The nitrogen balance of Raphanus sativus X raphanistrum plants. I. Daily nitrogen use under high nitrate supply

Abstract Growth-chamber cultivated Raphanus plants accumulate nitrate during their vegetative growth. After 25 days of growth at a constant supply to the roots of 1 mol m^?3 (NO^?₃) in a balanced nutrient solution, the oldest leaves (eight-leaf stage) accumulated 2.5% NO^?₃-nitrogen (NO₃-N) in their lamina, and almost 5% NO₃-N in their petioles on a dry weight basis. This is equivalent to approximately 190 and 400 mol^?3 m^?3 concentration of NO^?₃ in the lamina and the petiole, respectively, as calculated on a total tissue water content basis. Measurements were made of root NO^?₃ uptake, NO^?₃ fluxes in the xylem, nitrate uptake by the mesophyll cells, and nitrate reduction as measured by an in vivo test. NO^?₃ uptake by roots and mesophyll cells was greater in the light than in the dark. The NO^?₃ concentration in the xylem fluid was constant with leaf age, but showed a distinct daily variation as a result of the independent fluxes of root uptake, transpiration and mesophyll uptake. NO^?₃ was reduced in the leaf at a higher rate in the light than in the dark. The reduction was inhibited at the high concentrations calculated to exist in the mesophyll vacuoles, but reduction continued at a low rate, even when there was no supply from the incubation medium. Sixty-four per cent of the NO^?₃ influx was turned into organic nitrogen, with the remaining NO^?₃ accumulating in both the light and the dark.     

Nitrate reductase and nitrate accumulation in relation to nitrate toxicity in Boronia megastigma

Moderate levels of N were toxic to the native Australian plant boronia (Boronia megastigma Nees). As NO^-₃ is the major N form available for plants under cultivated conditions, NO^-₃ reduction and accumulation patterns in boronia were examined following the supply of various levels of NO^-₃ to understand the physiological basis of this toxicity. At a low level of supplied NO^-₃ [15 mmol (plant)^-1], NO^-₃ was reduced without any detectable accumulation and without nitrate reductase activity (NRA) reaching its maximum capacity. When higher NO^-₃ levels [≥25 mmol (plant)^-1] were supplied, both NRA and NO^-₃ accumulation increased further. However, NRA increased to a maximum of ca 500 nmol NO^-₃ (g fresh weight)^-1 h^-1, both in the roots and leaves, irrespective of a 4-fold difference in the levels of supplied NO^-₃, whereas NO^-₃ continued to accumulate in proportion to the level of supplied NO^-₃. Chlorotic toxicity symptoms appeared on the leaves at an accumulation of ca 32 μmol NO^-₃ (g fresh weight)^-1. High endogenous NO^-₃ concentrations inhibited NRA. The low level of NRA in boronia was not limited by NO^-₃ or electron donor availability. It is concluded that the low NR enzyme activity is a genetic adaptation to the low NO^-₃ availability in the native soils of boronia. Thus, when NO^-₃ supply is high, the plat cannot reduce it at high rates, leading to large and toxic accumulations of the ion in the leaf tissues.     

A method for the determination of nitrate reductase

A procedure for the assay of nitrate reductase based on Szekely's diaminodiphenylsulphone method of nitrate determination (Szekely, E. (1967) Talanta 14, 941–950) is described. The method is simple and sensitive, allowing determination of 1 μg of nitrate in a volume of 1 ml or less. It is particularly suited to the determination of nitrate reductase.     

菠菜叶片中硝态氮还原与叶柄中硝态氮累积的关系

测定了不同生长期在不同施氮水平下3个菠菜品种各器官的硝态氮含量、叶片的硝酸还原酶活性、叶片细胞硝态氮的贮存库和代谢库大小.结果表明:叶柄中硝态氮含量远高于其它器官,其含量与叶片内源/外源硝酸还原酶活性的比值呈负相关;叶片细胞中硝态氮代谢库的大小与叶柄中硝态氮含量之间没有确定的关系.     

