Different forms of molybdenum cofactor inVicia faba seeds: The presence of molybdenum cofactor carrier protein and its purification

There were significant differences in the contents of molybdenum cofactor (Mo-co), both in a low-molecular-mass form (free Mo-co) and in a protein-bound form, in seeds of sevenVicia faba genotypes. Low-molecular-mass Mo-co species present in the extracts were detected by their ability to reactivate, through a dialysis membrane, aponitrate reductase from theNeurospora crassa nit-1 mutant. In extracts of all genotypes tested, the amount of Mo-co capable of directly reactivating nitrate reductase of theN. crassa nit-1 mutant was always much higher than that of low-molecular-mass Moco. These data cannot be explained by considering, as traditionally, that Mo-co detected directly, i.e. without any previous treatment for its release from Mo-coproteins, corresponds to free low-molecular mass Mo-co. A protein which bound Mo-co was purified to electrophoretic homogeneity. This protein consisted of a single 70-kDa polypeptide chain and carried a Mo-co that could be efficiently released when in contact with aponitrate reductase.Abbreviations CP carrier protein - Mo-co molybdenum cofactor - NR nitrate reductase - XO xanthine oxidase     

The effect of cytokinins on nitrate reductase activity

Cytokinins in addition to nitrate induce nitrate reductase activity (NRA) in some plants. Effects of cytokinins onNRA was investigated in stem pith parenchyma of kale, intact wheat and barley seedlings and isolated cucumber cotyledons. The most profound effect onNRA was found in barley and wheat seedlings.NRA in seedlings sprayed with 100 μM 6-benzylaminopurine (BAP) for three subsequent days was increased in leaves and decreased in roots. These changes were further enhanced in seedlings grown in nutrient solution lacking nitrate:NRA in wheat and barley leaves was increased by 57% and 202%, respectively, in plants supplied with nitrate theNRA increase was not significant: in wheat and barley leaves by 22% and 9%, respectively. Similar effect of BAP and kinetin was found in kale stem parenchyma and cucumber cotyledons. The cytokinin kinetin or BAP alone increasedNRA about twice in kale and three times in cucumber. Addition of nitrate to the medium enhanced the effect of kinetin in kale discs, but the two effects were not additive. Additive effect of nitrate and BAP onNRA was found in cucumber cotyledons in light. In general NRA was more affected by cytokinins in intact seedlings of wheat and barley as compared to explanted tissue of kale and cucumber, and lack of nitrogen made their effect more expressive.     

Hydroxamic acids in Secale cereale L. and the relationship with their antifeedant and allelopathic properties

Contents of the hydroxamic acids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) in leaves and roots of 14 cultivars of rye, Secale cereale L., were determined. Dynamics of accumulation in three cultivars were evaluated. DIBOA was the main cyclic hydroxamic acid in leaves but the contents differed significantly between the cultivars. Both DIBOA and DIMBOA were present in the roots. Maximum concentration of DIBOA in leaves and DIMBOA in roots was reached between 48-54 h and 54-72 h after germination, respectively. Antifeedant activity of DIBOA towards the aphid Rhopalosiphum padi and the feeding behavior were studied by electronic recording in barley leaves treated with different contents of DIBOA. The deleterious activity of DIBOA could arise by starvation and/or a toxic effect. Additionally, allelopathic potential of pure DIBOA and aqueous extracts of leaves and roots of rye (Tetra-Baer) on the germination of lettuce (Lactuca sativa) and rye (Tetra-Baer) seeds was evaluated. A high percentage of germination inhibition of pure DIBOA and the extracts of leaves and roots was observed. The activity is in agreement with the contents of hydroxamic acids in the plants. The substrates had no allelopathic effect on rye seeds.     

Comparative studies of diurnal variations of nitrate reductase activity in wheat, oat and barley

Diurnal variations of in vitro and in vivo (intact tissue assay) nitrate reductase (EC 1.6.6.1) activity and stability were examined in leaves of wheat ( Triticum aestivum L. cv. Runar), oat ( Avcna saliva L. cv. Mustang) and barley ( Hordeum vulgure L. cv. Agneta and cv. Gunillu). Nitrate reductase activity was generally higher for wheat than for oat and barley. However, the diurnal variations of nitrate reductase activity and stability were principally the same for all species, e.g. the high activity during the photoperiod was associated with low stability. All species showed a rapid (30-60 min) increase in the in vitro and in vivo activity when the light was switched on. When light was switched off the in vitro activity decreased rapidly whereas decrease in in vivo activity was slower. These experiments support the hypothesis that an activation/ deactivation mechanism is involved in the regulation of diurnal variations in nitrate reductase activity. Red light enhanced nitrate reductase activity in etiolated wheat and barley leaves. In green leaves, however, the daily increase in nitrate reductase activity was not induced by a brief red light treatment. Indications of different regulation mechanisms for the diurnal variations of nitrate reductase activity among the cereals were not found.     

