Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.     

Chloroplast DNA variation and postglacial recolonization of common ash (Fraxinus excelsior L.) in Europe

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.     

Control and variation ofin vivo nitrate reductase activity inLolium perenne L. cv S24

Summary Variation in nitrate reductase activity associated with vegetative development and morphological differences inLolium perenne L. cv S24 was examined using anin vivo technique of enzyme assay. Little difference in enzyme activity per unit weight of leaf laminar tissue was found between tillers, between and within leaf laminae but activity in leaf laminae, was much higher than that of leaf sheaths; the activity of shoot material was greater than the apparent root activity. Senescence of leaf material was not accompanied by marked changes invivo nitrate reductase activity.     

Nitrate reductase in needles,roots and trunk wood of spruce trees [Picea abies (L.) Karst.]

Summary Nitrate reductase (EC 1.6.6.2) activity (NRA), as measured by an in vivo assay, is present in needle leaves and mycorrhizal fine root tips of adult Norway spruce [Picea abies (L.) Karst.] in at least equal amounts on a fresh weight basis, in both adult and 5-year-old trees. NRA could also be demonstrated in trunk wood of deroted trees after fertilization with 5 mM , exhibiting a longitudinal profile in the trunk. Inducibility in needles can more efficiently be achieved by NO₂ (100 g·m^-3) than by 5 mM nitrate, which is effective only in root-amputated trees. A remarkably high level of needle-NRA in unfertilized trees, which are characterized by a very low level of nitrate in the xylem sap, suggests that NRA in spruce needles may in part be constitutive. Organic-N is a major nitrogen source for the needles even in root-amputated trees, indicating pronounced exchange processes between ray parenchyma and trunk xylem, which in turn are modified by the nitrogen source fed to the trunk stump. Intact trees exhibit a very similar amino acid composition of the xylem sap, regardless of whether or has been fed. The amino acid pattern of the needles is not thrown out of balance by flooding with and , which occurs in fertilized derooted trees. This indicates a distinct potential for homoeostasis of nitrogen entrance-metabolism (i.e. NRA and glutamine synthetase activity) in the needles. In the ectomycorrhiza/fine root-system (EMC), marked differences in NRA were observed depending on root-tip diameter and along the longitudinal profile of the fine roots. EMC-nitrate reductase is strongly enhanced by . Needle-NRA exhibits a circannual rhythm. An early summer maximum is followed by a December minimum. This activity pattern matches well the transitory increase of soluble nitrogen in spring and the total protein maximum in winter. In an indirect way assimilatory NRA may well contribute to nitrogen overfertilization (by consumption of NO_X) as one possible cause of the contemporary decline of spruce populations.     

Characterization of a rice (Oryza sativa L.) mutant deficient in the heme domain of nitrate reductase

Summary Biochemical and genetical characterization of a rice nitrate reductase (NR)-deficient mutant, M819, which had been isolated as a chlorate-resistant mutant, was carried out. In M819, leaf NADH-NR activity was found to be about 10% of that of the wild-type cv Norin 8, while NADPH-NR activity was higher than that in the wild-type; FMNH₂-NR and MV-NR activities were also 10% of those of the wild type; BPB-NR activity was higher than that of the wild type; and xanthine dehydrogenase activity was revealed to be present in both. These results suggest that the mutant line M819 lacks the functional heme domain of the NADH-NR polypeptide due to a point mutation or a small deletion within the coding region of the structural gene. Chlorate resistance in M819 was transmitted by a single recessive nuclear gene.Abbreviations NR Nitrate reductase - NiR nitrite reductase - FMNH₂ reduced flavin nucleotide - MV reduced methyl viologen - BPB reduced bromphenol blue - XDH xanthine dehydrogenase     

Nitrate nutrition ofDeschampsia flexuosa (L.) Trin. in relation to nitrogen deposition in Sweden

Summary Current and maximally induced nitrate reductase activity (NRA), total-N, nitrate, K, P, Ca, Mg, Mo and sucrose in leaves ofDeschampsia flexuosa was measured three times during the vegetation period in forests along a deposition gradient (150 km) in south Sweden, in north Sweden where the nitrogen deposition is considerably lower, and at heavily N-fertilized plots. In addition, the interaction between nitrogen nutrition and light was studied along transects from clearings into forest in both south and north Sweden. Plants from sites with high nitrogen deposition had elevated current NRA compared to plants from less polluted sites, indicating high levels of available soil nitrate at the former. Current NRA and total N concentration in grass from sites with high deposition resembled those found at heavily N-fertilized plots. Under such circumstances, the ratio current NRA: maximally induced NRA as well as the concentration of nitrate was high, while the concentration of sucrose was low. This suggests that the grass at these sites was already utilizing a large portion of its capacity to assimilate nitrate. Light was found to play an important role in the assimilation of nitrate; leaf concentration of sucrose was found to be negatively correlated with both nitrate and total N. Consequently, grass growing under dense canopies in south Sweden is not able to dilute N by increasing growth. The diminished capacity of the grass to assimilate nitrate will increase leaching losses of N from forests approaching N saturation.     

