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Using in silico methods, several putative phytohormone-responsive cis-elements in the Oryza sativa non-symbiotic haemoglobin (NSHB) 1-4 and Arabidopsis thaliana NSHB1-2 promoters have been identified. An OsNSHB2 promoter::GUS reporter gene fusion shows tissue-specific expression in A. thaliana. GUS expression was observed in roots, the vasculature of young leaves, in flowers, and in the pedicel/stem junction. In transient assays, activity of the OsNSHB2 promoter was significantly up-regulated in the presence of the cytokinin, 6-benzylaminopurine (BA). Deletion analyses indicated that the full-length promoter was required for maximal trans-activation in the presence of cytokinin. Mutation of the single cytokinin-regulated ARR1-binding element abolished promoter activation in response to cytokinin. Constitutive expression of ARR1 under the control of the 35S cauliflower mosaic virus promoter enhanced wild-type OsNSHB2 promoter activity, but had no effect on the activity of the mutated promoter in the absence of cytokinin. However, overexpression of ARR1 in the presence of cytokinin resulted in super-activation of the wild-type promoter. The mutated promoter was only moderately activated in the presence of cytokinin and ARR1, indicating that the OsNSHB2 promoter can be regulated by the ARR1 protein, but requires other cytokinin-induced factors for optimal activation. This is the first report that identifies a trans-acting factor involved in the activation of a NSHB gene.

Hemoglobins (Hbs) have been characterized from a wide variety of eubacteria, but not from nitrogen- fi xing rhizobia. Our search for Hb-like sequences in the Sinorhizobium meliloti genome revealed that a gene coding for a fl avohemoglobin (fHb) exists in S. meliloti (SmfHb). Computer analysis showed that SmfHb and Alcaligenes eu- trophus fHb are highly similar and could fold into the same tertiary structure. A FNR-like box was detected upstream of the smfhb gene and mapping analy sis revealed that the smfhb gene is fl anked by nos and fi x genes. These observa- tions suggest that smfhb is regulated by the concentration of O 2 and that SmfHb functions in some aspects of nitro- gen metabolism.

The concentration of photosynthetic pigments (i.e., chlorophylls a and b, and carotenoids) and chlorophyll degradation enzyme (i.e., chlorophyllase, oxidative bleaching, and Mg-dechelatase) activities on aphid-damaged and non-damaged regions of the infested leaves were determined with two infestation periods (6 and 12 days). Russian wheat aphid [Diuraphis noxia (Mordvilko) (Hemiptera: Aphididae)] feeding caused significant losses of chlorophylls a and b and carotenoids in the damaged regions. However, bird cherry-oat aphid [Rhopalosiphum padi (L.) (Hemiptera: Aphididae)] feeding did not, except a significantly lower level of carotenoids was observed in the damaged regions from the short-infestation (6-day) samples. Interestingly, the non-damaged regions of D. noxia-infested leaves on both sampling dates had a significant increase of chlorophylls a and b and carotenoid concentrations when compared with the uninfested leaves. Although D. noxia feeding did not cause any changes in either chlorophyll a/b or chlorophyll (a + b)/carotenoid ratio between the damaged and non-damaged leaf regions on short-infestation (6-day) samples, a significantly lower chlorophyll a/b ratio was detected in long-infestation (12-day) samples. The assays of chlorophyllase and oxidative bleaching activities showed no significant differences between the damaged and non-damaged regions of the infested leaves on either sampling date. Mg-dechelatase activity, however, was significantly higher in D. noxia-damaged than non-damaged leaf regions from the short-infestation samples, while no differences were detected from the long-infestation samples. Furthermore, the long-infestation samples showed that Mg-dechelatase activity from both D. noxia-damaged and non-damaged regions increased significantly in comparison with the respective regions of either uninfested or R. padi-infested leaves. We infer that non-damaged regions of D. noxia-infested leaves compensate for the pigment losses in the damaged regions, and that Mg-dechelatase activity changed dynamically from a localized response to a systemic response as infestation duration extends. The findings from this study on cereal aphid-elicited chlorosis (or desistance) would help us to elucidate plant resistance mechanisms, in particular plant tolerance to non-defoliating herbivory.

A hemoglobin (hb) gene family was identified in rice, consisting of four functional hb copies located in BAC inserts OSM11719 and OSM11676, which contain the gene clusters hb1/hb3/hb4 and hb2, respectively. Sequence comparison with databases revealed that a number of potential promoters exist upstream from the hb genes suggesting that these genes are differentially expressed in rice.

Chlorophyll degradation is a complex phenomenon that often accompanies insect feeding damage to plants. Loss of chlorophyll can be initiated by several reactions, including oxidative bleaching, chlorophyllase activity, and Mg-dechelatase activity. Extracts from the Russian wheat aphid [Diuraphis noxia (Mordvilko)], the bird cherry-oat aphid [Rhopalosiphum padi (L.)], and aphid-infested and uninfested wheat plants were assayed in vitro for activities involved in chlorophyll degradation. Although the initial infestation was the same (10 apterous adults) for both aphid species, D. noxia weight was significantly higher than R. padi after feeding for 12 days. Consequently, D. noxia feeding caused greater fresh leaf weight reduction than R. padi feeding. Chlorophyll degradation assays showed no activity from either D. noxia or R. padi extracts. Plant extract assays showed a significant difference in Mg-dechelatase activity, while no difference was detected in either the chlorophyllase or oxidative bleaching pathways among the aphid-infested or uninfested plant extracts. Diuraphis noxia-infested leaf extracts showed a greater increase of Mg-dechelatase activity than either R. padi-infested or the uninfested plants. The findings suggest that leaf chlorosis elicited by D. noxia feeding is different from the chlorophyll degradation that occurs in natural plant senescence. Aphid-elicited chlorosis might be the result of a Mg-dechelatase-driven catabolism of chlorophyll in challenged wheat seedlings, however, the factor(s) from D. noxia that elicited the increase of Mg-dechelatase activity still remain to be determined.

