Influence of abscisic acid on nitrogen partitioning, sucrose metabolism and nitrate reductase activity of chicory suspension cells

Batch suspension cultures of chicory cells (Cichorium intybusL. var. Witloof) possess a NADH-specific nitrate reductase activitythat peaks on day 3 of a 10 d growth cycle. When both nitrateand ammonium are used as nitrogen sources, chicory cells absorbnitrate irst. Ammonium uptake becomes predominant at day 3,even though NO₃^– was still present in the medium. Althoughabscisic acid impairs growth as well as ¹⁵NO₃^– uptakeand reduction, it promotes nitrate reductase activity as measuredboth in vivo and in vitro. Specific activity is 50% higher inABA-treated cells than in controls. These conflicting data maybe explained either in erms of nitrate reductase levels or bythe availability of reducing power and energy. Since NRA isgenerally controlled by the availability of the reducing power,the energy status of the cell, the adenylate nucleotide pools,were measured simultaneously with the carbohydrate levels withinthe cell and the growth medium. The energy charge was not modifiedduring the growth cycle, regardless of the rowth conditions.Yet ABA modified the intracellular carbohydrate metabolism andinhibited the acidic invertase, the sucrose synthase and thesucrose phosphate synthase activities. Modified assimilationrates of nitrate in chicory cells grown in the presence of ABA,were probably correlated to modified carbohydrate metabolismpathways leading to increased availability of reducing power,energy and C-skeletons. Key words: Abscisic acid, Cichorium intybus L, nitrate reductase, reductase, invertase, sucrose synthase, sucrose phosphate synthase     

Nitrate reductase activity in chicory roots following excision

In young chicory plantlets (Cichorium intybus L. Witloof cv.Flash), nitrate assimilation takes place mainly in the roots.Nitrate reductase activity (NRA) was measured in roots deprivedof shoot control by excision and transferred into a sucrose-containingmedium. Such a treatment resulted in a drop of about 60% ofNRA within 3 h. The level of NR protein decreased after 12 hand the level of NR-mRNA after several days. This adaptationof nitrate assimilation to excision was affected by a phosphorylation-dephosphorylationmechanism as shown by increased sensitivity to magnesium ofin vitro NRA. Okadaic acid, a serinethreonine protein phosphatasesinhibitor, enhanced the decrease of NRA. Conversely, staurosporine,a serine-threonine protein kinases inhibitor, antagonized theinhibition of NRA. This suggests that excision caused a rapidinactivation of NRA in roots of chicory by modifying the phosphorylationbalance towards a phosphorylated NR form which could enter aninactive complex. Key words: Chicory, nitrate reductase, staurosporine     

Unusual regulatory nitrate reductase activity in cotyledons of Brassica napus seedlings: enhancement of nitrate reductase activity by ammonium supply

The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.     

Evidence for the nitrate-dependent spatial regulation of the nitrate reductase gene in chicory roots

Young chicory plants (Cichorium intybus L. var. Witloof) show a tenfold higher nitrate reductase NR activity in roots compared to leaves. Northern analysis revealed, besides the nitrate inducibility of the nitrate reductase gene (nia), a higher level of expression in the roots. By modifying the external nitrate concentration the NR activity in the leaves remained negligible whereas a maximal activity was observed in the roots when grown in the presence of 5 mM nitrate. Surprisingly, variation of the external nitrate concentration induced changes in the spatial regulation of nia within the root. In-situ hybridization mainly localized nia mRNA in the cortical cells of roots grown at low nitrate concentrations (0.2 mM). At high nitrate concentrations (5 mM), nia mRNA was more abundant in the vascular tissues. The root apex revealed a strong signal under both conditions. The isolation and characterization of the NR structural gene from chicory is also presented. Southern blot analysis revealed the presence of a single nia gene per haploid genome of chicory.     

Characterization of the association of nitrate reductase with barley (Hordeum vulgare L.) root membranes.

The nature of the association between nitrate reductase (NR) and membranes was examined. Nitrate reductase activity (NRA) associated with the microsomal fraction of barley (Hordeum vulgare L.) roots amounted to 0.6 to 0.8% of soluble NRA following sonication in the presence of 250 mM KI and repeated osmotic shock. This treatment removed all contaminating soluble NRA from microsomes of uninduced barley roots that had been homogenized in a soluble extract from roots of NO3(-)-induced plants. On continuous sucrose gradients, NRA co-migrated specifically with VO4(-)-sensitive ATPase activity, a plasma membrane (PM) marker; activity of glucose-6-phosphate dehydrogenase, assayed as cytosolic marker, co-migrated with NRA. Microsomal NRA was absent in barley deficient in soluble NR. Perturbation and trypsinolysis experiments with PM vesicles isolated by aqueous two-phase partitioning indicated that NR is associated with the periphery of the cytoplasmic face of the bilayer. These results demonstrate that PM and soluble NRs are essentially the same protein but that the membrane-associated form is tightly bound. Although it is possible that PM-associated NR exists in vivo, unequivocal evidence for this has yet to be shown. However, PM NR is definitely present in vitro.     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

Time-course of Nitrate Uptake and Nitrate Reductase Activity in Nitrogen-depleted Dwarf Bean

