Aldehyde oxidase in roots, leaves and seeds of barley (Hordeum vulgare L.)

Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves, roots and seeds of barley (Hordeum vulgare L.) plants were studied. Differences in substrate specificity and mobility in native PAGE between AO proteins extracted from roots, leaves and seeds have been observed. Four clear bands of AO reacting proteins were detected in barley plants capable of oxidizing a number of aliphatic and aromatic aldehydes such as indole-3-aldehyde, acetaldehyde, heptaldehyde, and benzaldehyde. Mouse polyclonal antibodies raised against purified maize AO cross-reacted with barley AO proteins extracted from roots, leaves and seeds. At least three different AO proteins were detected in roots on the basis of their mobility during PAGE after native Western blot analysis while in leaves and seeds only one polypeptide cross-reacted with the antibody. SDS-immunoblot analysis showed marked differences in molecular weight between subunits of the AO bands extracted from roots, leaves and seeds. Two distinct subunit bands were observed in roots, with relatively close molecular weights (160 kDa and 145 kDa), while a single subunit with a molecular weight of 150 kDa was observed in leaf and seed extracts.Menadione, a specific and potent inhibitor of animal AO did not affect barley AO proteins. Root and leaf AO differed in their thermostability and susceptibility to exogenous tungstate. The AO proteins in plants may be a group of enzymes with different substrate specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ.     

Regulation of aldehyde oxidase and nitrate reductase in roots of barley (Hordeum vulgare L.) by nitrogen source and salinity

The molybdenum cofactor (MoCo) is a component of aldehyde oxidase (AO EC 1.2.3.1), xanthine dehydrogenase (XDH EC 1.2.1.37) and nitrate reductase (NR, EC 1.6.6.1). The activity of AO, which catalyses the last step of the synthesis of abscisic acid (ABA), was studied in leaves and roots of barley (Hordeum vulgare L.) plants grown on nitrate or ammonia with or without salinity. The activity of AO in roots was enhanced in plants grown with ammonium while nitrate-grown plants exhibited only traces. Root AO in barley was enhanced by salinity in the presence of nitrate or ammonia in the nutrient medium while leaf AO was not significantly affected by the nitrogen source or salinity of the medium.Salinity and ammonium decreased NR activity in roots while increasing the overall MoCo content of the tissue. The highest level of AO in barley roots was observed in plants grown with ammonium and NaCl, treatments that had only a marginal effect on leaf AO. ABA concentration in leaves of plants increased with salinity and ammonium.Keywords: ABA, aldehyde oxidase, ammonium, nitrate, salinity.      

Ammonium induction of a novel isoform of glucose-6P dehydrogenase in barley roots

The effects of ammonium and glutamine supply on amino acid levels and the activity of glucose-6P dehydrogenase (G6PDH EC 1.1.1.49), the main regulated enzyme of the oxidative pentose phosphate pathway, were investigated in barley roots ( Hordeum vulgare cv. Alfeo). Feeding ammonium to barley plants increased the contents of glutamine, asparagine and G6PDH in roots. These effects were abolished by using inhibitors of glutamine synthetase. Glutamine-fed barley roots showed a similar increase in G6PDH activities to ammonium-fed plants. Two G6PDH enzymes (G6PDH 1 and 2) were partially purified and characterized from ammonium-fed and glutamine-fed roots. The isozymes had different pH optima and apparent Km values for glucose-6P. G6PDH 2 showed similar kinetic parameters to the G6PDH present in root extracts of barley grown without any nitrogen source, while G6PDH 1 exhibited different kinetic parameters, suggesting the appearance of a second G6PDH isoform in response to ammonium. Western blot analysis demonstrated the existence of two G6PDH subunits of different molecular mass in barley roots grown in the presence of ammonium or glutamine, while only one isoform could be detected in roots grown without any nitrogen source. The results suggest a primary role of ammonium and/or glutamine in the appearance of a novel G6PDH isoform; this enzyme (G6PDH 1) shows kinetic parameters similar to those measured previously for chloroplastic and plastidic isoforms and seems to be induced by changes in glutamine content or a related compound(s) in the roots.     