In rice (Oryza sativa var. Jackson) at least three copies of hemoglobin (hb) gene exist. Rice hb1 and hb2 genes are differentially expressed in roots and leaves from mature plants. We used polyclonal antibodies raised to recombinant rice Hb1 and Western blotting to analyze the synthesis of Hbs in rice plants growing under normal or stress conditions. Results showed that rice Hbs are synthesized in coleoptiles, seminal roots and embryos from seeds germinated for 6 days, and also in leaves and roots from plants 2-14 weeks after germination. Analysis of Hb synthesis in stressed rice showed that: (i) level of Hbs was higher in etiolated than control plants, (ii) level of Hbs increased in roots from flooded rice, and (iii) level of Hbs did not change under oxidative (H(2)O(2)), nitrosative (SNP) and hormonal (2,4-D) stresses. These results suggest that the effect of light withdrawal in etiolated leaves and O(2)-limiting conditions in flooded roots, but not oxidative, nitrosative and hormonal stresses, modulate the synthesis of rice Hbs.

Ferric leghemoglobin reductase (FLbR), an enzyme reducing ferric leghemoglobin (Lb) to ferrous Lb, was purified from cowpea (Vigna unguiculata) root nodules by sequential chromatography on hydroxylapatite followed by Mono-Q HR5/5 FPLC and Sephacryl S-200 gel filtration. The purified cowpea FLbR had a specific activity of 216 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for cowpea Lb(3+) and a specific activity of 184 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for soybean Lb(3+). A cDNA clone of cowpea FLbR was obtained by screening a cowpea root nodule cDNA library. The nucleotide sequence of cowpea FLbR cDNA exhibited about 88% similarity with soybean (Glycine max) FLbR and 85% with pea (Pisum sativum) dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) cDNAs. Conserved regions for the FAD-binding site, NAD(P)H-binding site, and disulfide active site were identified among the deduced amino acid sequences of cowpea FLbR, soybean FLbR, pea DLDH and other enzymes in the family of the pyridine nucleotide-disulfide oxido-reductases.

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.

Phage DNA isolation from genomic and cDNA libraries is time-consuming and cumbersome. Our work reports on the use of a pair of λ gt11 primers to amplify by PCR any insert DNA that is cloned into theEco RI restriction site. Using crude phage pools and direct phage plaques from a corn root cDNA library, we amplified and cloned DNA fragments. The amount of amplification products was similar when phage DNA and phage pools were used as templates. Amplified fragments could be purified, cloned and sequenced by a relatively simple procedure, thus allowing rapid detection and analysis of inserts in a large number of phage plaques.

We previously cloned and sequenced a cDNA encoding soybean ferric leghemoglobin reductase (FLbR), an enzyme postulated to play an important role in maintaining leghemoglobin in a functional ferrous state in nitrogen-fixing root nodules. This cDNA was sub-cloned into an expression plasmid, pTrcHis C, and overexpressed in Escherichia coli. The recombinant FLbR protein, which was purified by two steps of column chromatography, was catalytically active and fully functional. The recombinant FLbR cross-reacted with antisera raised against native FLbR purified from soybean root nodules. The recombinant FLbR, the native FLbR purified from soybean (Glycine max L.) root nodules, and dihydrolipoamide dehydrogenases from pig heart and yeast had similar but not identical ultraviolet-visible absorption and fluorescence spectra, cofactor binding, and kinetic properties. FLbR shared common structural features in the active site and prosthetic group binding sites with other pyridine nucleotide-disulfide oxidoreductases such as dihydrolipoamide dehydrogenases, but displayed different microenvironments for the prosthetic groups.

This is published as paper No. 10577, Journal Series, Agriculture Research Division, University of Nebraska-Lincoln., Hemoprotein derivatives of an abundant soybean (Glycine max Merr.) root nodule leghemoglobin, Lba, were studied for their modified spectral characteristics and physical properties. Three modified hemoprotein derivatives of Lba (Lbam1, Lbam2, and Lbam3) were purified by preparative isoelectric focusing. The ferric forms of these pigments were green and exhibited anomalous spectra in the visible region as compared to the Lba3+ forms. These modified pigments showed a hypochromic shift of 10 nm for the charge transfer absorption maximum; however, differences were not apparent in the Soret region. Upon binding with nicotinate, the [alpha] and [beta] bands were shifted significantly into the red region as compared to the Lba3+ nicotinate complex. The three Lbam fractions were reduced by dithionite or by NADH in the presence of riboflavin. Lbam2+ also bound nicotinate and displayed absorption spectra indistinguishable from those of Lba2+ nicotinate. In contrast to Lba2+, Lbam2+ displayed aberrant spectra when bound with either O2 or CO. These complexes exhibited a prominent charge transfer band at approximately 620 nm and failed to exhibit spectra characteristic of Lba2+O2 and Lba2+CO. The protein moiety of these modified pigments was intact because their tyrosine/tryptophan ratios and their amino acid compositions were identical with those of Lba, nor were differences observed in the peptide profiles resulting from trypsin digests of purified Lba and Lbams. Automated Edman degradation of selected peaks further confirmed the intactness of the protein backbone including the absence of deamination. Pyridine hemochromogen for heme from Lbams could be formed, and the spectra displayed distinct differences compared to those of Lba. A new peak at 580 nm and a loss of a peak at 480 nm were observed for all three Lbams., This work was supported by grants from the Nebraska Soybean Development, Utilization and Marketing Board (F.W.W.), The Center for Biotechnology, University of Nebraska-Lincoln (G.S.), and Dirección General de Investigación Científica y TecnolÓgica (grant PB92- 0058; M.B.).