Nitrate transport and signalling

Physiological measurements of nitrate (NO(3)(-)) uptake by roots have defined two systems of high and low affinity uptake. In Arabidopsis, genes encoding both of these two uptake systems have been identified. Most is known about the high affinity transport system (HATS) and its regulation and yet measurements of soil NO(3)(-) show that it is more often available in the low affinity range above 1 mM concentration. Several different regulatory mechanisms have been identified for AtNRT2.1, one of the membrane transporters encoding HATS; these include feedback regulation of expression, a second component protein requirement for membrane targeting and phosphorylation, possibly leading to degradation of the protein. These various changes in the protein may be important for a second function in sensing NO(3)(-) availability at the surface of the root. Another transporter protein, AtNRT1.1 also has a role in NO(3)(-) sensing that, like AtNRT2.1, is independent of their transport function. From the range of concentrations present in the soil it is proposed that the NO(3)(-)-inducible part of HATS functions chiefly as a sensor for root NO(3)(-) availability. Two other key NO(3)(-) transport steps for efficient nitrogen use by crops, efflux across membranes and vacuolar storage and remobilization, are discussed. Genes encoding vacuolar transporters have been isolated and these are important for manipulating storage pools in crops, but the efflux system is yet to be identified. Consideration is given to how well our molecular and physiological knowledge can be integrated as well to some key questions and opportunities for the future.     

Nitrate regulation of nitrate uptake and nitrate reductase expression in barley grown at different nitrate:ammonium ratios at constant relative nitrogen addition rate

Barley (Hordeum vulgare L. cv. Golf) was cultured using the relative addition rate technique, where nitrogen is added in a fixed relation to the nitrogen already bound in biomass. The relative rate of total nitrogen addition was 0.09 day^?1 (growth limiting by 35%), while the nitrate addition was varied by means of different nitrate: ammonium ratios. In 3- to 4-week-old plants, these ratios of nitrate to ammonium supported nitrate fluxes ranging from 0 to 22 μmol g^?1 root dry weight h^?1, whereas the total N flux was 21.8 ± 0.25 μmol g^?1 root dry weight h^?1 for all treatments. The external nitrate concentrations varied between 0.18 and 1.5 μM. The relative growth rate, root to total biomass dry weight ratios, as well as Kjeldahl nitrogen in roots and shoots were unaffected by the nitrate:ammonium ratio. Tissue nitrate concentration in roots were comparable in all treatments. Shoot nitrate concentration increased with increasing nitrate supply, indicating increased translocation of nitrate to the shoot. The apparent V_max for net nitrate uptake increased with increased nitrate fluxes. Uptake activity was recorded also after growth at zero nitrate addition. This activity may have been induced by the small, but detectable, nitrate concentration in the medium under these conditions. In contrast, nitrate reductase (NR) activity in roots was unaffected by different nitrate fluxes, whereas NR activity in the shoot increased with increased nitrate supply. NR-mRNA was detected in roots from all cultures and showed no significant response to the nitrate flux, corroborating the data for NR activity. The data show that an extremely low amount of nitrate is required to elicit expression of NR and uptake activity. However, the uptake system and root NR respond differentially to increased nitrate flux at constant total N nutrition. It appears that root NR expression under these conditions is additionally controlled by factors related to the total N flux or the internal N status of the root and/or plant. The method used in this study may facilitate separation of nitrate-specific responses from the nutritional effect of nitrate.     

Nitrate assimilation in leaf blades of wheat of different age

A field experiment on wheat (Triticum aestivum L.) ev. Shera grown at 120 kg N ha^?1 was conducted. Half of the dose of fertilizer N was applied at the pre-sowing stage and the other half when the seedlings were one month old. The leaf blades were examined for their NO₃^? content and NO₃^? assimilatory activity at various stages of growth and development. Soil nitrate level at 50 cm depth was determined throughout the wheat growing season in terms of cencentration (μg/ml) and total amount (kg ha^?1). The upper leaf blades were examined for their capacity to assimilate NO₃^?. Highly significant correlation between NR (nitrate reductase) activity and NO₃^? content in the leaf blades. NR activity and soil NO₃^?, and between soil NO₃^? and leaf blade NO₃^? was observed. Findings on low soil NO₃^? status during the reproductive phase and the capacity of the upper leaf blades to assimilate additional amounts of NO₃^?, point to the need for developing a programme of soil fertilizer application whereby all the leaf blades can utilize the NO₃^? optimally and thus result in greater N harvest.     