Effects of Exposure to Ammonium and Transplant Shock upon the Induction of Nitrate Absorption

In barley (Hordeum vulgare L. cv Steptoe) seedlings, the time course for induction of root nitrate absorption varied significantly with pretreatment. Net nitrate uptake of nitrogen-deprived plants more than doubled during the 12 hours after first exposure to nitrate. For these plants, gentle physical disturbance of the roots inhibited net nitrate absorption for more than 6 hours and potassium absorption for 2 hours. Pretreatment with ammonium appeared sufficient to induce nitrate absorption; plants either grown for 2 weeks on or exposed for only 10 hours to a medium containing ammonium as a sole nitrogen source showed high rates of net nitrate uptake when first shifted to a medium containing nitrate. Gentle physical manipulation of these plants inhibited nitrate absorption for 2 hours and potassium absorption for more than 12 hours. These results indicate (a) that experimental protocols should avoid physical manipulation of the roots when-ever possible and (b) that ammonium or a product of ammonium assimilation can induce nitrate absorption.     

Transport and accumulation rates of abscisic acid and aldehyde oxidase activity in Pisum sativum L. in response to suboptimal growth conditions.

Pea plants (Pisum sativum L.) grown initially in nutrient solutions with adequate nitrogen supply (4 mM NO3-) were transferred to solutions containing salt (50 or 100 mM NaCl), ammonium (4 mM) or a low nitrogen supply (0.4 mM NO3-). No changes of abscisic acid (ABA) content were found in roots of stressed pea plants 9 d after the beginning of the treatments; however, accumulation of ABA in the leaves was observed. Old leaves accumulated ABA to a higher extent than young leaves. Accumulation of ABA in leaves of ammonium-fed plants and plants grown under low nitrogen supply occurred in the absence of both increased ABA xylem loading rate and enhanced aldehyde oxidase (AO, EC 1.2.3.1) activity in roots. Enhanced leaf AO activity was observed in all treatments, with the highest increase in old leaves. Among the three AO isoforms (AO-1, AO-2 and AO-3) detected in extracts of pea leaves, the lowest one AO-3 (highest mobility in the gel) correlated with ABA production and showed the highest increment in response to the treatments. The increase of AO activity detected in leaves after 2 weeks of stress application was less prominent than after 9 d, suggesting a transient enhancement of ABA production following the onset of stress. An increase of ABA xylem loading rate as well as AO root activity 4 d and 9 d after application of the treatments was observed only in salt-treated plants followed by a decrease after 14 d in 100 mM NaCl. Decreased cytokinin (trans-zeatin riboside) delivery rate into the xylem sap was observed in all treatments. The role of abscisic acid and cytokinins as positive and negative growth signals, as well as the involvement of root-generated ABA on ABA accumulation in leaves is discussed.     

An investigation into the interaction between nitrogen nutrition,photosynthesis and photorespiration

Photosynthetic CO₂ assimilation, photorespiration and levels of glycollate oxidase and ribulose bisphosphate (RuBP) carboxylase were measured in barley, wheat and maize plants grown on media containing nitrate or ammonium or in plants transferred from nitrate to ammonium. The CO₂ compensation point and photorespiratory CO₂ release were not altered by the nitrogen growth regime nor by transfer from nitrate to ammonium. In barley and wheat plants grown on ammonium the levels of glycollate oxidase and RuBP carboxylase per unit leaf area were higher than in nitrate grown material. These differences were not evident when the results were expressed on a protein or chlorophyll basis. The ratio of glycollate oxidase activity to RuBP carboxylase activity was not altered by the nitrogen regime.     

