Effect of low nitrate supply to nodulated lucerne on time course of activites of enzymes involved in inorganic nitrogen metabolism

Plants of lucerne ( Medicago sativa L. cv. Aragón) inoculated with several strains of Rhizobium meliloti were supplied with a low level of nitrate (5 m M ). After 1 week, normalised nodule mass, obtained by dividing nodule weight by shoot weight, was decreased by one-fourth. This result closely paralleled the bacteroid protein content of nodules, whereas the cytosolic content remained constant. Nitrate reductase activity (NRA, EC 1.7.99.4) of bacteroids increased rapidly after nitrate supply, with actual rates being highly dependent on the Rhizobium strain. The expression of cytosolic NR (EC 1.6.6.1) also varied depending on the bacterial strain but was largely insensitive to nitrate feeding. Nitrite reductase activity (NiRA, EC 1.7.2.2) of either bacteroid or plant origin was independent of the R. meliloti strain. Activation occurred after 3 and 7 days, respectively, of nitrate feeding. Significant amounts of nitrite were obtained throughout the experimental period from buffered extracts of both bacteroids and cytosol of nodules. However, when these nodules were ground in the presence of inhibitors of enzyme activity, nitrite was only found in nodules containing strain 102-F-51 after 1 week of treatment. These results agree with the recent hypothesis that nitrite plays a role in a secondary stage of nodule damage by nitrate. We propose that NiRA rather than NRA can be used as an internal probe of nitrate access to the infected region of nodules.     

Organic nitrogen content and nitrate and nitrite reductase activities in tritordeum and wheat grown under nitrate or ammonium

Tritordeum is a fertile amphiploid derived from durum wheat (Triticum turgidum L. conv. durum) × a wild barley (Hordeum chilense Roem. et Schultz.). The organic nitrogen content of tritordeum grain (34 mg g^-1 DW) was significantly higher than that of its wheat parent (25 mg g^-1 DW). Leaf and root nitrogen content became higher in tritordeum than in wheat after four weeks of growth, independently of the nitrogen source (either NO₃ ^- or NH₄ ⁺). Under NO₃ ^- nutrition, tritordeum generally exhibited higher levels of nitrate reductase (NR) activity than wheat. Nitrite reductase (NiR) levels were however lower in tritordeum than in its wheat parent. In NH₄ ⁺-grown plants, both NR and NiR activities progressively decreased in the two species, becoming imperceptible after 3 to 5 weeks of growth. Results indicate that, in addition to a higher rate of NO₃ ^- reduction, other physiological factors must be responsible for the greater accumulation of organic nitrogen in tritordeum grain.     

Inorganic nitrogen assimilation in Chlamydomonas

Inorganic nitrogen is an essential nutrient for photosynthetic organisms. Its efficient use in nature involves adaptation of the organisms to the availability of the nitrogen supply, to changing environmental conditions, and to the provision of carbon and other nutrients. The unicellular alga Chlamydomonas provides a useful model to identify not only each of the components participating in the assimilative process in a species, but also the regulatory networks modulating their activity. A remarkable fact is the ample array of transporters for inorganic nitrogen compounds operating in this single cell: 13 putative nitrate/nitrite transporters and eight putative ammonium transporters. However, for nitrate, only a few of them participate as the main suppliers of nitrogen for cell growth, and others probably function to adapt nitrogen utilization efficiency to conditions depending not only on the nitrogen source available but also on other nutrients and environmental conditions. This paper summarizes recent findings in Chlamydomonas to provide an integrated perspective.     

