Search

‘Eve’ (Reg. No. CV‐350, PI 659067), a six‐row, winter hulless barley (Hordeum vulgareL.) developed and tested as VA01H‐68 by the Virginia Agricultural Experiment Station, was released in May 2007. Eve was derived from the cross SC860974/VA94‐42‐13 using a modified bulk‐breeding method. It was evaluated in the 2009–2011 Virginia Official Variety Trial in replicated tests at five to six locations. Eve had an average grain yield (3718 kg ha−1) that was higher than those of the hulless check cultivars Dan and Doyce. The average grain volume weight of Eve (73.8 kg hL−1) over the same period was higher than that of Doyce (68.9 kg hL−1). The head emergence of Eve is 6 d earlier than Dan and ‘Thoroughbred’, which is the predominant barley cultivar grown in the eastern United States. Earlier maturity is a primary factor determining whether barley or wheat (Triticum aestivumL.) is used in double‐cropping systems with soybean (Glycine maxL.). The grain of Eve has high starch (60.4%) and protein (10.1%) concentration. Eve is the first winter hulless barley released in the eastern United States having a high level of resistance to Fusarium head blight [FHB; caused by Fusarium graminearum(Schwabe)] and reduced accumulation of the mycotoxin deoxnivalenol in the grain. Eve's unique grain composition and resistance to FHB make it desirable as a commodity for food, feed, and ethanol production.

Meristematic tissues from rye (Secale cereale) and oat (Avena sativa) were studied in an isothermal calorimeter at -3 degrees C. When the frozen tissue was placed in the calorimeter, the pressure increased within 4 d to 25 and 9 kPa above ambient pressure in the sample vessels containing crowns of rye and oat, respectively. Concurrently, the thermal output went down to -194 microW in rye over the 4-d period; this negative thermal activity could be accounted for by ice melting in the plants. When the pressure was released, the output from the calorimeter went from -194 to 229 microW within 1 h, suggesting that water had frozen in the plants. We propose that CO(2) from respiration had dissolved in the water in the plants and caused melting of ice (heat absorption) due to the colligative properties of solutions. When the pressure was released, the CO(2) came out of solution and the water froze (heat evolution). These thermal observations were duplicated in a simplified, non-biological system using a glycol/water mixture that was partially frozen at -3 degrees C.

The effect of L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, on the formation of nitrate reductase in the wild-type strain of Neurospora in the presence of ammonium ions and of glutamine was studied. Under conditions in which glutamine synthetase was inactivated, it was found that only glutamine could repress nitrate reductase. In a mutant of Neurospora, gln-1b, which requires glutamine for growth, only glutamine could repress nitrate reductase. These results suggest a direct role for glutamine as corepressor of nitrate reductase in Neurospora.

The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory of the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.

S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4′,5′-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4′,5′-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.

This report describes the isolation and characterization of a Neurospora crassa mutant with an impaired regulation of nitrate reductase. Glutamine, which prevents the induction of nitrate reductase in N. crassa, did so relatively ineffectively in this mutant. The mutation did not affect the regulation of all enzymes regulated by "nitrogen metabolite regulation"; it did affect the regulation of nitrate reductase, nitrite reductase, histidase, and acetamidase, as well as that of thiourea sensitivity. The mutation was not allelic with nit-2, the gene controlling a general positive effector of nitrogen metabolite-regulated enzyme formation.

Transcription of the genes encoding molybdenum (Mo)‐independent nitrogenases 2 and 3 of Azotobacter vinelandiirequires the activators VnfA and AnfA, respectively. The effect of NH+4, Mo, or V (vanadium) was tested on the expression of vnfA‐lacZand anfA‐lacZtranscriptional fusions. Mo repressed expression of both fusions whereas NH+4and V repressed the anfA‐lacZfusion, but not the vnfA‐lacZfusion. Thus the repressive effect on transcription of the anfHDGKORoperon by NH+4, Mo, or V is mediated through their effect on transcription of anfAand the repressive effect of Mo on the vnfHFdand vnfDGKoperons is mediated through Mo repression of vnfAtranscription. Mo‐dependent repression of anfAtranscription is influenced but not entirely mediated by the Mo‐responsive regulator ModE.