Purification and characterization of a safener-induced glutathione S-transferase from wheat (Triticum aestivum)

The genome of cultivated wheat is hexaploid, and in consequence a large number of glutathione S-transferase (GSTs, EC 2.5.1.18) isozymes is expected in that organism. Wheat GST subunits were first analyzed by reverse-phase high performance liquid chromatography (RP-HPLC). In root and shoot tissues, subunits 4, 8, and 9 were constitutively expressed whereas subunits 2, 3, and 5 were inducible by the herbicide safener naphthalic anhydride (NA). Significant differences were observed, however, between the distributions of these six major subunits in roots and shoots. A major GST isozyme was purified from the shoots of plants treated by NA. A combination of ammonium sulphate precipitation, hydrophobic interaction chromatography (HIC) and affinity chromatography resulted in purification with an apparent yield of 4.6% and a 48-fold increase in specific activity toward 1-chloro-2,4-dinitrobenzene (CDNB). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band at 24.5 kDa. Molecular mass estimated by nondenaturing PAGE was 49.5 kDa. These results suggest that the enzyme exists as a dimer. A pI of 5.2 was determined by native isoelectric focusing (IEF). Analysis by 2-D electrophoresis showed a single spot, with a pI of 5.8–5.9. However, further analysis by RP-HPLC revealed that the two subunits were different. They were characterized and identified by electrospray ionization mass spectrometry (ESI-MS) as subunits 2 and 3, molecular masses 24 924±3 and 24 958±5 Da, respectively. Therefore, GST(2–3) is apparently a heterodimer consisting of subunits 2 and 3. Apparent K_M values were 424 μ M for CDNB and 228 μ M for glutathione (GSH). GST(2–3) metabolized the herbicide fluorodifen, and a K _M of 22 μ M was determined for the herbicide.     

Antioxidative system in maize roots as affected by osmotic stress and different nitrogen sources

The activities of antioxidative enzymes and contents of proline and total phenolics were assayed in roots of two maize (Zea mays L.) genotypes grown in a medium containing nitrate (NO₃ ⁻) or both nitrogen forms, nitrate and ammonium (NH₄ ⁺/NO₃ ⁻). An increase in the activities of class III peroxidases (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), ascorbate oxidase (AO) and proline content, and decrease in phenolic content were observed in NH₄ ⁺/NO₃ ⁻ in comparison with NO₃ ⁻ grown plants. When polyethylene glycol (PEG) was added to both nitrogen treatments, the content of total phenolics and proline was increased, especially in NH₄ ⁺/NO₃ ⁻ treatment. The PEG treatment decreased enzyme activities in NH₄ ⁺/NO₃ ⁻ grown plants, but in NO₃ ⁻ grown plants activities of POD and SOD were increased, opposite to decreased APX and AO. Isoelectric focusing demonstrated increased activities of acidic POD isoforms in PEG treated NO₃ ⁻ grown plants, and lower activities of both, acidic and basic isoforms in NH₄ ⁺/NO₃ ⁻grown plants.     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: I. COMPARTMENTAL ANALYSIS FROM MEASUREMENTS OF 13N EFFLUX

The short-lived radio-isotope nitrogen-13 (half-life 10 min)was used as a tracer in studying fluxes of N in the roots ofintact barley plants. After supplying the plants with ¹³N-nitratefor 30 min, efflux of ¹³N into an unlabelled (wash) solutionwas followed under steady-state conditions for a further 10min. Tests with ion exchange resins suggested that all of the¹³N released during this period was in the form of nitrate. In addition to nitrate from a surface film of solution and fromthe free space of the roots, efflux from another compartmentwas detected, tentatively identified as the cytoplasmic nitratepool. In plants grown with nitrate as the only external N-source,efflux from this compartment decreased with a rate constantabout 0·17 min^–1 (half-time 4 min). Adding ammoniumsulphate to the wash solution alone did not significantly affecteither the initial rate, or the rate constant, of efflux of¹³N from these roots. However, ¹³N efflux decreased more rapidly(rate constant about 0·32 min^–1, half-time 2·2min) in roots grown in, and subsequently washed with, solutioncontaining ammonium nitrate. In barley plants grown with 1·5 mol m^–3 nitrate,the cytoplasmic nitrate pool was estimated to contain about2% of the total nitrate in the roots, corresponding to a cytoplasmicnitrate concentration 26 mol m^–3. Nitrate efflux was equivalentto almost 40% of nitrate influx in the roots of these plants. Key words: Ion transport, nitrate, ammonium, efflux analysis, compartmentation     

The effect of ammonium ions on membrane potential and anion flux in roots of barley and tomato