Can genetic manipulation of plant nitrogen assimilation enzymes result in increased crop yield and greater N-use efficiency? An assessment

The literature on the relations between plant nitrogen (N) assimilation enzymes and plant/crop N assimilation, growth and yield is reviewed to assess if genetic manipulation of the activities of N assimilation enzymes can result in increased yield and/or increased N use efficiency. The available data indicate that (I) levels of N assimilation enzymes do not limit primary N assimilation and hence yield; (II) root or shoot nitrate assimilation can have advantages under specific environmental conditions; (III) for cereals, cytosolic glutamine synthetase (GS1) is a key enzyme in the mobilisation of N from senescing leaves and its activity in senescing leaves is positively related to yield; and (TV) for rice (Oryza sativd), NADH-glutamate synthase (NADH-GOGAT) is important in the utilisation of N in grain filling and its activity in developing grains is positively related to yield. In our opinion, selection of plants, from either a genetically manipulated population or genetic resources, with expression of nitrate reductase/nitrite reductase primarily in the root or shoot should increase plant/crop growth and hence yield under specific environmental conditions. In addition for cereals the selection of plants with high GS1 in senescing leaves and in some cases high NADH-GOGAT in developing grains could help maximise the retrieval of plant N in seeds.     

Inorganic nitrogen assimilation in plants of Austrlian rainforest communities

In a study of the plant communities of two Australian rainforests, it was found that pioner species had high levels of nitrate reductase (EC 1.6.6.1) and were predominantly leaf nitrate assimilators. Under- and over-storey species had low levels of shoot and root nitrate reductase activity, and many of them showed little capacity for nitrate reduction even when nitrate ions were freely available. Although closed-forest species have lower levels of nitrate reductase than those of gaps and forest margins, their total nitrogen contents were similar, suggesting the former utilize nitrogen sources other than nitrate ions. Glutamine synthetase (EC 6.3.1.2) was present in the leaves of all species examined. In the leaves of pioneer species the chloroplastic isoform of glutamine synthetase predominted, while in most of the species typical of closed-forest the cytosolic isoform accounted for at least 40% of total leaf activity. Low levels of chloroplastic glutamine synthetase were correlated with a low capacity for leaf nitrate reduction, and both are characteristic of many species that regenerate and grow for some time in shade. Low levels of chloroplastic glutamine synthetase imply that, in some of these woody plants, photorespiratory ammonia is re-assimilated via cytosolic glutamine synthetase.     

Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)

Since the recognition of iron‐limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non‐steady‐state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron‐limited and iron‐replete conditions using artificial seawater medium. Iron‐limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU‐induced variable fluorescence signal (F_v/F_m). Under iron‐replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50‐fold higher. Under iron limitation, cultures excreted up to 100 fmol NO₂^?·cell^?1·d^?1 (about 10% of incorporated N) and NiR activities declined by 50‐fold while internal NO₂^? pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron‐limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO₂^? reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate‐limiting step in nitrogenassimilation.     

Microcosm investigation of growth and phytoremediation potential of Azolla japonica along nitrogen gradients

Although Azolla species are among the most promising plants for use in phytoremediation, more studies on their growth and nitrogen (N) uptake along the N gradients of growing media are required. In this study, N concentration-dependent growth in growing media and phosphorus (P) and N accumulation by Azolla japonica were studied by estimating direct N uptake from media by molybdenum-iron proteins. The doubling time of A. japonica was less than a week, regardless of the N concentration (0, 5, and 25 mg N/L) present in the growth media, indicating that this plant is suitable for remediation. Plants showed a high uptake of P, probably via plant-bacteria symbiosis, indicating their potential for effective P remediation. A. japonica also showed more than 4% N content regardless of the treatment and accumulated more than 40 mg of N per microcosm in 3 weeks. iron and molybdenum levels in plants were strongly associated with N fixation, and N uptake from media was estimated to be more than 25 mg per microcosm in 3 weeks, indicating that A. japonica has N remediation potential. As A. japonica is a rapidly growing plant, capable of efficient P and N remediation, it has great potential for use in phytoremediation of nutrient-enriched waters such as agricultural or urban wastewater and eutrophicated aquatic ecosystems.     