Choline Kinase and Phosphorylcholine Phosphatase in Plants

Choline kinase was present in barley and wheat roots and leaves of barley, wheat, tobacco, spinach and squash plants. The kinase was purified 25-fold from spinach leaves. The enzyme had a broad pH optimum between 7.5 and 10.0. Mg(++) was required for activity and in the presence of Mg(++) the enzyme was relatively stable. Maximum enzyme activity was obtained when the Mg(++): ATP ratio was 1:1. The K(m) was 1 x 10(-4)m. The kinase from leaves was similar to that from rapeseed or from yeast, except that the leaf and seed enzymes were not inhibited by compounds which attach sulfhydryl groups.Only a very slow hydrolysis of phosphorylcholine by similar plant extracts was observed. This phosphatase activity was purified 200- or 300-fold and appeared to be caused by a nonspecific acid phosphatase.The activity of both the kinase and the phosphatase did not seem sufficient to account for the rapid equilibration of the large phosphorylcholine reservoir of plants with exogenous P(32)-labeled orthophosphate.     

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.     

多裂骆驼蓬提取物对玉米幼苗生长和细胞保护酶系的影响

以不同浓度的多裂骆驼蓬提取液浸种处理玉米,研究对幼苗生长和细胞保护酶系的影响。结果表明,多裂骆驼蓬提取液浸种处理种子的萌发和种子中α-淀粉酶活性受到明显抑制,抑制作用随处理浓度提高而增强,随培养时间延长而减弱。随着培养时间的延长,浸种处理可显著提高幼苗根系活力和叶片硝酸还原酶活性,促进植株生长,根和茎叶生长量增加,根冠比增大。多裂骆驼蓬提取液浸种后显著降低叶片过氧化氢酶(CAT)、抗坏血酸氧化酶(ACO)活性,提高过氧化物酶(POD)活性,促进根系和叶片过氧化物同工酶谱的数量表达。     

Inheritance of nitrite reductase and regulation of nitrate reductase, nitrite reductase, and glutamine synthetase isozymes

Banding patterns of nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS) from leaves of diploid barley (Hordeum vulgare), tetraploid wheat (Triticum durum), hexaploid wheat (Triticum aestivum), and tetraploid wild oats (Avena barbata) were compared following starch gel electrophoresis. Two NR isozymes, which appeared to be under different regulatory control, were observed in each of the three species. The activity of the more slowly migrating nitrate reductase isozyme (NR1) was induced by NO3- in green seedlings and cycloheximide inhibited induction. However, the activity of the faster NR isozyme (NR2) was unaffected by addition of KNO3, and it was not affected by treatments of cycloheximide or chloramphenicol. Only a single isozyme of nitrite reductase was detected in surveys of three tetraploid and 18 hexaploid wheat, and 48 barley accessions; however, three isozymes associated with different ecotypes were detected in the wild oats. Inheritance patterns showed that two of the wild oat isozymes were governed by a single Mendelian locus with two codominant alleles; however, no variation was detected for the third isozyme. Treatment of excised barely and wild oat seedlings with cycloheximide and chloramphenicol showed that induction of NiR activity was greatly inhibited by cycloheximide, but only slightly by chloramphenicol. Only a single GS isozyme was detected in extracts of green leaves of wheat, barley, and wild oat seedlings. No electrophoretic variation was observed within or among any of these three species. Thus, this enzyme appears to be the most structurally conserved of the three enzymes.     