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb(+3)), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O(2) was removed from the reaction system, but all were restored upon readdition of O(2). In the absence of exogenous electron carriers and in the presence of O(2) and excess NADH, FLbR catalyzed NADH oxidation with the generation of H(2)O(2) functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 mug/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb(+3) reduction. The formation of H(2)O(2) during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H(2)O(2). The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb(+3) reduction were not constant but changed with time and with concentrations of NADH and O(2) in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb(+3) by reaction intermediates. A study of the affinity of FLbR for O(2) showed that the enzyme required at least micromolar levels of dissolved O(2) for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.

Reactions involving changes that affect the function of leghemoglobin (Lb) are reviewed. The chemical nature of Lb and conditions inside nodules, such as slightly acid pH and the presence of metal ions, chelators, and toxic metabolites (nitrite, superoxide radical, peroxides), are conducive for oxidation of ferrous Lb (Lb(2+)) or its oxygenated form (LbO(2)) to nonfunctional ferric Lb (Lb(3+)) and ferryl Lb. Because Lb(3+) is nearly nonexistent in nodules and undergoes observable reduction in vivo, mechanisms must operate in nodules to maintain Lb in the Lb(2+) state. Redox reactions of Lb are mediated, for the most part, by activated oxygen species: (a) oxidation of LbO(2) to Lb(3+) involves superoxide; (b) excess peroxide oxidizes LbO(2) and Lb(3+) to ferryl Lb and may cause breakdown of heme, release of iron, and generation of hydroxyl radicals (protein radicals may be formed in this process); (c) enzymatic reduction of Lb(3+) requires active flavin and thiol groups and involves formation of peroxide; and (d) direct reduction of Lb(3+) by NADH is mediated by superoxide and peroxide. Transition metal ions and certain small molecules of nodules such as flavins may act as intermediate electron carriers between NADH and Lb(3+), increasing the rate of reaction, which then proceeds via superoxide or flavin radicals, respectively.

A ferric leghemoglobin reductase from the cytosol of soybean (Glycine max) root nodules was purified to homogeneity and partially characterized. The enzyme is a flavoprotein with flavin adenine dinucleotide as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kilodaltons. The pure enzyme shows a high activity for ferric leghemoglobin reduction with NADH as the reductant in the absence of any exogenous mediators. The enzyme also exhibits NADH-dependent 2,6-dichloroindophenol reductase activity. A sequence of the first 50 N-terminal amino acids of the purified protein was obtained. Comparisons with known protein sequences have shown that the sequence of the ferric leghemoglobin reductase is highly related to those of the flavin-nucleotide disulfide oxido-reductases, especially dihydrolipoamide dehydrogenase of the pyruvate dehydrogenase complex.

Nicotinate has been postulated to interfere with the binding of O2 to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O2. Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O2 in soybean root nodules.

Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots and dicots; however, very little is known about the tissue and cell type localization as well as the physiological function(s) of these oxygen-binding proteins. We report the immunodetection and immunolocalization of ns-Hbs in rice (Oryza sativa L.) by Western blotting and in situ confocal laser scanning techniques. Ns-Hbs were detected in soluble extracts of different tissues from the developing rice seedling by immunoblotting. Levels of ns-Hbs increased in the germinating seed for the first six days following imbibition and remained relatively constant thereafter. In contrast, ns-Hb levels decreased during leaf maturation. Roots and mesocotyls contained detectable, but low levels of ns-Hbs. Split-seed experiments revealed that ns-Hbs are synthesized de novo during seed germination and are expressed in the absence of any signal originating from the embryo. Immunolocalization of ns-Hbs by confocal microscopy indicated the presence of ns-Hbs primarily in differentiated and differentiating cell types of the developing seedling, such as the aleurone, scutellum, root cap cells, sclerenchyma, and tracheary elements. To our knowledge, this is the first report of the specific cellular localization of these proteins during seedling development.

This is journal paper no. 12,643, Agricultural Research Division, University of Nebraska., Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH., This work was supported by the National Science Foundation (grant no. OSR-92552255) and the U.S. Department of Agriculture-Cooperative State Research Education and Extension Service (grant no. 95-37305-2441). Access to the BioCad workstation was provided by the Center for Biotechnology at the University of Nebraska, Lincoln, funded through the Nebraska Research Initiative. J.F.M. was the recipient of a postdoctoral contract from the Ministry of Education and Culture (Spain).

With the exception of barley and rice, little is known about the existence of hemoglobins (Hbs) in cereals. This work reports the cloning and analysis of hb genes from maize (Zea mays ssp. mays) and teosinte (Zea mays ssp. parviglumis). Coding sequences of maize and teosinte hb genes (hbm and hbt, respectively) are highly similar to each other and are interrupted by three introns located at identical positions as other plant hb genes. Sequences of predicted Hbm and Hbt proteins are identical. The hydropathic profile of Hbm and Hbt is highly similar to that of rice Hb1, suggesting that Hbm, Hbt and Hb1 have the same tertiary structure and biochemical properties. Expression analysis showed that low levels of Hb transcripts, but considerable levels of Hb proteins exist in maize embryonic organs. No Hb transcripts and proteins were detected in teosinte embryonic organs. Low levels of Hb proteins, but no Hb transcripts, were detected in maize and teosinte vegetative organs. These observations suggest that the regulation of hb genes is different in maize and teosinte embryonic organs, and that the expression of hb genes is down- or up-regulated in maize and teosinte, respectively, from germination to vegetative growing.