The application of ethephon (an ethylene releaser) increases growth, photosynthesis and nitrogen accumulation in mustard (Brassica juncea L.) under high nitrogen levels

Ethephon (2-chloroethyl phosphonic acid), an ethylene-releasing compound, influences growth and photosynthesis of mustard (Brassica juncea L. Czern & Coss.). We show the effect of nitrogen availability on ethylene evolution and how this affects growth, photosynthesis and nitrogen accumulation. Ethylene evolution in the control with low N (100 mg N kg(-1) soil) was two-times higher than with high N (200 mg N kg(-1) soil). The application of 100-400 microl x l(-1) ethephon post-flowering, i.e. 60 days after sowing, on plants receiving low or high N further increased ethylene evolution. Leaf area, relative growth rate (RGR), photosynthesis, leaf nitrate reductase (NR) activity and leaf N reached a maximum with application of 200 microl x l(-1) ethephon and high N. The results suggest that the application of ethephon influences growth, photosynthesis and N accumulation, depending on the amount of nitrogen in the soil.     

Characteristics of inorganic nitrogen assimilation of plants in fire-prone Mediterranean-type vegetation

A range of approaches was used to investigate how species within a fire-prone Banksia woodland in South West Australia exploited inorganic soil nitrogen sources and how this changes through the development of the fire chronosequence. Nitrate and ammonium were present in soil solution throughout the chronosequence but nitrate predominated in recently burnt sites. Mean shoot nitrate reductase activities were high for all species in recently burnt sites and showed little increase when nitrate was supplied via the transpiration stream. Nitrate reductase of shoots of most species was low at sites not burnt for several years, but following transpirational induction with nitrate, developed activities similar to those at recently burnt sites. The principal amino compounds transported in the xylem were species specific, including asparagine, glutamine and citrulline-dominated species, and changed little in relative composition across the chronosequence. Species most active in leaf nitrate reduction transported the largest amounts of nitrate in their xylem sap and proportional amounts of nitrate in xylem tended to be greatest in recently burnt sites. Most of the species examined appeared to be shoot rather than root nitrate assimilators, but marked differences were recorded in potential of leafy shoots of different species to reduce nitrate. As a general rule, shallow-rooted herbaceous, non-mycorrhizal or VAM-positive species had the highest capacity to reduce nitrate, whereas woody species with ericoid mycorrhizae or combined vesicular arbuscular/ectomycorrhizal associations exhibited little capacity to reduce nitrate in roots or shoots. It seems likely that this latter group utilize ammonium or even organic forms of nitrogen rather than nitrate. Some putative nitrogen-fixing species were active in reducing and transporting nitrate, others were virtually inactive in these respects.     

植物内生菌促进宿主氮吸收与代谢研究进展

内生菌与植物共生能够提高宿主的氮吸收与氮代谢水平,这可能是由于内生菌在植物体内引发的多种效应的综合结果.植物内生菌能够通过促进植物根系发育和固氮作用为宿主植物提供更多的无机氮素;能够通过分泌多种胞外酶系如漆酶、蛋白水解酶等使宿主植物更好地利用有机氮素;能够提高宿主氮代谢关键酶如硝酸还原酶(NR)、谷氨酰胺合成酶(GS)等酶的活性;能够提高宿主植物激素水平和维生素含量从而促进宿主氮代谢;能够通过影响宿主植物氮代谢促进宿主植物分蘖、提高宿主植物叶绿素含量和光合速率等等.综述了国内外关于植物内生菌促进宿主氮代谢的相关报道,归纳了植物内生菌影响宿主氮素吸收与代谢的可能机制,并展望了关于植物内生菌促进宿主氮代谢机制方面的研究方向.     

Alterations in nitrogen assimilation and partitioning in nitrogen stressed plants

Although nutrient stress is known to alter partitioning between shoots and roots, the physiological basis for the phenomenon is unresolved. Experiments were conducted to examine assimilation of ¹⁵NO₃ by N-stressed plants and to determine whether apparent changes in assimilation in the root contributed to alterations in whole-plant partitioning of reduced-N. Tobacco plants (Nicotiana tabacum L. cv. NC 2326) were exposed to a low concentration of NO₃^? in solution (80 μM) for 9 days to effect a N-stress response. Exposure of plants to 1000 μM¹⁵NO₃^? for 12 h on selected days revealed that roots of N-stressed plants developed an increased capacity to absorb NO₃^?, and accumulation of reduced-¹⁵N in the root increased to an even greater extent. When plants were exposed to 80 or 1000 μM¹⁵NO₃^? in steady-state, ¹⁵NO₃^? uptake over a 12 h period was noticeably restricted at the lower concentration, but a larger proportion of the absorbed ¹⁵N still accumulated as reduced-¹⁵N in the root. The alteration in reduced-¹⁵N partitioning was maintained in N-stressed plants during the subsequent 3-day “chase” period when formation of insoluble reduced-¹⁵N in the root was quantitatively related to the disappearance of ¹⁵NO₃^? and soluble reduced-¹⁵N. The results indicate that increased assimilation of absorbed NO₃^?, in the root may contribute significantly to the altered reduced-N partitioning which occurs in N-stressed plants.     