A conditional-lethal molybdopterin-defective mutant of barley

Summary The molybdenum cofactor of the barley mutant R9401 is not able to reconstitute NADPH nitrate reductase activity from extracts of the N. crassa nit-1 mutant nor is it able to effect dimerisation of the nitrate reductase subunits present in the R9401 mutant. Unphysiologically high levels of molybdate cannot restore nitrate reductase and xanthine dehydrogenase activity to mutant R9401 in vivo nor reactivate the Mo-co factor in vitro. The results indicate that the defect in mutant R9401 lies in the pathway leading to the formation of a functional molybdopterin moiety and that the same nuclear gene is involved in the synthesis of both shoot and root molybdenum cofactor.Abbreviations BSA bovine serum albumen - GSH glutathione (reduced) - NEM N-ethylmaleimide     

A root-specific O-methyltransferase gene expressed in salt-tolerant barley

A cDNA encoding an O-methyltransferase (OMT) was isolated from salt-tolerant barley roots by subtraction hybridization with cDNAs of salt-tolerant barley roots as a tester cDNA and cDNAs of the salt-sensitive barley roots as a driver cDNA. The deduced amino acid sequence showed significant identity with plant caffeic acid/5-hydroxyferulic acid OMTs. Southern blot analysis showed that the OMT gene was a single copy in both salt-tolerant and -sensitive barley. The cloned gene was expressed in a wheat germ cell-free system to produce the OMT, which had methylating activity for caffeic acid. Northern blot analysis showed that the OMT gene was expressed constitutively in the salt-tolerant barley roots and the expression level was increased 1.5 times by salt stress, but the salt-sensitive barley showed no expression of the gene in roots and leaves.     

Uptake and Movement of 14C-Chlormequat Chloride Applied to Leaves of Barley and Wheat

Chlormequat chloride labelled with ¹⁴C was applied to the thirdleaf of Proctor barley and Maris Dove wheat; after 1 d lessthan a fifth could be washed off with water. More ¹⁴C-labelledmaterial moved from treated areas of wheat than of barley toaccumulate in tips of treated leaves, younger main-stem leaves,main stems, and ears. Relatively small amounts of radioactivematerial accumulated in roots, but less in wheat than barley.About 80% of the radioactivity was recovered from plants upto 1 week after application, but the amounts recovered in aqueouswashings and extracts decreased faster from wheat than frombarley.     

Nitrate Reductase Acivity in Leaves of Barley (Hordeum vulgare) and Durum Wheat (Triticum durum) during Field and Rapidly Applied Water Deficits

Smirnoff, N., Winslow, M. D. and Stewart, G. R. 1985. Nitratereductase activity in leaves of barley (Hordeum vulgare) anddurum wheat (Triticum durum) during field and rapidly appliedwater deficits.-J. exp. Bot 36: 1200-1208. The effect of field and rapidly applied water deficits on nitratereductase activity in the leaves of two barley varieties andone durum wheat variety was investigated. In field experimentsplants were subjected to irrigation at different rates in threeMediterranean environments by means of a line source sprinklerirrigation system. The environments differed in rainfall andnitrogen fertility. Plant water potentials decreased from –1.5MPa to between –2.5 and –3.0 MPa as the irrigationrate decreased. Nitrate reductase activity in the leaves ofthese plants during heading was either unaffected or sometimesincreased where the least water was supplied. Nitrate reductaseactivity was highest in the plants growing with an ample nitrogensupply irrespective of water regime. In contrast, seedlingssubject to rapidly applied water stress over 6 d lost 30-85%of their nitrate reductase activity when leaf water potentialfell from between –0.33 and –0.75 MPa to between–O.93 and –2.04 MPa. The decrease was less in theyoung leaves than in the old leaves. Polyethylene glycol inducedosmotic stress resulted in a drop in leaf water potential from–0.20 MPa to between –1.05 and –1.20 MPa alongwith a loss of 40-85% of leaf nitrate reductase activity after48 h. It is suggested that maintenance of nitrate reductase activityin field grown barley and durum wheat plants reflects an acclimationto water deficit Maintenance of nitrate assimilation duringwater stress may allow continued synthesis of nitrogenous compatiblesolutes using the excess photochemical energy available duringstomatal closure. Key words: Nitrate reductase, water stress, barley, durum wheat     

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.      