Microautoradiographic detection of CO2 fixation in lenticel chlorenchyma of youngFraxinus excelsior L. stems in early spring

Microautoradiography indicated that 1-year-oldFraxinus excelsior L. stem chlorenchyma assimilated external¹⁴CO₂ in mid-April, when buds were swollen, but before bud-break. The lenticel regions showed the highest amount of radioactively labeled assimilates. Labeled assimilates declined in the tangential direction with increasing distance from lenticels, suggesting that¹⁴CO₂ entered the stem through the open intercellular spaces of lenticels. In the radial direction, the amount of radioactively labeled assimilates did not constantly decline with growing distance from the lenticel entrance. It was high in all lenticel phelloderm cells, which had high chlorophyll autofluorescence and very small starch grains, highest in the adjacent 4–6 rows of chlorenchyma, which had larger starch grains that increased in size towards the interior rows, and much lower in the inner cortex chlorenchyma, which had large starch grains. We suggest that the main function of the lenticel chlorenchyma (lenticel phelloderm plus 4–6 rows of adjacent cortex chlorenchyma) is the refixation of respiratory CO₂ which could easily leave the stem intercellular spaces, rather than the fixation of external CO₂. The lenticel chlorenchyma could reduce the loss of respiratory CO₂ by its photosynthetic activity.     

Interaction between electron transport at the plasma membrane and nitrate uptake by maize (Zea mays L.) roots

Summary In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 M nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 M dicumarol but was not altered by 100 M dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.Abbreviations ADH alcohol dehydrogenase - HCF III hexacyanoferrate III (ferricyanide) - ME NADP-dependent malic enzyme - NR nitrate reductase - PM plasma membrane - PM NR nitrate reductase copurifying with plasma membranes     

The presence of extended phragmosomes containing cytoskeletal elements in fusiform cambial cells ofFraxinus excelsior L.

Summary Fusiform cambial cells of the ash (Fraxinus excelsior L.), which are strongly elongated and vacuolated, contain a phragmosome which traverses the whole length of the cells during preprophase and karyokinesis and which remains present during cytokinesis until it is integrated in cell plate with adjacent cytoplasm.The phragmosome consists of a thin perforated cytoplasmic layer located in the plane of the future cell plate. Otherwise oriented transvacuolar cytoplasmic layers or strands are not present in these cells.The phragmosome contains cytoskeletal elements, namely microtubules and also microfilament bundles both of which are oriented mainly in longitudinal direction.The phragmosomal microtubules are a new category of microtubules associated with cell division; presumably they guide the centrifugally growing cell plate to the parental cell wall site previously marked by the preprophase band of microtubules.     

Molecular biodiversity and population structure in common ash (Fraxinus excelsior L.) in Britain: implications for conservation

Current forestry policy promotes the use of local seed for new plantings, on the assumption that local material may be better adapted to local conditions. However, landscape‐scale genetic studies which are necessary to underpin conservation and breeding strategies are often lacking. We investigated molecular diversity in common ash (Fraxinus excelsior L.) sampled from 42 British and six French sites with microsatellites. Chloroplast haplotype H04 was the most common and widespread in Britain, although rare and localized individuals with H02 and H09 were also detected. In addition, three new chloroplast haplotypes were identified, and these were rare and highly localized. In terms of nuclear microsatellite markers, allelic richness differed between sites and decreased in an east to west direction. Differentiation between sites was often very low (mean F_ST 0.025), indicating few differences between the majority of sites. There was a clear excess of homozygotes (mean H_O 0.669, mean H_E 0.818) and a relatively high F_IS (mean 0.182), suggests a consistent level of inbreeding or a widespread Wahlund effect in many F. excelsior sites. Gene pool ancestry analysis suggested that the majority of British F. excelsior belongs to a single meta‐population which covers mainland western and central Europe. Three northern and western sites diverged markedly from the dominant population, and may represent remnants of two late potential Ice Age refugia in northern Britain. The data provide new information which will aid development of appropriate conservation policies for ash and other wind pollinated tree species.     