Background: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O2 conditions. Results: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A resolution us ing a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b 5 . The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. Conclusions: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O 2 reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O 2 affinity is ultra-high ( K D ≈ 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

Soybean root nodules possess a developmentally regulated acid phosphatase (ACP) that exhibits the highest specificity for purine 5′-nucleoside monophosphates. The enzyme is a glycosylated dimer of 28- and 31-kDa subunits, which appear to be products of the same gene but differ in posttranslational modifications. In order to perform directed mutagenesis and more extensive biochemical characterization, a means of producing recombinant ACP was needed. Several attempts were made to express ACP in Escherichia coli , but all conditions employed resulted in protein that was found entirely in inclusion bodies, and resolubilization experiments were unsuccessful. Therefore, the methyltrophic yeast Pichia pastoris was chosen as a eukaryotic expression host. The coding sequence of ACP was cloned into the pPIC9 vector to create a fusion with the yeast α mating factor secretion signal. The ACP:pPIC9 construct was integrated into P. pastoris strain GS115. Expression of ACP was under the control of an alcohol oxidase methanol-inducible promoter. Methanol induction resulted in secretion of ACP to a level of 10 mg/L. The recombinant ACP was purified 550-fold to homogeneity by phenyl-Sepharose, hydroxyapatite, and MonoS chromatography. The purified enzyme had K m values of 0.08 and 0.12 for 5′-AMP and 5′-GMP. These values were similar to those obtained for the native ACP heterodimer purified from soybean (0.08 and 0.15 mM for 5′-AMP and 5′-GMP). The specific activity of the recombinant enzyme for all substrates tested was 1.6- to 1.8-fold higher than the values for the purified soybean heterodimer.

Conversion of XMP to xanthosine is critical step in the ureide biosynthetic pathway. The enzyme responsible for the removal of the phosphate group of XMP has been postulated to be an acid phosphatase and/or a 5′ nucleotidase. A soluble acid phosphatase with highest specificity for 5-nucleotides was isolated from effective nodules of Glycine max and sujected to N-terminal and internal peptide microsequencing. PCR primers were designed from the peptide sequences and used to screen and soybean root nodule cDNA library. Three independent clones were isolated from the library and found to be identical. A comparison of the mature protein sequence and the translated cDNA sequence revealed the presence of an uncharacterized N-terminal signal sequence. Three potential N-linked glycosylation sites were found in the translated cDNA. The 5′-nucleotidase cDNA was found to be 73 and 58% identical at the nucleotide level to the soybean vegetative storage protein α and the tomato acid phosphatase, respectively. Northern and western blotting and RT-PCR indicate a differential regulation of the nodule 5′-nucleotidase and the closely-related vegetative storage proteins.

6 Pags.- 3 Tabls.- 1 Fig., on-exchange high performance liquid chromatography (HPLC) with an Aminex column and photodiode-array detection was used to purify, from soybean root nodules, highly polar phenolic acid conjugates that are recalcitrant to isolation by conventional techniques based on reversed-phase chemistry. HPLC, acid and enzymatic hydrolysis, ultraviolet spectroscopy, high resolution fast atom bombardment mass spectrometry (MS) and single and tandem electrospray MS were used to identify the 4-O-β-glucosides of p-hydroxybenzoic, protocatechuic and vanillic acids (which are reported in legumes for the first time) and the 5-O-β-glucoside of gentisic acid. The four phenolic acids, as well as gallic acid and its methyl ester, were also found in soybean nodules., This work was financed by Acciones Hispano-Britá́nicas HB91-218 and HB92-185, and grant PB95-0091 from the Dirección General de Enseñanza Superior (Ministry of Education and Culture, Spain).

24 Pags. The definitive version, with Figs. and Tabls., is available at: http://www.sciencedirect.com/science/journal/08915849, The low-molecular-mass fraction of the soybean nodule cytosol contains Fe capable of catalyzing free radical production through Fenton chemistry. A large portion of the pool of catalytic Fe, measured as bleomycin-detectable Fe, was characterized as complexes of Fe with phenolic compounds of three classes: phenolic acids, cinnamic acids, and flavonoids. Many of these compounds, along with other phenolics present in legume tissues, were used for a systematic structure–activity relationship study. All phenolics tested were able to chelate Fe, as judged from their inhibitory effect on site-specific deoxyribose degradation (minus EDTA assay). However, only those having catechol, pyrogallol, or 3-hydroxy-4-carbonyl groupings were potent chelators and reductants of Fe3+ at pH 5.5. The same phenolics promoted oxidative damage to DNA (bleomycin assay) and to deoxyribose (plus EDTA assay), but inhibited linolenic acid peroxidation by chelating and reducing Fe3+ and by neutralizing lipid radicals. Also, phenolics having a pyrogallol nucleus attenuated the free radical-mediated inactivation of glutamine synthetase, which was used as a model system, by chelating Fe2+. It is reasoned that under the microaerobic (10–20 nM O2) and acidic (pH 5.5–6.4) conditions prevailing in nodules, phenolics are likely to act primarily as antioxidants, decreasing oxidative damage to biomolecules., This work was supported by grants from Dirección General de Investigación Científica y Tecnológica (PB92-0058) to M.B., Acciones Integradas Hispano-Británicas (HB91-218 and HB92-185) to M.B. and R.J. G., and U.S. Department of Agriculture (93-0318) to R.V.K.