The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

Mutants of Azotobacter vinelandii affected in N2 fixation in the presence of 1 microM Na2MoO4 (conventional system), 50 nM V2O5, or under Mo deficiency (alternative system) have been isolated after Tn5 mutagenesis with the suicide plasmid pSUP1011. These mutants can be grouped into at least four broad phenotypic classes. Mutants in the first class are Nif- under Mo sufficiency but Nif+ under Mo deficiency or in the presence of V2O5. A nifk mutant and a mutant apparently affected in regulation of the conventional system belong to this class. Mutants in the second class are Nif- under all conditions. An FeMo-cofactor-negative mutant (NifB-) belongs to this class, implying an involvement of nifB in both the conventional and the alternative N2 fixation systems. The third mutant class consists of mutants incapable of N2-dependent growth under Mo deficiency. Most of the mutants in this class are also affected in N2 fixation in the presence of 1 microM Na2MoO4, with acetylene reduction rates ranging from 28 to 51% of the rates of the wild type. Strains constructed by genetic transfer of the Kanr marker of mutants from this class into nifHDK or nifK deletion mutants showed N2-dependent growth only in the presence of V2O5, suggesting that growth in the presence of V2O5 and growth under Mo deficiency are independent phenomena. The only mutant in the fourth class shows wild-type nitrogenase activity under Mo sufficiency, but only 10% of the acetylene reduction activity of the wild type in the presence of 50 nM V2O5. The acetylene reduction rates of whole cells of this mutant are identical in Mo-deficient medium and in medium containing V2O5. The conventional nitrogenase subunits are expressed in this mutant even under Mo deficiency or in the presence of V2O5; however, the NH4+- and Mo-repressible proteins normally seen under these conditions could not be detected on two-dimensional gels. The Tn5 insertion carried by this mutant makes N2 fixation dependent solely on the conventional system and consequently abolishes the vanadium effect.

A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.

Multiple genomic regions homologous to nifH were found in the diazotroph Azotobacter vinelandii. The nifHDK gene cluster, located on a 12.8-kilobase (kb) XhoI fragment and two additional XhoI fragments (7.4 and 8.4 kb) hybridized to a nifH-specific DNA template but the 7.4- and 8.4-kb fragments did not hybridize to nifD- or nifK-specific DNA probes. In vivo transcription of the nifHDK gene cluster was ammonia-repressible and required the presence of at least 50 nM molybdenum in the derepression medium. Three mRNA species were transcribed from the nifHDK gene cluster, a 4.2-kb transcript homologous to nifH-, nifD-, and nifK-specific DNA templates, a 2.6-kb transcript homologous to nifH- and nifD-specific DNA templates, and a 1.2-kb transcript homologous only to the nifH-specific DNA template. In strain CA11, a nifHDK deletion mutant, the nifHDK-specific transcripts were not produced and the strain was unable to grow in N-free medium in the presence of Na2MoO4 at concentrations of 50 nM or higher. However, at concentrations of 25 nM Mo or less, growth occurred in N-free medium. Under these conditions two nifH-homologous (but not nifD- or nifK-homologous) transcripts were observed (1.2 and 1.8 kb). Presumably these were transcribed from the additional nifH-homologous sequences present in the genome. These results are consistent with the existence of two N2 fixation systems in A. vinelandii which are regulated by molybdenum at the level of transcription.

Studies were carried out to determine the factors governing the induction of anthranilate hydroxylase and other enzymes in the pathway for the dissimilation of anthranilate by Aspergillus niger(UBC 814). The enzyme was induced by growth in the presence of tryptophan, kynurenine, anthranilate, and, surprisingly, by 3-hydroxyanthranilate, which was not an intermediate in the conversion of anthranilate to 2,3-dihydroxybenzoate. There was an initial lag in the synthesis of anthranilate hydroxylase when induced by tryptophan, anthranilate, and 3-hydroxyanthranilate. Cycloheximide inhibited the enzyme induction. Comparative studies on anthranilate hydroxylase, 2,3-dihydroxybenzoate carboxy-lyase, and catechol 1:2-oxygenase revealed that these enzymes were not coordinately induced by either anthranilate or 3-hydroxyanthranilate. Structural requirements for the induction of anthranilate hydroxylase were determined by using various analogues of anthranilate. The activity of the constitutive catechol oxygenase was increased threefold by exposure to anthranilate, 2,3-dihydroxybenzoate, or catechol. 3-Hydroxyanthranilate did not enhance the levels of catechol oxygenase activity.