Effect of nitrate on nitrogen fixation and nodule carbohydrate and organic acid concentrations in pea mutants deficient in nitrate reductase

Nitrate inhibits symbiotic N₂ fixation and a number of hypotheses concerned with NO₃⁻ assimilation have been suggested to explain this inhibition. These hypotheses were tested using a pea ( Pisum sativum L. cv. Juneau) with normal nitrate reductase NR; (EC 1,6,6,4) activity and two mutants of cv. Juneau, A317 and A334, with impaired NR activity. The plants were inoculated with three strains of Rhizobium leguminosarum and grown for 3 weeks in N-free medium, followed by 1 week in medium supplemented with 0, 5 or 10 m M KNO₃ before harvesting. NO₃ was taken up at comparable rates by the parent and the mutants and accumulated in leaf and stem tissue of the latter. Acetylene reduction rates were inhibited similarly in both the parent and mutants in the presence of KNO₃ but there were differences among rhizobial strains. Starch concentration of the nodules decreased by 46% in the presence of KNO₃ and there were differences among rhizobial strains but not among pea genotypes. Malate and succinate accumulated in nodules in the presence of KNO₃. These data are not consistent with the photosynthate deprivation hypothesis as a primary mechanism for NO₃ inhibition of N₂ fixation since NO₃ affected the nodule carbohydrate composition of all three pea genotypes in a similar manner. The lack of correlation between NR activity and NO₃ inhibition of N₂ fixation suggests that NO₃ assimilation may be only indirectly involved in the inhibition phenomenon.     

施氮对不同品种冬小麦植株硝态氮和硝酸还原酶活性的影响

以黄土高原南部半湿润区土垫旱耕人为土为供试土壤进行盆栽试验,以NR 9405、9430、偃师9号、小偃6号、陕229号和西农2208冬小麦品种为供试材料,研究施氮对不同品种冬小麦植株硝态氮含量和硝酸还原酶活性(NRA)的影响.结果表明,施氮能明显增加叶片NRA.不施氮时除小偃6号和偃师9号外,其余品种NRA在全生育时期的动态变化均呈双峰曲线,2个高峰期分别在返青期和开花期,且开花期高峰值(36.17 NO2-μg.-g 1FW.h-1)明显比返青期峰值(15.407 NO2-μg.-g 1FW.h-1)大;施氮时不同品种叶片NRA在全生育期呈单峰曲线变化,最高峰在开花期,平均峰值为80.93 NO2-μg.-g 1FW.h-1),比同期不施氮处理增加1倍以上.施氮后地上部硝态氮含量在各时期均显著提高,在小麦生育前期(出苗到拔节)表现最为显著.氮肥对不同品种硝态氮含量的影响程度基本上与对NRA的影响程度相反,即施氮后硝态氮增加幅度小的品种,NRA却增加幅度大.     