22 Pags. The definitive version, with Figs. and Tabls., is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-3040, Iron (Fe) deficiency in pea leaves caused a large decrease (44–62&) in chlorophyll a, chlorophyll b and carotenoids, and smaller decreases in soluble protein (18&) and net photosynthesis (28&). Catalase, non-specific peroxidase and ascorbate peroxidase activities declined by 51& in young Fe-deficient leaves, whereas monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities remained unaffected. Ascorbate peroxidase activity was highly correlated (r2= 0. 99, P < 0. 001) with the Fe content of leaves, which allows its use as an indicator of the Fe nutritional status of the plant. Fe deficiency resulted in an increase of CuZn-superoxide dismutase but not of Mn-superoxide dismutase. The content of ascorbate decreased by only 24& and those of reduced and oxidized glutathione and vitamin E did not vary. The low-molecular-mass fraction of Fe-sufficient leaves contained 30–65 μg (g dry weight)−1 Mn. This concentration was 15–60 times greater than that of Fe and Cu in the same fraction, and was further enhanced (1. 5- to 2. 5-fold) by Fe deficiency without causing Mn toxicity. The concentration of catalytic Fe, that is, of Fe active for free radical generation, was virtually zero and that of catalytic Cu did not change with severe Fe deficiency. Because catalytic metals mediate lipid and protein oxidation in vivo, the above findings would explain why oxidatively damaged lipids and proteins do not accumulate in Fe-deficient leaves., I.I.O. and J.F.M. were the recipients of predoctoral fellowships from the Comunidades Autónomas del País Vasco and Aragón, respectively, and Y. Gogorcena was the recipient of a postdoctoral contract from the Mininsterio de Educación y Ciencia. This work was supported by grant PB92-0058 from the Dirección General de Investigación Científica y Técnica to M.B. and grant 93-0318 from the U.S. Department of Agriculture to R.V.K.

22 Pags.- 3 Tabls. The definitive version, with Figs., is available at: http://www.pnas.org/, The cytosol from root nodules of soybean, bean, and cowpea contained Fe and Cu capable of catalyzing the formation of highly reactive free radicals. Specific and sensitive assays based on free radical-mediated DNA degradation revealed that most catalytic Fe and Cu were present as small chelates (300-600 Da). The involvement of catalytic Fe in free radical production during nodule senescence, which was induced by exposure of plants to continuous darkness for 2-4 days, was investigated. (i) Free heme remained at a constant and low concentration (1-4% of total nodule heme) during senescence, indicating that it is not an important constituent of the catalytic Fe pool of nodules. (ii) Catalytic Fe of nodule cytosol promoted deoxyribose degradation and linolenic acid peroxidation in reaction mixtures containing physiological concentrations of ascorbate and H2O2. Deoxyribose degradation but not lipid peroxidation required hydroxyl radicals to proceed. (iii) The cytosol from senescent nodules, particularly of bean and cowpea, sustained in vitro higher rates of deoxyribose degradation and lipid peroxidation than the cytosol from unstressed nodules. Both degradative processes were inhibited by the Fe chelator desferrioxamine and were correlated with the content of catalytic Fe in the nodule cytosol. (iv) Although other transition metals (Cu, Mn, Mo, and Ni) were present in significant amounts in the low molecular mass fraction (, This work received support from the Diputación General de Aragón (PCB10- 90) and the Comisión Interministerial de Ciencia y Tecnología (AGR91-0857- C02-2) to M.B., and from the NRI Competitive Grants Program-USDA (grant No. 91-37305-6705) to R.V.K.

22 Pags. The definitive version, with Figs., is available at: http://link.springer.com/journal/425, The reduction of ferric leghemoglobin (Lb3+) from soybean (Glycine max (L.) Merr.) nodules by riboflavin, FMN and FAD in the presence of NAD(P)H was studied in vitro. The system NAD(P)H + flavin reduced Lb3+ to oxyferrous (Lb2+ · O2) or deoxyferrous (Lb2+) leghemoglobin in aerobic or anaerobic conditions, respectively. In the absence of O2 the reaction was faster and more effective (i.e. less NAD(P)H oxidized per mole Lb3+ reduced) than in the presence of O2; this phenomenon was probably because O2 competes with Lb3+ for reductant, thus generating activated O2 species. The flavin-mediated reduction of Lb3+ did not entail production of superoxide or peroxide, indicating that NAD(P)H-reduced flavins were able to reduce Lb3+ directly. The NAD(P)H + flavin system also reduced the complexes Lb3+ · nicotinate and Lb3+ · acetate to Lb2+ · O2, Lb2+ or Lb2+ · nicotinate, depending on the concentrations of ligands and of O2. In the presence of 200 μM nitrite most Lb remained as Lb3+ in aerobic conditions but the nitrosyl complex (Lb2+ · NO) was generated in anaerobic conditions. The above-mentioned characteristics of the NAD(P)H + flavin system, coupled with its effectiveness in reducing Lb3+ at physiological levels of NAD(P)H and flavins in soybean nodules, indicate that this mechanism may be especially important for reducing Lb3+ in vivo., M. Becana thanks the Spanish Ministry of Education and Science/Fulbright Commission for financial support.