Presence of natural genetic resistance in Fraxinus excelsior (Oleraceae) to Chalara fraxinea (Ascomycota): an emerging infectious disease

Fraxinus excelsior, common ash native to Europe, is threatened by a recently identified pathogenic fungus Chalara fraxinea, which causes extensive damage on ash trees across Europe. In Denmark, most stands are severely affected leaving many trees with dead crowns. However, single trees show notably fewer symptoms. In this study, the impact of the emerging infectious disease on native Danish ash trees is assessed by estimating presence of inherent resistance in natural populations. Disease symptoms were assessed from 2007 to 2009 at two different sites with grafted ramets of 39 selected clones representing native F. excelsior trees. A strong genetic variation in susceptibility to C. fraxinea infections was observed. No genetic or geographic structure can explain the differences, but strong genetic correlations to leaf senescence were observed. The results suggest that a small fraction of trees in the Danish population of ash possess substantial resistance against the damage. Though this fraction is probably too low to avoid population collapse in most natural or managed ash forests, the observed presence of putative resistance against the emerging infectious disease in natural stands is likely to be of evolutionary importance. This provides prospects of future maintenance of the species through natural or artificial selection in favour of remaining healthy individuals.     

In nature dormant buds and in vitro dormant-like buds ofFraxinus excelsior L.

Summary Microcuttings ofFraxinus excelsior, sampled from adult trees during the period of cell cycle blockage of bud in G_0–1, developed long rejuvenated sprouts on the Woody Plant Medium (WPM) with benzylaminopurine (BAP) (4.0 mg/l) and indolebutyric acid (IBA) (0.03 mg/l). These sprouts had the ability to enter a resting period, building dormant-like buds when maintained on the original WPM. Sprouts developed from subcultures also entered a resting period without any transfer. Comparison of in nature buds in active growth and dormancy with buds of growing sprouts and in vitro dormant-like buds revealed similarity in behaviour at the shoot apical level. In particular, in dormant-like buds in a constant environment, shoot apical functioning was suppressed while the cell cycle of the shoot apex was blocked at the G_0–1 phase, like in nature dormant buds.Abbreviations a.u. arbitrary unit - BAP benzylaminopurine - CF cumulative frequency - d₁, d₂ diameters of the shoot apex - IBA indolebutyric acid - P_n last opposite primordia of range n - WPM woody plant medium     

The seasonal variation of 20 elements in 1st and 2nd year needles of Norway spruce,Picea abies (L.) Karst

Summary The concentration of 20 elements (including eight nutritional elements) in spruce needles was monitored by taking samples at nine different dates in a single year. On each date the youngest and the 1-year-old needles were collected at four different sites. The needles were washed with an organic solvent to remove surface contamination before analysis by instrumental neutron activation. Although the sites showed widely different concentrations for the same element, they could be treated as a uniform population using normalized concentration values. Taking the time dependence of the concentration and the ratio of the concentrations in new and old needles as criteria, the following three groups of elements could be distinguished: 9 group I —Ca, Sr, Ba, and Mn; group II — Al, Br, Co, Fe, Hg, La, Sc, Sb, and Zn; group III — K, Rb, Cs, P, and Cl. Mg and Na did not fit into any of these groups. Within group I and III elements the strongest variations occur during the growing season (late spring and early summer) and the weakest during the dormant season; the variation is greater in the younger needles. Group II elements show an approximately linear increase throughout the year in both the younger and older needles. The concentrations of elements of groups I and II increase with time, while those of group III decrease. Na is unique insofar as its concentration is influenced by foliar uptake of road de-icing salt. The concentration of elements belonging to the same group shows strong inter-element correlations. The grouping of elements deduced here shows similarities with the chemical nature of the elements and with their transportability within the plant.     