The effects of ammonium (0–5 mol m^?3) on root hair membrane potential and on the influx of nitrate and phosphate were investigated in roots of intact barley and tomato plants. In both species, addition of ammonium to the medium bathing the roots caused an almost immediate depolarization of the membrane potential; the depolarization was greater at higher concentrations of ammonium. Influx of ¹³NC₃^? and ³²Pi was inhibited over the same time scale and concentration range. In tomato roots, there was little further depolarization of the membrane potential or inhibition of anion influx at ammonium concentrations above 0.4 mol m^?3. In barley roots, the inhibition of nitrate influx and the depolarization of the membrane potential did not saturate below 5 mol m^?3 ammonium.     

Local NO3- or NH4+ supply modifies the root system architecture of Cedrus atlantica seedlings grown in a split-root device

To study the effects of local nitrate or ammonium supply on the architecture of the Cedrus atlantica root system, cedar seedlings were grown in split-root boxes in a growth chamber. In each box-compartment, roots were fertilized with a solution containing nitrogen, either as nitrate [Ca(NO₃)₂] or ammonium (NH₄Cl), supplied at 0.1 or 5.0 mM. For each seedling, the shoot growth was measured twice a week for 3 months. The root system architecture was also recorded twice a week by tracing the root elongation through the transparent face of the root observation boxes. The apical diameter of the tap-root relay and that of a representative sample of lateral roots were recorded once a month using a monocular magnifier.

The increase of ammonium or nitrate concentration in the nutrient solution has significantly enhanced the production of lateral roots on the tap-root relay. After 90 days of culture, percentages of short lateral roots obtained with nitrate were higher than those obtained using ammonium. A preferential carbon allocation to the shoots was also obtained with an increasing nitrogen supply. Until the 40th day of culture, the elongation of lateral roots was similar for all treatments and ranged from 0.25 to 0.5 cm day⁻¹. From the 40th day to the 95th day, significant differences were observed between the compared modes and maximum elongation rates were obtained with 5 mM NH₄⁺ (2.18 cm day⁻¹) and 5 mM NO₃⁻ (1.18 cm day⁻¹). Local applications of nitrate and ammonium at a low or a high concentration had local effects on elongation and branching of the root system in the fertilized compartment. Contrasting effects of ammonium and nitrate were observed on the apical diameter of tap-roots and lateral roots. The root-split culture device confirmed that nitrate had local effects on the architecture of the C. atlantica root system.     

The effect of Nitrogen on the size of spring barley root system as determined by means of its electric capacity

The size of the spring barley root system was studied on the basis of its electric capacity in plants grown in nutrient solutions either lacking or containing nitrogen in the form of nitrate or ammonium. Root electric capacity changed in dependence on nutrition from Day 12 after emergence, when F values increased in the root systems of plants exposed to nitrate and ammonium salts. In plants grown in H₂O, the values of electric capacity statistically significantly decreased on Days 15 to 17, in plants grown in PK solution lacking nitrogen on Day 20. Root electric capacity of plants grown in full nutrient solution gradually increased on Day 18 after emergence. Then a marked increase in root electric capacity values followed with no statistically significant differences between NH₄ ⁺ and NO₃ ^- nutrition. Nitrate nutrition of barley plants only resulted in an increased root to shoot mass ratio.     

Nitrogen assimilation and cysteine biosynthesis in barley: Evidence for root sulphur assimilation upon recovery from N deprivation