Evidence is presented for the operation in nodules of at least four systems for restoring functional ferrous leghemoglobin (Lb2+) from its inactive, ferric form. (i) Reduction of ferric leghemoglobin (Lb3+) by a reductase. The enzyme is a flavoprotein of 100 kDa with two equally sized subunits and exhibits aK(m) of 9 ¿M for soybean Lb3+ component a and aK(m) of 51 ¿M for NADH. NADPH is only 30% (initial velocities) as effective as NADH. Lb3+ reductase converts 215 nmol of Lb3+ to Lb2+·CO (or Lb2+·O2) per mg of protein per min and does not require an exogenous electron carrier. The enzyme shows similar affinity for soybean, bean, and cowpea Lb3+, but different V(max) values. The reductase is inactive when Lb3+ is bound to nicotinate or NO2-. (ii) Direct reduction of Lb3+ by NAD(P)H, ascorbate, and cysteine. Reduction by NAD(P)H is greatly stimulated by trace amounts of metals such as Mn2+. (iii) Reduction of Lb3+ by the flow of electrons from NAD(P)H to free flavins to Lb3+. The reaction does not occur via O2-/· or H2O2, and thus NAD(P)H-reduced flavins can directly reduce Lb3+. The efficiency of the reaction follows the order riboflavin > FMN > FAD. (iv) Reduction of Lb3+ by an unknown compound, B, of nodules. B has a molecular mass < 1 kDa and is heat-stable. The reaction mediated by B differs from those mediated by flavins and metals in several ways, requires NAD(P)H, and generates O2-/·., M.B. received financial support from the Organisation for Economic Co-operation and Development (Project on Food Production and Preservation) and from the Spanish Ministry of Education/Fulbright Commission.

Plant Gene Register titles for PGR 99–174 to PGR 99–187 appear below. The sequences have beendeposited in GenBank and the articles listed online through the World Wide Web.To cite an electronic Plant Gene Register article as a bibliographic reference, follow the stylegiven below:Park S, Thornburg RW (1998) Characterization of UMP kinase cDNAs from rice (accession nos.AF187062 and AF187063) (PGR 99–174). Plant Physiol

Soybean Glycine max. L. Merr. nodule senescence was studied using the loss of acetylene reduction by intact tap root nodules as its indication. Tap root nodules from two varieties (Calland and Beeson) of field-grown soybeans were used. The specific activities of nitrogenase (micromoles/minute gram fresh weight of nodules) as measured by the acetylene reduction assay decreased abruptly between 58 to 65 and 68 to 75 days after planting the Beeson and Calland soybeans, respectively. Major changes were not detected in dry weight, total nitrogen, and leghemoglobin levels during the period when in vivo nitrogenase activity declined. Ammonium levels in the cytosol of nodules and poly-beta-hydroxybutyrate increased moderately just prior to or coincidental with the loss of nitrogenase activity. Neither enzymes that have been postulated to be involved in ammonium assimilation nor NADP(+)-specific isocitrate dehydrogenase exhibited any large changes in specific activities during the initial period when nitrogenase activity declined.

Nitrogen-fixing bacteria were isolated from surface-sterilized seeds and roots of turf grasses growing in Nebraska. The percentage of successful isolations from surface-sterilized seeds ranged from 0 to 100 depending upon the source of the seeds. Based upon morphological and physiological characterization, some of the root and seed isolates appeared to be Azospirillum spp. Deoxyribonucleic acid homology studies on four selected isolates indicated that two root and one seed isolates were related to Azospirillum brasilense SP-7 with 70% or greater DNA homology, and one seed isolate was related to reference strain Azospirillum amazonense Y1 with 100% DNA homology. After repeated culturing on semisolid malate medium, some of the isolates lost their capacity to fix nitrogen. However, when certain non-nitrogen-fixing isolates were used as inoculants on turf grasses grown from surface-sterilized seeds which possessed no detectable indigenous nitrogen-fixing bacteria, nitrogenase activity as measured by acetylene reduction was detected.

An NADH: (acceptor) oxidoreductase from the cytosol of soybean root nodules was purified by ammonium sulfate fractionation, hydroxylapatite adsorption, and Sephacryl S-200 Superfine chromatography. The native molecular weight of the reductase was found to be 100,000 by analytical gel filtration and 83,000 by equilibrium ultracentrifugation. The subunit molecular weight was 54,000 as determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The pI of the enzyme was 5.5. With ferric leghemoglobin (Lb) as the substrate, nearly identical initial velocities were obtained using either CO or O2 to ligate the enzymatically produced ferrous leghemoglobin. With CO as the ligand in the reaction, the product of the enzyme-catalyzed, NADH-dependent reduction of ferric Lb was spectrally identified as LbCO. Initial velocity was a linear function of increasing enzyme concentration. NADPH was only 31% as effective an electron donor as NADH as determined by initial velocity. The Michaelis constants (Km) for ferric Lba and NADH were 9.5 and 18.8 microM, respectively. Myoglobin, Lba, Lbc1, Lbc2, Lbc3, and Lbd were reduced at similar rates by the reductase. At pH 5.2, acetate-bound ferric Lb and nicotinate-bound ferric Lb were reduced by the enzyme at 83 and 5%, respectively, of rates observed in the absence of these ligands. The rate of enzymatic reduction of ferric Lb was constant between pH 6.5 and 7.6 but increased approximately threefold at pH 5.2. The results indicate that the NADH: (acceptor) oxidoreductase could be identified as a ferric Lb reductase.

Senescence of soybean (Glycine max L. Merr.) tap root nodules was investigated by comparing changes in various physiological and biochemical activities with changes in capacity to fix nitrogen. Field-grown Beeson and Calland varieties of soybeans of various ages were sources of tap root nodules. With both varieties, the number of tap root nodules per plant remained constant between 56 and 86 days after planting but fresh weight, dry weight, and mass of tap root nodules increased during this period. Nitrogen (C2H2) fixation by attached tap root nodules was maximum on a fresh weight, dry weight, or nitrogen basis about 56 days after planting for either variety. Metabolic activities of bacteroids as measured by carbon dioxide evolution from glucose and succinate did not appear to vary among nodules of different ages. There was also no indication of mobilization or deposition of iron, molybdenum, calcium, zinc, and nitrate in aging tap root nodules. Nitrate levels in the aerial portion of the plants decreased significantly after the initial decline in acetylene reduction. Nicotinamide deamidase activity in the cytosol and in extracts of bacteroids did not change significantly as tap root nodules aged. However, significant and consistent changes were observed in initial pH values of nodule breis and the initial decline occurred before (Calland) or concurrently (Beeson) with the initial decline of nitrogen fixation.