Species and Organ Diversity in the Effects of Hydrogen Peroxide on Superoxide Dismutase Activity In Vitro

Superoxlde dlsmutase （SOD） is ubiquitous in aerobic organisms and constitutes the first link In the enzyme scavenging system of reactive oxygen species. In the present study, species and organ diversity of SOD activity In a solution and In an in-gel assay system, as well as the effects of hydrogen peroxide （H202） on SOD activity, were Investigated. In a solution assay system, SOD activity of jackfruIt root, shoot, leaves, axes, and cotyledons, of maize embryos and endosperms, of mung bean leaves and seeds, of sacred lotus axes and cotyledons, and of rice and wheat leaves was Increased by 1-15 mmol/L H2O2. However, SOD activity In rice root and seeds, maize roots and leaves, mung bean roots and shoots, and wheat seeds was decreased by 1-15 mmol/L H2O2. The SOD activity of wheat root and soybean roots, leaves, axes, and cotyledons was Increased by 1-4 mmol/L H2O2, but was decreased by concentrations of H2O2 〉4 mmol/L. The SOD activity of soybean shoots was not affected by 1-15 mmol/L H2O2. The SOD activity In crude mltochondrla of jackfruIt, maize, and upas seeds, as well as In purified mitochondria of jackfruIt, was also Increased by 1-15 mmol/L H2O2. In the In-gel assay system, the SOD In jackfruIt cotyledons was comprised of Mn-SOD, Cu/Zn-SOD, and Fe-SOD, the crude mltochondria of jackfruit seeds and maizes embryo was comprised of Mn-SOD and Cu/ Zn-SOD, and the crude mltochondria of maize seeds was comprised of Mn-SOD only. In the present study, H2O2 markedly Inhibited Cu/Zn-SOD and Fe-SOD activity.     

Nitrate reductase is not accumulated in chloroplast-ribosome-deficient mutants of higher plants

The activities of nitrite reductase (EC 1.7.7.1) are 60–70% of wild-type activity in pigment-deficient leaves of the chloroplast-ribosomedeficient mutants albostrians (Hordeum vulgare) and iojap (Zea mays). The activity and apoprotein of nitrate reductase (EC 1.6.6.1.) are lacking in the barley mutant. Only very low activities of nitrate reductase can be extracted from leaves of the maize mutant. The molybdenum cofactor of nitrate reductase and xanthine dehydrogenase (EC 1.2.3.2) is present in maize and barley mutant plants. However, it is not inducible by nitrate in pigment-deficient leaves of albostrians. From these results we conclude: (i) Nitrite reductase (a chloroplast enzyme) is synthesized in the cytoplasm and does not need the presence of nitrate reductase for the induction and maintenance if its activity. (ii) The loss or low activity of nitrate reductase is a consequence of the inability of the mutants to accumulate the apoprotein of this enzyme. (iii) The chloroplasts influence the accumulation (i.e. most probably the synthesis) of the nonchloroplast enzyme, nitrate reductase. The accumulation of nitrate reductase needs a chloroplast factor which is not provided by mutant plastids blocked at an early stage of their development.Abbreviations CRM cross-reacting material - Mo-co molybdenum cofactor - NiR nitrite reductase - NR nitrate reductase     

Absence of nitrate reductase activity in San 9789 bleached leaves of barley seedlings (Hordeum vulgare cv. Midas)

Abstract San 9789 (norflurazone) blocks carotenoid synthesis which allows chlorophyll bleaching in the light, and has been used recently as a tool to study phytochrome responses without interference from photosynthetic pigments. By using this herbicide, we have found that nitrate reductase activity and light dependent nitrite reduction were lost simultaneously from achlorophyllous areas of barley leaves, with the green areas of the leaf tip still showing high activities. By contrast nitrate reductase is still present in the roots of herbicide treated plants. We suggest that intact chloroplasts are required for the presence of nitrate reductase in barley leaves.     

Regulation of Corn Leaf Nitrate Reductase : I. Immunochemical Methods for Analysis of the Enzyme's Protein Component

NADH:nitrate reductase was extracted from corn leaves (Zea mays L. W64A × W182E) and purified on blue Sepharose. After the nitrate reductase was further purified by polyacrylamide gel electrophoresis, it was used to immunize mice and a rabbit. Western blots of crude leaf extracts were used to demonstrate monospecificity of the mouse ascitic fluids and the rabbit antiserum. The electrophoretic properties of purified corn and squash NADH:nitrate reductases in both native and denatured states were shown to be similar using western blotting with mouse ascitic fluid. The corn leaf enzyme has a 115,000 polypeptide subunit like that of squash. Western blots could detect 3 to 10 nanograms of nitrate reductase protein. But the detection of proteolytic degradation products using western blotting was inconsistent and remains to be established. An enzyme-linked immunosorbent assay (ELISA) was developed for quantifying nitrate reductase protein in the crude extracts of corn leaves. Using a standard curve based on nitrate reductase activity, the ELISA for corn nitrate reductase could detect 0.5 to 10 nanograms of nitrate reductase protein and was adequately sensitive for quantitative analysis of nitrate reductase in crude extracts of leaves even when activity levels were very low. When the ELISA was used to compare the nitrate reductase protein content of corn roots and leaves, these tissues were estimated to contain 0.24 to 0.5 and 4 to 5 micrograms nitrate reductase protein/gram root and leaf, respectively.     