The mixed effects of nitrogen nutrition and sulphate assimilation were investigated in barley plants (Hordeum vulgare var. Alfeo) that were subjected to long-term sulphur and/or nitrogen starvation, by measuring the O-acetylserine(thio)lyase (OASTL-EC 4.2.99.8) activity, changes in -SH compounds and amino acid levels.The growth of barley plants cultured in the hydroponic vessels was severely affected by altered nutrient levels. The barley plants grown in medium deprived of nitrogen and/or sulphur sources for 21 days showed increase in both root length and weight. In contrast, the shoot growth was reduced in nitrogen-starved plants and was unaffected by sulphur deprivation. Sulphur starvation affected the level of proteins in barley plants more than nitrogen deprivation. The decline in the protein levels observed under sulphur-deficient conditions was coupled with the accumulation of glutamine, asparagine and serine, mainly in the roots; additionally, a nitrogen deficiency in the roots promoted a decrease in both glutathione and cysteine levels.The simultaneous deprivation of nitrogen and sulphur in plants leads to an alteration in their metabolism; high levels of glutathione (GSH) in the shoots could signify the induction of a mechanism intended for coping with stressful conditions.Sulphate deprivation enhanced OASTL activity, mainly in the roots; on the other hand, OASTL increases observed under S deprivation were clearly dependent on the nitrogen availability in the culture medium. In fact, the nitrate supply to the N and S starved plants that showed OASTL activity very low, rapidly recovered the OASTL activities to the levels typical of control plants. Nevertheless, the ammonium supply had negligible effects on the OASTL activity only observed after three days in the roots.Our results support the hypothesis that in barley plants, a portion of S assimilation (up to cysteine biosynthesis) occurs in the roots, and a reciprocal influence of nitrogen assimilation on cysteine synthesis occurs.     

Nitrogen Assimilation in Barley (Hordeum vulgare L. cv. Mazurka) in Response to Nitrate and Ammonium Nutrition

Barley plants (Hordeum vulgare L. cv. Mazurka) were grown inaerated solution cultures with 2 mM or 8 mM inorganic nitrogensupplied as nitrate alone, ammonium alone or 1:1 nitrate+ammonium.Activities of the principal inorganic nitrogen assimilatoryenzymes and nitrogen transport were measured. Activities ofnitrate and nitrite reductases, glutamine synthetase and glutamatesynthase were greater in leaves than in roots but glutamatedehydrogenase was most active in roots. Only nitrate and nitritereductases changed notably (4–10 times) in response tothe different nitrogen treatments. Nitrate reductase appearedto be rate-limiting for nitrate assimilation to glutamate inroots and also in leaves, where its total in vitro activitywas closely related to nitrate flux in the xylem sap and wasslightly in excess of that needed to reduce the transportednitrate. Xylem nitrate concentration was 13 times greater thanthat in the nutrient solution. Ammonium nitrogen was assimilatedalmost completely in the roots and the small amount releasedinto the xylem sap was similar for the nitrate and the ammoniumtreatments. The presence of ammonium in the nutrient decreasedboth export of nitrate to the xylem and its accumulation inleaves and roots. Nitrate was stored in stem bases and was releasedto the xylem and thence to the leaves during nitrogen starvation.In these experiments, ammonium was assimilated principally inthe roots and nitrate in the leaves. Any advantage of this divisionof function may depend partly on total conversion of inorganicnitrogen to amino acids when nitrate and ammonium are givenin optimal concentrations. Hordeum vulgare L., barley, nitrate, ammonium, nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, nitrogen transport     

Purification and properties of alcohol oxidase from <Emphasis Type="Italic">Pichia putida</Emphasis>

Alcohol oxidase (AO) was extracted from the methylotrophic yeast Pichia putida and purified using various methods. AO purified by crystallization was homogeneous based on analytical centrifugation with subsequent gel filtration and SDS-PAGE. The molecular weight of the enzyme was around 600 kDa. SDS-PAGE revealed a single protein band (74 ± 4 kDa), and 8–9 bands of native protein with similar specific AO activities and substrate specificities were identified by PAGE without SDS. Electron microscopy of a single molecule revealed eight subunits located on the top of a regular tetragon with dotted symmetry of 422D4 providing evidence that AO consists of eight subunits. Apparently, each molecule of AO has two types of subunits with very similar molecular weights and differing from each other by the number of acidic and basic amino acid residues. Each subunit includes one molecule of FAD and 2–3 cysteine residues. The pH optimum was within 8.5–9.0. Specific activity of the enzyme varied from 10 to 50 μmol methanol/min per mg protein from batch to batch depending on separation methods and had linear relationship with protein concentration. The AO was quickly inactivated at 20°C and seemed to be stable in phosphate-citrate buffer with 30–50% (w/v) of sucrose. Different forms of 0.1–1 mm crystals of the enzyme were obtained. However the crystals did not yield X-ray reflections, apparently as a result of their molecular microheterogeneity.     