Response of 'Park' Kentucky bluegrass to inoculation with Klebsiella pneumoniae strain W-6 was tested under field conditions. Field inoculation did not increase nitrogenase activity measured in situ, but did increase the nitrogenase activity as measured using an excised root assay which included a 10-h incubation before the addition of acetylene. Fifteen lines composing 'Park' were grown in fritted-clay medium, inoculated with soil, and compared for nitrogenase activities using the excised roots assay. Significant differences were observed between two lines. Six lines were selected from the 15 lines, grown hydroponically, inoculated with soil, and assayed for nitrogenase activity using intact 105-day-old plants. Nitrogenase activities were immediately detectable and increased curvilinearly. Differences in nitrogenase activities among the six lines were detected within 1 h and significant differences were evident in 4 h. The six selected lines were also tested for levels of anthrone-reactive sugars in roots and root exudates of hydroponically grown plants. Significant differences were detected in levels of sugars in roots but not in root exudates. Possible correlations were sought among and within the three different experiments involving the six selected lines. With hydroponically grown plants, nitrogenase activities during the first 4 h were highly correlated with those after 24 h incubation. Correlations were found between nitrogenase activities in excised roots and soluble sugar concentrations in root tissue and root exudates.

Summary: Plasmids were isolated from strains of Rhizobium japonicum, predominantly serogroup 135, obtained from soybean nodules collected at 15 sites in Nebraska, U.S.A. In addition to their serotype, these strains were indistinguishable from R. japonicum strain 3I1b135 in growth rate, sensitivity to phage Rhj781, antibiotic sensitivities, general colony characteristics and rates of nitrogen fixation per plant. All strains occupied soil habitats with similar characteristics, including a high pH (7.2 to 8.3), relatively high conductivity (0.04 to 0.32 mS), relatively high sodium saturation (0.32 to 12.7%), low iron content (3.2 to 14.8 p.p.m.) and low manganese content (5.1 to 18.7 p.p.m.). However, agarose gel electrophoresis analysis of plasmids enabled subdivision of these extra-slow-growing strains into four groups on the basis of differences in plasmid number and size. These strains carried combinations of two or more of four plasmids, ranging in mass from 49 to 118 megadaltons and comprising approximately 20% of the total DNA per cell. Biological and symbiotic data, along with plasmid analysis, were useful in identifying a wild-type strain (RJ23A) that shows potential as a soybean inoculant in alkaline soils.

Ferric leghemoglobin isolated from soybean root nodules was reduced nonenzymatically to ferrous leghemoglobin in vitro at pH 5.2 using either 1.0 mM NADH or NADPH as the reductant. In the pH range of 5.2 to 7.0, the highest rates of reduction occurred below pH 6.5 with a maximum rate observed at pH 5.2. Rates of nonenzymatic ferric leghemoglobin reduction above pH 6.5 or at reduced-pyridine nucleotide concentrations below 0.4 mM were insignificant. Oxygen was required for the nonenzymatic reduction. Inhibition of ferric leghemoglobin reduction by superoxide dismutas and catalase indicated that superoxide and hydrogen peroxide may be intermediates in the reaction.

A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N2 fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H2 to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein.

The nitrogenase system in cell-free extracts of soybean nodule bacteroids was fractionated into 2 components by use of protamine sulfate or polypropylene glycol precipitation followed by chromatography on DEAE-cellulose. Iron and molybdenum were concentrated in 1 fraction and iron in the other. Combination of fractions resulted in a striking stimulation in activity relative to the activity of individual fractions. The effect of different proportions of the 2 fractions on specific activities was studied. The ratios of the rates of reduction of acetylene and N(2) by extracts or fractions of different purities were relatively constant. Extracts or fractions retained most of their nitrogenase activities when stored in liquid N(2).

The catalysis of nitrogen reduction by extracts of nitrogen-fixing organisms requires an ATPgenerating system and a source of electrons (1, 2, 5, 11, 13). The phosplhoroclastic reaction provides botlh ATP anid electrons for the reduction of nitrogen by cell-free extracts of Clostridium1t pasteurianumn (2, 5, 13) and ferredoxin functions as the natural electron carrier from the phlosphoroclastic reaction to nitrogenase ('12). Some evidence has been reported (2) that boro,hydride, hydrogen, NADH or NAiDPH, in presence of ferredoxin, will serve as a reductant for nitrogen fixation by the clostridial system. Cell-free extracts of Azotobacter vinelandiz (1) and bacteroids from soybean nodules (11) also catalvze the reduction of N. to NH, but an exogenous souirce of both reductant and ATP must be furnisihed. Probablv the phvsiological source of reductant and ATP for nitrogen fixation by leguime nodules and azotobacter is the respiratory electron transport chain with its associated oxidative phosphorvlation. All efforts however to coutple nitrogen fixation by cell-free extracts of these organisms to naturally occutrring oxidation-redutction reactions have failed. There are no reports of the identification in azotobacter or legume nodules of a low potential electron carrier analogous to ferredoxin. Using the reduction of acetylene to etlhy-lene as a measure of nitrogenase activity (3, 14), evidence has been obtaiined that NADH genierated by the fl-hydroxybutyrate dehvdrogenase reaction or high concentrations of NADH supplied directly function as a source of electrons for acetvlene redutction by extracts of nodule bacteroids or azotobacter. Activity also is dependent 1110pon an ATP-generating system and a suiitable dye. Cell-free extracts of soybean nodlules were prepared by the nmetho(d of Kochi, Evan,s, and Ruissell (10, 11) with minor changes to improve the nitrogenase activity. Ethyrlene production from acetylene was measuired by a method described by Kelly