Nitrate reductase and its role in nitrate assimilation in plants

Nitrate reductase (EC 1.6.6.1) is an enzyme found in most higher plants and appears to be a key regulator of nitrate assimilation as a result of enzyme induction by nitrate. The biochemistry of nitrate reductase has been elucidated to a great extent and the role that nitrate reductase plays in regulation of nitrate assimilation is becoming understood.     

A strain of Arthrobacter that tolerates highconcentrations of nitrate

A gram-positive strain identified as Arthrobacter globiformis CECT 4500, tolerant to up to 1 M nitrate, was isolated from the grounds of a munitions factory. Under strict aerobic conditions, this bacterium used a wide variety of C-sources to obtain the energy required for growth, which took place when the nitrate concentration in the medium was below150 mM. Cells of this bacterium growing in the absence of nitrate were seen as individual cells or forming pairs,whereas cells grown in the presence of nitrate formed short filaments. With ethylene glycol as the C-source, optimal conditions for the full nitrate removal by Arthrobacter were established under laboratory conditions with wastewaters from the synthesis of dinitroethylene glycol. This revised version was published online in August 2006 with corrections to the Cover Date.     

小麦NO3-转运蛋白基因TaNRT2.3的克隆和表达分析

用RT-PCR和RACE技术在NO3-诱导处理的小麦(Triticum aestivum L.)根中克隆到一个硝酸根转运蛋白基因的cDNA,命名为TaNRT2.3(GenBank登录号AY053452).序列分析表明,TaNRT2.3全长1 744 bp,其中含有1 521bp的ORF,编码507个氨基酸,具有12个跨膜区,属于MFS超基因家族中的NNP家族.TaNRT2.3与其他植物中已知的NRT2具有很高的同源性.Northern杂交表明:TaNRT2具有在根中表达的组织特异性,而在叶中未检测到.TaNRT2的表达受NO3-诱导,在含NH4 介质中不表达.NO3-在低浓度(5～200μmol/L)和高浓度(2.0 mmol/L)时均起作用.通过研究小麦在0.2 mmol/LNO3-条件下TaNRT2的表达水平及对NO3-的吸收效率,表明TaNRT2在小麦高效吸收NO3-方面起着重要的作用.分根实验表明植物中N循环本身可以作为吸收N的调节信号.     

Increase in respiratory cost at high growth temperature is attributed to high protein turnover cost in Petunia x hybrida petals

It is widely believed that turnover of nitrogenous (N) compounds (especially proteins) incurs a high respiratory cost. Thus, if protein turnover costs change with temperature, this would influence the dependence of respiration rate on growth temperature. Here, we examined the extent to which protein turnover cost explained differences in N-utilization costs (nitrate uptake/reduction, ammonium assimilation, amino acid and protein syntheses, protein turnover and amino acid export) and in respiration rate with changes in growth temperature. By measurements and literature data, we evaluated each N-utilization cost in Petunia x hybrida petals grown at 20, 25 or 35 degrees C throughout their whole lifespans. Protein turnover cost accounted for 73% of the integrated N-utilization cost on a whole-petal basis at 35 degrees C. The difference in this cost on a dry weight basis between 25 and 35 degrees C accounted for 75% of the difference in N-utilization cost and 45% of the difference in respiratory cost. The cost of nitrate uptake/reduction was high at low growth temperatures. We concluded that respiratory cost in petals was strongly influenced by protein turnover and nitrate uptake/reduction, and on the shoot basis, C investment in biomass was highest at 25 degrees C.     