Changes in dormancy levels ofFraxinus excelsior L. embryos at different stages of morphological and physiological maturity

The dormancy status ofFraxinus excelsior embryos at different developmental stages under environmental conditions was examined over a period of 2 years. For each sampling date the length of the fruit, of the seed, and of the embryo were measured, and the embryological stage determined. The depth of dormancy was assayed by the germination behaviour of isolated embryos under aseptic conditions on an agar medium without nutrients. As an approach towards a quantitative estimate of the dormancy status, the degree of inhibiton of germinative growth in the embryonic organs was evaluated on the basis of four categories from none to full germinative growth. From these ratings a dormancy index was calculated, expressing the mean dormancy status of the embryos at a given date. Embryo dormancy already became apparent during embryogenesis and reached its highest level during the later phase of reserve deposition in the seed. A marked loss of embryo dormancy occurred during the phase of maturation drying in autumn, followed by a moderate increase in winter. In hydrated seeds in spring the embryo was gradually released from dormancy and enlarged further. In maintaining the embryo ofF. excelsior in a developmental but not germinative mode, dormancy mechanisms within the embryo and the endosperm, combined with environmental factors, may be involved.     

Nitrate reductase induction and molecular characterization in rice (Oryza sativa L.)

Summary Barley nitrate reductase cDNA clone bNRp10 was used as a hybridization probe to screen a genomic DNA library of rice (Oryza sativa L.) cultivar M201. Two different lambda clones were isolated, subcloned to plasmids, and partially characterized. The subclone pHBH1 was tentatively identified as encoding a NADH nitrate reductase. Southern and dot blot analysis suggest that, in rice, nitrate reductase is encoded by a small gene family. Regulation of NADH nitrate reductase was investigated in rice cultivars Labelle and M201 representing the subspecies indica and japonica, respectively. In the absence of nitrate, only trace levels of nitrate reductase activity and mRNA were detected in seedling leaves. Upon addition of nitrate to seedling roots, nitrate reductase activity and mRNA increased rapidly in leaves. Nitrate reductase activity continued to increase over a 24 h period, but the mRNA accumulation peaked at about 6 h and then declined. Western blot analysis with a barley NADH nitrate reductase antiserum showed the presence of two bands of approximately 115 and 105 kDa. These protein bands were not detected in extracts of tissue grown in the absence of nitrate.     

Appearance of nitrite reductase in cotyledons of the mustard (Sinapis alba L.) seedling as affected by nitrate,phytochrome and photooxidative damage of plastids

Nitrite reductase (NIR; EC 1.7.7.1) is a central enzyme in nitrate assimilation and is localized in plastids. The present study concerns the regulation of the appearance of NIR in cotyledons of the mustard (Sinapis alba L.) seedling. It was shown that light exerts its positive control over the nitrate-mediated induction of NIR via the farred-absorbing form of phytochrome. Without nitrate the light effect cannot express itself; even though the light signal is accumulated in the cotyledons it remains totally cryptic in the absence of nitrate. Moreover, it was recognised that intact plastids are important in the control of the appearance of NIR. If the plastids are damaged by photooxidation the action of nitrate and phytochrome on NIR appearance is abolished. The appearance of nitrate reductase (NR; EC 1.6.6.1) responds similarly to photooxidative damage even though this enzyme is cytosolic. While the data strongly indicate that some plastidic signal is a prerequisite for the nitrate-induced and phytochrome-modulated appearance of NIR and NR, the possibility could not be ruled out that photooxidative damage affects the accumulation of NIR in the organelle.Abbreviations c continuous - D darkness - FR far-red light - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.1.13) - NF Norflurazon - NIR nitrite reductase (EC 1.7.7.1.) - NR nitrate reductase (EC 1.6.6.1) - Pfr phytochrome (far-red light obtained with RG9 glass filter - R red light - RG9-light long wavelenght far-red light obtained with RG9 glass filter - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - WL white light - WL_s strong white light (28 W m^-2)     

Nitrate reductase and glutamine synthetase activities,nitrate and ammonia assimilation,in the unicellular alga Cyanidium caldarium

Nitrogen-limited continuous cultures of Cyanidium caldarium contained induced levels of glutamine synthetase and nitrate reductase when either nitrate or ammonia was the sole nitrogen source. Nitrate reductase occurred in a catalytically active form. In the presence of excess ammonia, glutamine synthetase and nitrate reductase were repressed, the latter enzyme completely. In the presence of excess nitrate, intermediate levels of glutamine synthetase activity occurred. Nitrate reductase was derepressed but occurred up to 60% in a catalytically inactive form.Cell suspensions of C. caldarium from nitrate- or ammonialimited cultures assimilated either ammonia or nitrate immediately when provided with these nutrients. In these types of cells, as well as in cells grown with excess nitrate, the rate of ammonia assimilation was 2.5-fold higher than the rate of nitrate assimilation. It is proposed that the reduced rate at which nitrate was assimilated as compared to ammonia might be due to regulatory mechanisms which operate at the level of nitrate reductase activity.