Soybean plants and Rhizobium japonicum 122 DES, a hydrogen uptake-positive strain, were cultured in media purified to remove Ni. Supplemental Ni had no significant effect on the dry matter or total N content of plants. However, the addition of Ni to both nitrate-grown and symbiotically grown plants resulted in a 7- to 10-fold increase in urease activity (urea amidohydrolase, EC 3.5.1.5) in leaves and significantly increased the hydrogenase activity (EC 1.18.3.1) in isolated nodule bacteroids. When cultured under chemolithotrophic conditions, free-living R. japonicum required Ni for growth and for the expression of hydrogenase activity. Hydrogenase activity was minimal or not detectable in cells incubated either without Ni or with Ni and chloramphenicol. Ni is required for derepression of hydrogenase activity and apparently protein synthesis is necessary for the participation of Ni in hydrogenase expression. The addition of Cr, V, Sn, and Pb in place of Ni failed to stimulate the activity of hydrogenase in R. japonicum and urease in soybean leaves. The evidence indicates that Ni is an important micronutrient element in the biology of the soybean plant and R. japonicum .

Reduction of ferric leghemoglobin to ferrous leghemoglobin in soybean nodules (Glycine max [L.] Merr. cv Woodworth) was studied using a spectrophotometer equipped with an in-cell space diffuse reflectance accessory. Nodule slices prepared and scanned under nitrogen gas showed a ferrous leghemoglobin absorption spectrum. Nodule slices equilibrated with 100% O(2) or air exhibited two absorption bands characteristic of oxygenated leghemoglobin. The addition of CO shifted those bands to CO leghemoglobin absorption bands. Potassium ferricyanide was not effective in oxidizing ferrous to ferric leghemoglobin in nodule slices. However, ferric leghemoglobin was formed by treating the nodule slices with hydroxylamine, and this was confirmed by complexing the ferric leghemoglobin to acetate, fluoride, or nicotinic acid. The diminution of ferric leghemoglobin was monitored as a function of time, and in the presence of nicotinic acid, the conversion of ferric to ferrous leghemoglobin was monitored by the appearance of ferrous leghemoglobin nicotinate complex as a function of time. Ferric leghemoglobin reduction was also confirmed by direct transmission spectrophotometry. The evidence presented here suggests that ferrileghemoglobin reduction occurs in nodule slices.

Glutamine amide–2-oxoglutarate aminotransferase NAD+ oxidoreductase (GOGAT), glutamine synthetase (GS), glutamate dehydrogenase (GD), and alanine dehydrogenase (AD) were studied in soybean root nodules. GS, GOGAT, and AD were present in bacteroids at levels that could account for ammonium assimilation, but GD activity was quite low. The total activities of GS and GD were higher in the cytosol than in the bacteroids by factors of 20 and 7, respectively, whereas GOGAT was not detected in the cytosol. GS (transferase activity) was inhibited by alanine, CTP, glycine, and tryptophan at 5 mM but was relatively unaffected by asparagine, aspartic acid, CMP, glucosamine, and histidine at 5 mM. GOGAT activity was unaffected by ATP, ADP, 8-hydroxyquinoline, and 1,10-phenanthroline but was inhibited by EDTA, citrate, and parachloromercuribenzoate. GOGAT activity (reductive amination) was also inhibited 97% by preincubation with 10−4 M azaserine for 30 min but GD activity was inhibited only 13%. The apparent Km values for NH4+ by AD was 7.4 × 10−3 M and by GD was 7.3 × 10−2 M while for glutamine by GOGAT it was 9.3 × 10−5 M. Activities and kinetic properties for these enzymes may suggest potential routes of nitrogen assimilation in vivo.

Several facultative asymbiotic N2-fixing bacteria exhibited nitrogenase activity when grown in the presence of air and a limiting amount of fixed nitrogen. One isolate (S-4) identified as Klebsiella was examined in more detail for oxygen effects. Nitrogenase was not detectable in highly aerated submerged cultures but was detectable in shaken and continuous cultures at dissolved oxygen tensions of 3 and 10 mm of Hg respectively. No nitrogenase activity was found in cells from continuous cultures maintained at a dissolved oxygen tension of 15 mm of Hg or above. The nitrogenase activity in whole cells was not oxygen dependent but was oxygen tolerant.

Publisher Summary This chapter discusses the preparation of nitrogenase from nodules and separation into components. The chapter reviews Bergerson's utilization of a specially designed anaerobic press for the preparation of nodule brei, which exhibited nitrogen-fixing capacity. Cell-free extracts of nodule bacteroids could not be prepared until it is realized that the phenolic compounds that are prevalent in legume nodules react with, and denature, the nitrogenase when nodules are macerated in air. In the method, nodules are macerated and extracts of bacteroids are prepared under conditions where contact with oxygen is minimized. Ascorbate is used in the preparative medium to prevent the oxidation of phenols, and phenolic compounds in nodule breis are removed as an insoluble complex with polyvinylpolypyrrolidone (PVP). By use of these procedures, crude extracts of nodule bacteroids with specific activities comparable to those of extracts of free-living nitrogen-fixing bacteria may be prepared consistently. The preparations are purified and fractionated into two components, both of which are essential for nitrogenase activity.

Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.