Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO₃ (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.     

Effects of norflurazon (San 9789) on light-increased extractable nitrate reductase activity in soybean [Glycine max (L.) Merr.]seedlings

Abstract. The effects of norflurazon (San 9789) on light-increased extractable NADH nitrate reductase activity (NRA) in soybean seedlings were studied. Continuous white light (W) increased NRA steadily in root and cotyledonary tissues over a 5 d period. Morflurazon, a pyridazinone herbicide which causes chlorophyll bleaching in W, reduced the initial NRA induction rate in roots and cotyledons. However, in cotyledons of norfiurazon-treated plants NRA increased at a more rapid rate than in the control after 24 h of W, with activity levels reaching three times those of control seedlings after 5 d. NRA induced by W in control and norflurazon-treated cotyledons was fluence-rate dependent. Continuous FR induced equal amounts of NRA in control and norflurazontreated tissues, suggesting that the superinduceable NRA of norflurazon-treated plants under W is not phytochrome induced. The FR-induced NRA of control and norflurazon-treated cotyledons had pH optima of 6.6, but during development under W the pH optimum of control cotyledons changed from 6.3 to between 6.6 and 7.1. The pH optimum of the norflurazon-induced NRA of the cotyledon under W was about 7.5. The NADH/NADPH NRA ratio after 4 d of W was 1.3 in control and 2.5 in norflurazontreated cotyledons. These data indicate that photosynthelic pigments are involved only secondarily in light-induction of NRA in this system.     

Effect of glutamine on the induction of nitrate reductase

Nitrate reductase (NR. EC 1.6.6.1/2) is a substrate inducible enzyme that could be repressed by its end product glutamine or amino acids. To test this hypothesis, 6-day-old maize seedlings ( Zea mays cv. W64A × W182E) were grown hydroponically in a 1/10 strength Hougland's salt solution modified to contain no nitrogen. Previous experiments had established that after a 24-h induction with NO₃⁻ (5 mM KNO₃⁻) the level of NR activity and protein had reached a constant level. In the present experiments when glutamine (5 mM) was included together with NO₃⁻, there was a significant reduction in NR activity (34% of the control values). NR protein and NR mRNA accumulation in the root. In the shoot, on the other hand, glutamine additions had little or no effect on the levels of either NR activity (81% of control) or NR protein. Inhibition of glutamine synthetase by methionine sulfoximine (MSX) resulted in reduced levels of glutamine in both root and shoot tissues. Contrary in our prediction, however, it had no effect on NR activity and mRNA content in roots. In the shoot, on the other hand, there was a marked reduction of NR activity (34% of the control value) and NR protein, but no apparent effect on NR mRNA. When detached shoots were treated with MSX and other inhibitors of glutamine synthetase (tabtoxinine-β-lactam or phosphinothricin) the induction of NR activity by NO₃⁻ was also inhibited. Glutamine additions 15 or 50 mM to detached shoots had essentially no effect on the induction of NR activity (90% of control). These results demonstrate that the influence of glutamine and MSX on the induction of NR in maize root and shoot tissues, respectively, is very different.     

The effects of endogenous cytokinin content on benzyladenineenhanced nitrate reductase induction

Foliar application of benzyladenine (BA) has been shown to enhance nitrate-dependent induction of nitrate reductase (NR; EC 1.6.6.1) in etiolated wheat ( Triticum aestivum L.) seedlings. Whether similar enhancement occurs in light-grown plants, or whether endogenous cytokinin content affects this enhancement is unknown. Since the cytokinin content of etiolated plants probably differs from that of light-grown seedlings, the NR response of each to exogenous root- or shoot-applied BA in wheat (cv. Red Bob) was examined. Endogenous cytokinins present in untreated control tissues prior to BA application and changes that occurred after a 22 h (12 h dark followed by 10 h of light) period were determined using a combined HPLC-immunoassay method. Shoot application of BA enhanced the induction of NR in etiolated seedlings in a concentration-dependent manner but failed to enhance NR induction in light-grown plants. Root-applied BA enhanced NR induction in both etiolated and light-grown seedlings. Endogenous root cytokinin levels were similar in both etiolated and light-grown plants. In contrast, shoots of 6 day-old light-grown seedlings contained at least 20 times the amount of total cytokinins measured in shoots from etiolated plants of the same age. Total cytokinin content of the light-grown plants diminished after the 22-h period while that measured in etiolated seedlings increased. The responsiveness of seedlings to BA was correlated with endogenous cytokinin levels in that enhancement of NR induction by exogenous BA was low in tissues which contained high concentrations of cytokinin at the time of BA application. These results may prove useful in interpretation of gene responses to exogenous plant growth regulators.     

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.     

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.     