The effect of NO3- and NH4 + ions on enzymes involved in nitrogen assimilation inPisum sativum L

Nitrate reductase level in leaves of pea plants is higher than in roots despite of the lower content of endogenous nitrate. Addition of ammonium ions to nutrient solution containing nitrate decreases nitrate reductase level in leaves estimatedin vivo while its level estimatedin vitro is increased. Glutamine synthetase (GS) level in roots decreases during short (24 and 48 h) and long (14 d) term cultivation of seedlings in solutions containing ammonium ions. This decrease occurs in leaves only after the long term influence of ammonium ions. Level of this enzyme is higher in plants grown in the presence of nitrogen (ammonium and nitrate) as compared to those grown without the nitrogen. Level of glutamate dehydrogenase in roots is increased after both short and long term cultivation of plants in the presence of ammonium ions.     

Ammonium and nitrate nutrition inPlantago lanceolata L. andPlantago major L. ssp.major

With the aims (1) to test whether the different natural occurrence of twoPlantago species in grasslands is explained by a different preference of the species for nitrate or ammonium; (2) to test whether the different occurrence is explained by differences in the flexibility of the species towards changes in the nitrogen form; (3) to find suitable parameters as a tool to study ammonium and nitrate utilization of these species at the natural sites in grasslands, plants ofPlantago lanceolata andP. major ssp.major were grown with an abundant supply of nitrate, ammonium or nitrate+ammonium as the nitrogen source (0.5 mM). The combination of ammonium and nitrate gave a slightly higher final plant weight than nitrate or ammonium alone. Ammonium lowered the shoot to root ratio inP. major. Uptake of nitrate per g root was faster than that of ammonium, but from the mixed source ammonium and nitrate were taken up at the same rate. In vivo nitrate reductase activity (NRA) was present in both shoot and roots of plants receiving nitrate. When ammonium was applied in addition to nitrate, NRA of the shoot was not affected, but in the root the activity decreased. Thus, a larger proportion of total NRA was present in the shoot than with nitrate alone. In vitro glutamate dehydrogenase activity (GDHA) was enhanced by ammonium, both in the shoot and in the roots.In vitro glutamine synthetase activity (GSA) was highest in roots of plants receiving ammonium. Both GDHA and GSA were higher inP. lanceolata than inP. major. The concentration of ammonium in the roots increased with ammonium, but it did not accumulate in the shoot. The concentration of amino acids in the roots was also enhanced by ammonium. Protein concentration was not affected by the form of nitrogen. Nitrate accumulated in both the shoot and the roots of nitrate grown plants. When nitrate in the solution was replaced by ammonium, the nitrate concentration in the roots decreased rapidly. It also decreased in the shoot, but slowly. It is concluded that the nitrogen metabolism of the twoPlantago species shows a similar response to a change in the form of the nitrogen source, and that differences in natural occurrence of these species are not related to a differential adaptation of nitrogen metabolism towards the nitrogen form. Suitable parameters for establishing the nitrogen source in the field are thein vivo NRA, nitrate concentrations in tissues and xylem exudate, and the fraction of total reduced nitrogen in the roots that is in the soluble form, and to some extent thein vitro GDHA and GSA of the roots. Grassland Species Research Group. Publ. no 118.     

Nitrate Reductase in Barley Roots under Sterile, Low Oxygen Conditions

Levels of nitrate reductase activity (EC 1.9.6.1.) as high as 11 μmoles nitrite produced/hour gram fresh weight were found in barley (Hordeum vulgare cv. Compana) roots grown under low oxygen conditions. Roots of plants given identical treatment under sterile conditions did not develop the high levels of nitrate reductase activity. The results suggest that the buildup of particulate, reduced viologen-utilizing nitrate reductase reported in barley roots may be caused by bacterial contamination. The nitrate reductase activity in roots grown under low oxygen conditions was not specific for reduced nicotinamide adenine dinucleotide like the assimilatory nitrate reductase (EC 1.6.6.1.) normally found in aerated plant roots.