The rate of nitrate uptake by N-depleted French dwarf bean (Phaseolus vulgaris L. cv. Witte Krombek) increased steadily during the first 6 h after addition of NO₃ -After this initial phase the rale remained constant for many hours. Detached root systems showed the same time-course of uptake as roots of intact plants. In vivo nitrate reductase activity (NRA) was assayed with or without exogenous NO₃^- in the incubation medium and the result ing activities were denoted potential and actual level, respectively. In roots the difference between actual and potential NRA disappeared within 15 min after addition of nitrate, and NRA increased for about 15 h. Both potential and actual NRA were initially very low. In leaves, however, potential NRA was initially very high and was not affected by ambient nitrate (0.1–5 mol m^-3) for about 10 h. Actual and potential leaf NRA became equal after the same period of time. In the course of nitrate nutrition, the two nitrate reductase activities in leaves were differentially inhibited by cycloheximide (3.6 mmol m^-3) and tungstate (1 mol m^-3). We suggest that initial potential NRA reflects the activity of pre-existing enzyme, whereas actual NRA depends on enzyme assembly during NO₃^- supply. Apparent induction of nitrate uptake and most (85%) of the actual in vivo NRA occurred in the root system during the first 6 h of nitrate utilization by dwarf bean.     

Nitrate assimilation in the forage legume Lotus japonicus L.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.     

The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised pisum sativum roots

Nitrate reductase (NR) induction is enhanced by exogenously supplied sucrose in excised pea roots exposed to both exogenous nitrate and exogenous nitrite. NR synthesis is preferentially supported by sugars transported to the cells at the moment, however NR induction can take place for some time without exogenous sugar influx if roots are saturated with sugars during precultivation. Steady high NR levels are dependent on steady sugar and nitrate influxes. NR induction is low in roots precultivated for 20 h without sucrose although sugar content is still high in them. This suggests that compartmentation of sugars in the cells is of major importance during NR induction. Total nitrate content in roots exposed to nitrate is not influenced by sucrose supplied together with nitrate. Some nitrite is oxidized to nitrate in roots exposed to exogenous nitrite ; we assume that this nitrate is responsible for NR induction. Our results indicate that sugars, besides many indirect effects on NR induction, may also directly influence NR synthesis either as coinducers or as derepressors of NR synthesis. Our results further show that NR is not a product-inducible enzyme.     

干旱胁迫下紫花苜蓿幼苗非结构性碳水化合物代谢对NO的响应

以紫花苜蓿（Medicago sativa）为材料,采用盆栽试验方法,用聚乙二醇（PEG-6000）作为渗透介质模拟干旱胁迫,外源喷施NO供体硝普钠,NO清除剂（carboxy-PTIO,cPTIO）,对紫花苜蓿幼苗叶片、根系中非结构性碳水化合物含量及相关酶活性的变化进行研究,探讨NO对紫花苜蓿耐旱机制的作用。结果表明：外源NO促进了紫花苜蓿叶片中淀粉的分解、根系中淀粉的积累,提高叶片及根系中可溶性糖（蔗糖、果糖和葡萄糖）含量,降低了渗透势,促进细胞吸水,缓解干旱造成的损伤。此外,外源NO能提高干旱胁迫下紫花苜蓿叶片中蔗糖合成酶（SS）、酸性转化酶（AI）和中性转化酶（NI）活性,降低了蔗糖磷酸合成酶（SPS）的活性,提高根系中SS、SPS和转化酶活性,使蔗糖的合成与分解处于高水平的动态平衡,增强了紫花苜蓿的抗旱性。而NO清除剂cPTIO则会不同程度的抑制紫花苜蓿幼苗中非结构性碳水化合物（NSC）及其相关酶活性。因此,NO可以通过调控NSC的代谢响应干旱胁迫,缓解干旱胁迫造成的不利影响,在紫花苜蓿的抗旱中扮演着重要的角色。     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

Concentration-Dependent inhibition and enhancement of glutamine synthetase,glutamate dehydrogenase and nitrate reductase activities in pea roots by some respiratory inhibitors and uncouplers

The effect of 2,4-dinitrophenol (2,4-DNP), NaN₃, and iodoacetic acid (IDA) on glutamine synthetase (GS) and the effect of arsenate on GS, glutamate dehydrogenase (GDH) and nitrate reductase (NR) was studied in isolated pea roots. In sucrose supplied roots, GS level is depressed by higher concentrations of all the inhibitors tested and increased by lower (2 × and 3 × 10⁻ M) concentrations of 2,4-DNP; the decrease in GS level caused by sugar starvation is enhanced by all but IDA. GDH is enhanced by arsenate in a wider range of concentrations in sucrose-supplied roots than in roots cultivated without sucrose. NR is affected by arsenate similarly as GS.     

硝酸盐对硝酸还原酶活性的诱导及硝酸还原酶基因的克隆

硝酸盐在植物体内的积累过多已成为影响蔬菜品质并影响人类健康的重要因素。硝酸还原酶（NR）是硝酸盐代谢中的关键酶,提高其活性有利于硝酸盐的降解。为了解植物不同组织中NR的活性,用活体测定法检测了经50mmol/L的KNO₃诱导不同时间后的油菜、豌豆和番茄幼苗根茎叶中NR活性,同时为了明确外源诱导剂浓度与植物体内NR活性的关系,检测了经不同浓度KNO₃诱导2h后的矮脚黄、抗热605、小白菜和番茄叶片中的NRA。结果表明,不同植物组织NR活性有很大差异,叶中NR活性较高,根其次,茎最低;不同植物的NR活性随诱导时间呈不同的变化趋势,相同植物不同组织的NR活性变化趋势相似;不同植物叶片NRA为最高时KNO₃浓度不同。用30mmol/L的KNO3诱导番茄苗2h后,从番茄根和叶中提取总RNA,用RT-PCR方法获得NR cDNA,全长2736bp,编码911个氨基酸。为进一步利用该基因提高植物对硝酸盐的降解能力打下基础。