Nitrate reductase in cotyledons of cucumber seedlings as affected by nitrate, phytochrome and calcium

Regulation by the active form of phytochrome (P_FR) and the effect of Ca²⁺ was examined with nitrate reductase (NR) in etiolated cucumber ( Cucumis sativus cv. Beilpuig). Nitrate reductase activity (NRA) was studied in excised cotyledons of cucumber seedlings grown in distilled water and in darkness for seven days at 24 ± 0.5°C. All experiments were performed in the dark and a dim green safelight was used during analyses. In etiolated cucumber cotyledons NRA was induced by nitrate and a brief irradiation (15 min) with red light (R) resulted in 62% increase in NRA. This effect was nullified when R was followed immediately by a brief (5 min) far-red light (FR). NRA also showed a semidian (12 h) rhythmicity. Both P_FR, and nitrate effects were age dependent. Calcium seemed to be involved since the phytochrome effect was only observed when calcium was supplied in the external solution. The effect of R on NRA depended on the period of calcium nitrate incubation. An external supply of calcium ionophore mimicked the effect of R and, if supplied to R-irradiated cotyledons, produced a higher NR level than that caused by R alone. This suggested that intracellular free calcium was involved.     

Effects of Light Illumination on the Population of Translatable mRNA in Radish Cotyledons during Dark-Induced Senescence

The population of translatable mRNA in radish cotyledons hasbeen shown to change strikingly during dark-induced senescenceby analyzing cell-free translation products from polyadenylatedRNA using two-dimensional electrophoresis (Kawakami and Watanabe1988, Plant Cell Physiol. 29: 33–42). In the present study,the translatable mRNAs in the cotyledons were studied afterre-illumination of seedlings which had been induced to senesceby dark treatment. All of the mRNA species which showed relativeincrease or decrease during a 24-h dark treatment returned totheir initial levels after a subsequent 24-h illumination withwhite light. When seedlings were kept in the dark for 48 h,21 mRNA species increased, but the increase in some of themwas no longer reversible; 9 out of the 21 mRNA species remainedat the same level even after 48 h of re-illumination. All ofthe 18 mRNA species which decreased during 48 h in the darkreturned to their initial levels upon re-illumination. The mRNAspecies that showed irreversible increase were also found toaccumulate in cotyledons senesced after a lengthy growth periodunder light. There were also some mRNA species whose contentwas not affected by dark treatment, but increased transientlyafter re-illumination of the seedlings. These observations arediscussed in relation to the progress of senescence in the cotyledons. (Received September 24, 1987; Accepted December 19, 1987)     

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。     

Induction and Turnover of Nitrate Reductase in Zea mays (Influence of NO3-)

Zea mays (cv W64A x W182E) was used to investigate the induction and turnover of nitrate reductase (NR). In our system, 5 or 10 mM KNO3 gave the best growth over a 6-d growing period. With these NO3- levels, NR reached steady-state levels after 24 h. For the turnover experiments, the seedlings were transferred to a NO3--free medium after a 24-h induction. Shoot NR was less sensitive to the removal of NO3- than root NR, which declined almost as soon as NO3- was removed when the seedlings were induced with 5 or 10 mM NO3-. With 1 mM NO3-, however, removal of NO3- from medium resulted in declines in both NR activity and NO3- in shoot and root. Although there was a delay in the degradation of NR protein relative to the loss of NR activity, this protein was not reactivated when NO3- was resupplied. These results indicate that NO3- regulates NR by influencing the de novo synthesis of the NR protein and not by a reversible activation-inactivation of that protein.     

Endogenous alpha-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil

Alpha-ketol linolenic acid [KODA, 9,10-ketol-octadecadienoic acid, that is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] is a signal compound found in Lemna paucicostata after exposure to stress, such as drought, heat or osmotic stress. KODA reacts with catecholamines to generate products that strongly induce flowering, although KODA itself is inactive [Yokoyama et al. (2000) Plant Cell Physiol. 41: 110; Yamaguchi et al. (2001) Plant Cell Physiol. 42: 1201]. We examined the role of KODA in the flower-induction process of Pharbitis nil (violet). KODA was identified for the first time in seedlings of P. nil grown under a flower-inductive condition (16-h dark exposure), by means of LC-SIM and LC-MS/MS. In addition, the changes in endogenous KODA levels (evaluated after esterification of KODA with 9-anthryldiazomethane) during the flower-inductive phase in short day-induced cotyledons were closely related to flower induction. The KODA concentration sharply increased in seedlings during the last 2 h of a 16-h dark period, while the KODA level showed no significant elevation under continuous light. The increase of KODA level occurred in cotyledonal blades, but not in other parts (petiole, hypocotyls and shoot tip). When the 16-h dark period was interrupted with a 10-min light exposure at the 8th h, flower induction was blocked and KODA level also failed to increase. The degree of elevation of KODA concentration in response to 16-h dark exposure was the highest when the cotyledons had just unfolded, and gradually decreased in seedlings grown under continuous light for longer periods, reaching the basal level at the 3rd day after unfolding. Flower-inducing ability also decreased in a similar manner. These results suggest that KODA may be involved in flower induction in P. nil.     

