the growth of barley

Productivity of cereal crops is restricted in saline soils but may be improved by nitrogen nutrition. In this study, the effect of ionic nitrogen form on growth, mineral content, protein content and ammonium assimilation enzyme activities of barley (Hordeum vulgare cv. Alexis L.) irrigated with saline water, was determined. Leaf and tiller number as well as plant fresh and dry weights declined under salinity (120 mM NaCl). In non-saline conditions, growth parameters were increased by application of NH(4)(+)/NO(3)(-) (25:75) compared to NO(3)(-) alone. Under saline conditions, application of NH(4)(+)/NO(3)(-) led to a reduction of the detrimental effects of salt on growth. Differences in growth between the two nitrogen regimes were not due to differences in photosynthesis. The NH(4)(+)/NO(3)(-) regime led to an increase in total N in control and saline treatments, but did not cause a large decrease in plant Na(+) content under salinity. Activities of GS (EC 6.3.1.2), GOGAT (EC 1.4.1.14), PEPC (EC 4.1.1.31) and AAT (EC 2.6.1.1) increased with salinity in roots, whereas there was decreased activity of the alternative ammonium assimilation enzyme GDH (EC 1.4.1.2). The most striking effect of nitrogen regime was observed on GDH whose salinity-induced decrease in activity was reduced from 34% with NO(3)(-) alone to only 14% with the mixed regime. The results suggest that the detrimental effects of salinity can be reduced by partial substitution of NO(3)(-) with NH(4)(+) and that this is due to the lower energy cost of N assimilation with NH(4)(+) as opposed to NO(3)(-) nutrition.     

Juvenile nitrogen uptake capacities and root architecture of two open-pollinated families of Picea abies. Effects of nitrogen source and ectomycorrhizal symbiosis

This study was carried out to find early physiological differences occurring in young seedlings between two contrasting Picea abies open-pollinated families (OPF), one with high- and one with low-growth performance in the field by, determining their N uptake capacities and their root architecture. We used three potential N-sources in forest soil solution, NO3-, NH4+ and amino acids, to establish N uptake rates by the plants, whether or not associated with a fungus isolated from the field and identified as Paxillus involutus. NO3- fluxes were determined locally at the root surface using NO3(-)-selective microelectrodes whereas NH4+ and amino acid (L-glutamate and L-aspartate) uptake rates were calculated from their depletion of the incubation solution by the whole root system. Root systems were digitised in order to determine the number and the length of different root types. In non-mycorrhizal plants, the results showed that the most distinguishing parameters between OPF were NO3- uptake rates measured in the white tip of the secondary roots and the root architecture, with higher values determined in high-growth than in low-growth field performance OPF. The presence of the mycorrhizal fungus decreased NO3- uptake rates in both OPF and had an opposite effect on root architecture by increasing it in low-growth and decreasing it in high-growth field performance OPF, respectively. In non-mycorrhizal plants, NH4+ and amino-acid uptake rates were not different between OPF. Mycorrhizal symbiosis did not change NH4+ uptake rates whereas it increased that of amino acids, specifically that of L-aspartate in the low-growth field performance OPF. Taken together these results suggest that the measurement of local fluxes in roots of young plants could be a good potential tool for the early evaluating of growth capacity of Picea abies OPF.     

Relation of light and nitrogen source to growth, nitrate reductase and glutamine synthetase activity of jack pine seedlings

Two-month-old jack pine ( Pinus banksiana Lamb.) seedlings were placed in a greenhouse where both nitrogen source and light level were varied. After 4 months, whole seedling biomass, leaf biomass and relative growth rate were greatest in seedlings grown with NH⁺₄/NO/NO⁻₃-N and full light (FL) and least in seedlings grown with NO ⁻₃-N and low light (LL). NO ⁻₃-seedlings grown under full light and NH⁺₄/NO⁻₃-seedlings grown under low light were approximately equal. This indicates that the extra carbon costs of assimilating only NO⁻₃-N were similar to the reduction of carbon fixation resulting from a 50% decrease in photon flux density. Percentage and total nitrogen content of needles were greater in seedlings grown under low light independent of nitrogen fertilization. Percentage and total nitrogen content of roots were higher under low light and lower when fertilized with NO⁻₃.
Nitrate reductase (NR) activity was higher in roots than in needles, while glutamine synthetase (GS) activity was higher in needles than in roots. Low light resulted in decreased NR activity (mg N)⁻¹ in needles, but not in roots. However, no nitrate was detected in the needles in any treatment. GS activity, on the other hand, was greater under low light in both needles and roots. GS activity in needles is most likely involved with the reassimilation rather than the initial assimilation of ammonium. Some implications of these shifts in enzymatic activity for ecological phenomena in forests are discussed.     

Assimilation of 13NH4+ by Azospirillum brasilense grown under nitrogen limitation and excess.

The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.