Properties of nitrate reductase from seedling leaves of selected barley genotypes

Crude extracts from leaves of 6-day barley seedlings of parental genotypes (cv. Aramir and primitive line R567) and selected doubled haploid (DH) lines were not found to have significant differences in the NADH:NR activity, while considerable differences between these genotypes were shown by the NAD(P)H:NR activity. The cv. Aramir and DH lines did not differ by nitrate accumulation in the leaves. However, the primitive line R567, as compared to the remaining genotypes, was characterized by an appreciably lower ability to accumulate nitrates. In partially purified leaf extracts, significant differences in total NADH:NR activity and in distal activity dependent on methyl viologen (MV:NR) were found between the parental genotypes and selected DH lines. The studied genotypes differed also in dehydrogenase NR activity, i.e. cytochrome c reductase activity in crude extracts. In the studied genotypes, the NADH:NR activity in partially purified leaf extracts did not substantially differ by Km values for nitrates. Calculated Vmax values for NADH:NR in these genotypes were similar to total NR activity in partially purified extracts. Significant differences between the parental genotypes and selected DH lines were found in the thermal NADH:NR stability in crude and partially purified leaf extracts. From the performed studies it follows that different NR stability was one of the reasons of revealed differences in total activity and in partial NR activities in the leaf extracts between the studied genotypes of spring barley. Besides, it is suggested that varied NR gene expression in the leaves of these barley genotypes could also influence NR activity.     

Phenylalanine Ammonia-lyase Activity in Barley After Infection with Bipolaris sorokiniana or Treatment with its Purified Xylanase

Phenylalanine ammonia-lyase (PAL) activity was determined from leaves and roots of two barley (Hordeum vulgare L.) cultivars after infection with a necrotrophic pathogen, Bipolaris sorokiniana (Sacc.) Shoem., and treatment with its purified xylanase. PAL activity increased in leaves of both cultivars 16 h after fungal inoculation but two phases, with activity peaks at 24–32 h and 40 h, were recorded only for the more resistant cultivar, Agneta. Attempts to use a PAL inhibitor, χ-amin, ooxyacetic acid, to increase susceptibility to B. sorokiniana in barley leaves were unsuccessful. Treatments of leaves with purified xylanase resulted in more rapid (4–12 h after injection), although reduced, induction of PAL compared with fungal injection. The higher the concentration of xylanase applied the earlier the activity peaks were detected. Fungal inoculation only slightly increased PAL activity in barley roots while xylanase treatment had no effect. The basal level of PAL was however much higher in roots than in leaves. In wheat, Triticum aestivum L. resistant to B. sorokiniana, the time-course of PAL induction after fungal infection and xylanase treatment resembled that for cv. Agneta, while in oats, Avena sativa L. (non-host) PAL activity did not change after the treatments. The results suggest that the second phase of PAL induction, associated only with responses of barley cv. Agneta and wheat, is linked with their resistance to B. sorokiniana infection. The possible role of xylanase as an elicitor of PAL is discussed.     

Tissue-Specific Expression of Germin-Like Oxalate Oxidase during Development and Fungal Infection of Barley Seedlings

Oxalate oxidase activity was detected in situ during the development of barley seedlings. The presence of germin-like oxalate oxidase was confirmed by immunoblotting using an antibody directed against wheat germin produced in Escherichia coli, which is shown to cross-react with barley (Hordeum vulgare) oxalate oxidase and by enzymatic assay after electrophoresis of the protein extracts on polyacrylamide gels. In 3-d-old barley seedlings, oxalate oxidase is localized in the epidermal cells of the mature region of primary roots and in the coleorhiza. After 10 d of growth, the activity is detectable only in the coleorhiza. Moreover, we show that oxalate oxidase is induced in barley leaves during infection by the fungus Erysiphe graminis f. sp. hordei but not by wounding. Thus, oxalate oxidase is a new class of proteins that responds to pathogen attack. We propose that oxalate oxidase could have a role in plant defense through the production of H2O2.