Effect of lead on nitrate reductase activity and alleviation of lead toxicity by inorganic salts and 6-benzylaminopurine

A concentration dependent decrease in root nitrate reductase (E.C.1.6.6.1) activity (NRA) by 0.1 to 2.0 mM lead acetate was noticed in three cultivars of Vigna radiata (L.) Wilczek (K851, MH8320 and ML337). Leaf NRA, on the other hand, increased significantly with increasing lead concentration which was more pronounced in cvs. MH8320 and ML337 than in cv. K851. Total nitrogen content of root and shoot was generally increased due to supply of the lead acetate, whereas the total nitrogen content of the colyledons was hardly affected in cvs. MH8320 and ML337 and decreased in cv. K851 during the early growth phase. The inhibition of root NRA could be alleviated by addition of inorganic salts (K2HPO4 and KNO3, but not CaCl2) or 6-benzylaminopurine (BAP) in the incubation medium. Lead mediated inhibition of root NRA was similar in light and dark grown seedlings, but lead induced increase in leaf NRA was more pronounced in the light than in the dark. This revised version was published online in August 2006 with corrections to the Cover Date.     

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     

Changes in cotyledon mRNA during floral induction of Pharbitis nil CV. Violet

Floral induction in seedlings of Pharbitis nil Choisy cv. Violet, with one cotyledon removed, was manipulated by applying various photoperiodic treatments to the remaining cotyledon. Populations of polyadenylated RNA from treated cotyledons were examined to identify messages specifically involved in floral induction. The RNA was translated in vitro using a wheat-germ system, and the resulting translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Substantial qualitative and quantitative differences were found between mRNA from cotyledons of seedlings kept in continuous light (non-induced) and of seedlings given a 16-h dark period (induced). In contrast, inhibition of flowering with a night-break resulted only in one detectable, quantitative difference in mRNA.Abbreviations CL continuous light - kDa kilodalton - NB 16 h darkness+10 min red-light break, 8 h into the dark period - poly(A)⁺ RNA polyadenylated RNA (isolated by binding to a cellulose oligodeoxythymidine affinity column) - SD short day (16 h dark) - SDP short-day plant - SDS sodium dodecyl sulfate     

The role of cotyledons in the establishment of Suaeda physophora seedlings

The role of cotyledons in seedling establishment of the euhalophyte Suaeda physophora under non-saline and saline conditions (addition of 1 mM or 400 mM NaCl) was investigated. Survival and fresh and dry weights were greater for seedlings grown in the light (12-h light/12-h dark) than in the dark (24-h dark). The shading of cotyledons tended to decrease shoot height, shoot organic dry weight, number of leaves, and survival of seedlings regardless of NaCl treatment, but the effect of cotyledon shading was greater with 400 mM NaCl. Concentrations of Na⁺ were higher in cotyledons than in leaves, regardless of NaCl treatment. The K⁺/Na⁺ ratio was lower in cotyledons than in leaves for seedlings treated with 1 mM NaCl but not for seedlings treated with 400 mM NaCl. Addition of 400 mM NaCl decreased oxygen production in cotyledons but especially in leaves. These results are consistent with the hypothesis that, by generating oxygen via photosynthesis and by compartmentalizing Na⁺, cotyledons are crucial for the establishment of S. physophora seedlings in saline environments.     

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.     

Rapid modulation of nitrate reductase in leaves and roots: Indirect evidence for the involvement of protein phosphorylation/dephosphorylation

Nitrate reductase activity (NRA; NADH-nitrate reductase, E. C. 1.6.6.1) has been measured in extracts from leaves of spinach ( Spinacia oleracea L.) in response to rapid changes in illumination, or supply of CO₂ or oxygen. Measured in buffers containing magnesium, NRA from leaves decreased in the dark and increased again upon illumination. It decreased also, when CO₂ was removed in continuous light, and was reactivated when CO₂ was added. Nitrate reductase (NR) from roots of pea ( Pisum sativum L.) was also rapidly modulated in vivo. It increased under anaerobiosis and decreased in air or pure oxygen. The half time for inactivation or reactivation in roots and leaves was 5 to 30 min.
When spinach leaves were harvested during a normal day/night cycle, extractable NRA was low during the night, and high during daytime. However, at any point of the diurnal cycle, NR could be brought to a similar maximum activity by preincubation of the desalted leaf extract with AMP and/or EDTA. Thus, the observed diurnal changes appeared to be mainly a consequence of enzyme modulation, not of protein turnover. In vivo, the reactivation of the inactivated enzyme from both leaves and roots was prevented by okadaic acid, and inhibitor of certain protein phosphatases. Artificial lowering of the ATP-levels in leaf or root tissues by anaerobiosis (dark), mannose or the uncoupler carbonyl cyanide m -chlorophenyl hydrazon (CCCP), always brought about full activation of NR.
By preincubating crude leaf or root extracts with MgATP, NR was inactivated in vitro. Partial purification from spinach leaves of two enzymes with molecular masses in the 67 kD and 100 kD range, respectively, is reported. Both participate in the ATP-dependent inactivation of NR.
Alltogether these data indicate that NR can be rapidly modulated by reversible protein phosphorylation/dephosphorylation, both in shoots and in roots.