Nitrate assimilation and nitrogen circulation in Austrian pine

Nitrate uptake, reduction and translocation were examined in 5-week-old Austrian pines ( Pinus nigra Arnold var. nigricans Host.) during exposure to 5 m M NaNO₃. The rate of nitrate uptake was linear during the 7 h light period. ¹⁵N-NO₃⁻ was detected in all parts of the pine except in the needles. By the 7th hour, 43% of the absorbed nitrate had been reduced, and this increased to 64% by the 24th hour. The major part of the total reduction occurred in the roots at this growth stage. Accumulation of ¹⁵N in reduced soluble and insoluble fractions was more prevalent in roots than in shoots. In the needles, the translocated nitrogen was mainly incorporated into the insoluble fraction. It is likely that most of the nitrogen from nitrate was transported from the roots to the aerial organs as organic nitrogen; however part of the upward nitrogen flux took place as nitrate ions.
An experiment in which an exposure for 24 h to 5 m M Na¹⁵NO₃ was followed by 13 days exposure to Na¹⁴NO₃ (pulse chase experiment) revealed a half time of about 1 day for depletion of root nitrate. A large part of this depletion was due to the loss of ¹⁵N-NO₃⁻ to the nutrient solution. The remaining pool of ¹⁵N-nitrate was partitioned between a metabolically inactive and an active pool. During the chase period, the simultaneous decrease of ¹⁵N-incorporation in the soluble N fraction and increase in the insoluble N fraction in different pine parts, particularly in the needles, suggested that protein synthesis occurred mostly in young tissues of the shoot and was the major sink of the newly absorbed ¹⁵N-NO₃.     

Effect of N source during soybean pod filling on nitrogen and sulfur assimilation and remobilization

During pod filling, a grain legume remobilizes vegetative nitrogen and sulfur to its developing fruit. This study was conducted to determine whether different nitrogen sources affected N and S assimilation and remobilization during pod filling. Well-nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea assimilated 0%, 37%, or 114% more N, respectively, and 25%, 46%, or 56% more S, respectively, than did the average non-nodulated control plant fed 5.0 mM KNO₃. Thus, N source during pod filling greatly affected both N and S assimilation. Depending upon N source, plant N concentration during pod filling decreased from 2.96% to between 1.36% and 1.82%. Non-nodulated control plants fed 5.0 mM KNO₃ had the highest residual N at harvest. During the same treatments, plant S concentration decreased from 0.246% to a relatively uniform 0.215%. Thus, during pod filling, vegetative N was seemingly remobilized more efficiently (38–54%) than was S (13%). N source also affected seed yield and seed quality. Non-nodulated control plants fed 5.0 mM KNO₃ produced the lowest yield (21.1 g seeds plant^-1), whereas well nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea produced yields of 26.2 g, 31.8 g, and 36.7 g seeds plant^-1, respectively. Non-nodulated plants fed 2.5 mM urea yielded 28.6 g of seeds plant^-1. Seed N concentrations of non-nodulated plants and nodulated plants fed 2.5 mM urea were high, 6.30% and 6.11% N, respectively, whereas their seed S concentrations were low, 0.348% and 0.330% S, respectively. N sources that produced both a relatively high seed yield and seed N concentration (i.e., a relatively high total seed N plant^-1) produced a proportionately smaller increase in total seed sulfur. Consequently, seed quality, as judged solely by seed S concentration, was lowered.     

Effect of low rhizosphere oxygen on growth, nitrogen fixation and nodule morphology in lucerne

Oxygen shortage in soils can occur following a wide range of natural circumstances, affecting the plant's physiology. In this paper the performance of nodulated lucerne plants under severe hypoxia is examined and the mechanisms involved to achieve this adaptation are discussed. Nodulated lucerne plants ( Medicago sativa L.) were grown with their rooting medium exposed to 1 or 21 kPa oxygen. Final yield, as expressed on a shoot dry weight basis, was unaffected but root and nodule dry weights were reduced by 50%. Water content in roots and nodules was higher at 1 kPa as a result of the formation of aerenchyma. Specific acetylene reduction activity was higher in hypoxic nodules as a consequence of modified nodule structure, although they were more sensitive to the presence of acetylene or nitrate. Root respiration was insensitive to changes in external oxygen supply, therefore providing adequate support for mineral uptake. Nodule respiration rates were 5 times higher in control plants when measured as CO₂ evolution, whereas no differences were observed in O₂ uptake. It is suggested that adaptation of nodulated lucerne to low oxygen concentrations involves changes in photosynthate allocation and nodule morphology, which provide a more efficient nitrogen fixation.