Remobilisation of vacuolar stored nitrate in barley root cells

Double-barrelled nitrate-selective microelectrodes have been used to measure the time course of the remobilisation of vacuolar stored nitrate in barley (Hordeum vulgare L. cv. Klaxon) root cells during 24 h of nitrate deprivation. These measurements showed that there are different time courses for this process in epidermal and cortical cells of the same root. The remobilisation was much slower from cortical cell vacuoles and had a time course which was similar to that obtained for tissue digests of the roots. The microelectrodes were also used to measure the nitrate concentration in sap exuding from detopped seedlings. These measurements showed that there was a gradual decrease in the delivery of nitrate to the shoot during this time. Root nitrate reductase activity of neither shoots nor roots changed significantly during the first 24 h. Direct measurement of the cytosolic nitrate in a root epidermal cell showed that during short-term changes, such as a 20-min exposure to zero external nitrate supply, cytosolic nitrate was maintained relatively unchanged. Net nitrate efflux from the roots was measurable during the initial 5 h of the zero-nitrate incubation period; after this time no further nitrate efflux was detectable. These measurements are discussed in relation to the nitrate budget of a root cell and we conclude that during the first 24 h of nitrate withdrawal vacuolar nitrate can be readily mobilised to supply the nitrogen demands of the seedling and to maintain the cytosolic nitrate concentration. Received: 31 July 1997 / Accepted 11 December 1997     

Alleviation of nitrate inhibition of soybean nodulation by high inoculum does not involve bacterial nitrate metabolism

Inoculation of soybean (Glycine max. cv. Bragg) plants with high level inoculum partially alleviated the nitrate inhibition of nodule formation (3 to 4 fold), but not nodule growth. This alleviation did not require the bacterial nitrate reductase asBradyrhizobium japonicum mutant strains 110CR1 and 110CR2 (both lacking assimilatory nitrate reductase activity) gave the same results as the wild type parent 311b110. The study was carried out in the glasshouse, thereby confirming preliminary field data by Herridgeet al. (1984) using a wild type bacterial inoculant.     

Nitrate reduction and compartmentation in tomato leaves. I. Nitrate accumulation in leaf sections in aqueous and gaseous medium

Nitrate pools in tomato ( Lycopersicon esculentum Mill. cv. Azes) leaf sections were estimated. Nitrite accumulation in aqueous medium was found to be an inadequate estimate of nitrate pools in tomato leaves. The main reason for the cessation of nitrite accumulation was not depletion of nitrate in the metabolic pool but rather a rapid decay of nitrate reductase (NR) activity as measured by nitrite accumulation in vivo and in vitro. Nitrate diffuses out of the tissue into the medium at a rate higher than the accumulation of nitrite in the tissue. Nitrate leakage from the tissue accelerates the loss of NR activity. Nitrite accumulation in leaf sections kept in an anaerobic gaseous atmosphere ceased earlier than in aqueous medium, at a time when NR activity was still relatively high. Measuring nitrite accumulation in gaseous atmosphere is preferable since NR is more stable and movements of nitrate between pools more restricted.     

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.     

Characterization of NADH: nitrate reductase from the coccolithophorid Emiliania huxleyi (Lohman) Hay & Mohler (Haptophyceae)

Abstract Nitrate reductase was purified from and characterized in a bloom-forming unicellular calcifying alga, Emiliania huxleyi (Haptophyceae). The molecular masses of the native form and the subunit were 514 and 85 kDa, respectively, showing that the enzyme is a hexamer composed of 6 homologous subunits. The K _m values for NADH and NO3− were 40 μM and 104 μM, respectively. Activity of the reduction of nitrate was very high with reduced methylviologen and NADH, but no activity was observed with NADPH or reduced flavin mononucleotide; oxidation of NADH was very high with cytochrome c but did not occur with ferricyanide. These results indicate that Emiliania nitrate reductase is NADH-specific (EC 1.6.6.1), and that among algae and plants its subunit structure and kinetic properties are unique.