Shoot versus Root Signal Involvement in Nodulation and Vegetative Growth in Wild-Type and Hypernodulating Soybean Genotypes.

Grafting studies involving Williams 82 (normally nodulating) and NOD1-3 (hypernodulating) soybean (Glycine max [L.] Merr.) lines and Lablab purpureus were used to evaluate the effect of shoot and root on nodulation control and plant growth. A single- or double-wedge graft technique, with superimposed partial defoliation, was used to separate signal control from a photosynthate supply effect. Grafting of hypernodulated soybean shoots to roots of Williams 82 or L. purpureus resulted in increased nodule numbers. Grafting of two shoots to one root enhanced root growth in both soybean genotypes, whereas the nodule number was a function of shoot genotype but not of the photosynthetic area. In double-shoot, single-root-grafted plants, removing trifoliolate leaves from either Williams 82 or NOD1-3 shoots decreased root and shoot dry matter, attributable to decreased photosynthetic source. Concurrently, Williams 82 shoot defoliation increased the nodule number, whereas NOD1-3 shoot defoliation decreased the nodule number on both soybean and L. purpureus roots. It was concluded that (a) soybean leaves are the dominant site of autoregulatory signal production, which controls the nodule number; (b) soybean and L. purpureus have a common, translocatable, autoregulatory control signal; (c) seedling vegetative growth and nodule number are independently controlled; and (d) two signals, inhibitor and promoter, may be involved in controlling legume nodule numbers.

Identification of cDNA clones corresponding to two inducible nitrate reductase genes in soybean: analysis in wild-type and nr1 mutant.

Among higher plants, soybean is unique in that biochemically it has been characterized as having two constitutive nitrate reductase (cNR) isoforms and one substrate-inducible nitrate reductase (iNR) isoform in leaves. All three NR isoforms are expressed in cv. Williams 82 while the nr1 mutant expresses only the iNR isoform. The genetic and molecular mechanisms for regulation of these isoforms have not been elucidated. We describe here the isolation, by reverse transcription-polymerase chain reaction (RT-PCR), of two cDNA clones encoding soybean NR. They were designated as iNR1 and iNR2, respectively, since both were inducible by nitrate. The iNR1 and iNR2 cDNAs cover total encoding regions of 2661 and 2673 nucleotides, respectively. The iNR1 clone shows a 12 bp deletion at the 5' end, relative to iNR2. They show overall similarity of 89% at the nucleotide level, and 87% at the amino acid level. Like all plant NRs cloned so far, deduced amino acid sequences between iNR1 and iNR2 show greatest variation at the N-terminal region while no difference was observed at the C-terminus. Soybean iNR mRNAs were found to be different from those of maize and tobacco in response to tungsten inhibitor treatment, since the inhibitor decreased the steady-state levels of mRNA for soybean iNR and for NiR. Using the same 5' regions of both cDNAs as the probes, Southern blot analysis of genomic DNA revealed differences in organization between iNR1 and iNR2. The genomic DNA from wild-type Williams 82 soybean was shown to have three Eco RI fragments while the nr1 mutant lacked an 8 kb fragment when probed with iNR1 cDNA. Likewise, the nr1 mutant lacked three Hae III restriction fragments when probed with iNR1 cDNA. When probed with iNR2, both wildtype and nr1 mutant showed one identical Eco RI band and two identical Hae III bands. In northern blots, the steady-state level of iNR1 mRNA was similar for the nr1 mutant and the wild-type parent after 20 to 48 h induction by nitrate. Based on the Eco RI and Hae III restriction enzyme digestion patterns observed in Southern blot analysis of soybean DNA, it is concluded that in soybean iNR1 is encoded by a small multiple gene family and iNR2 is a single gene.

Multiple activities of the human splicing factor ASF.

The effects of human alternative splicing factor, ASF, on in vitro splicing of adenovirus E1A pre-mRNA were examined. E1A pre-mRNA is a complex substrate, and splicing in HeLa cell nuclear extracts produces six different RNAs using three alternative 5' splice sites and two 3' splice sites. Addition of excess ASF to splicing reactions produced a simplified splicing pattern, in which only one spliced product, 13S RNA, was detected. Inhibition of 12S and 9S splicing, which use 5' splice sites upstream of the 13S 5' splice site, extends previous observations that when multiple 5' splice sites compete for the same 3' splice site, ASF causes preferential selection of the proximal 5' splice site. However, inhibition of the other splices, which use a different upstream 3' splice site, represents a novel activity of ASF, as competition between 5' splice sites is not involved. The effect of ASF on 12S splicing was found to depend on its position relative to competing 5' splice sites, indicating that the ability of ASF to activate proximal 5' splice sites is position- but not sequence-dependent. Finally, addition of small amounts of ASF to ASF-lacking S100 extract was able to activate distal as well as proximal 5' splice sites in two of three pre-mRNAs tested, indicating that in these cases changes in the concentration of ASF alone can be sufficient to modulate alternative 5' splice site selection.

A novel protein factor is required for use of distal alternative 5' splice sites in vitro.

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

Root Isoflavonoid Response to Grafting between Wild-Type and Nodulation-Mutant Soybean Plants.

It was previously reported that the hypernodulating soybean (Glycine max [L.] Merr.) mutants, derived from the cultivar Williams, had higher root concentration of isoflavonoid compounds (daidzein, genistein, and coumestrol) than did Williams at 9 to 12 days after inoculation with Bradyrhizobium japonicum. These compounds are known inducers of nod genes in B. japonicum and may be involved in subsequent nodule development. The current study involving reciprocal grafts between NOD1-3 (hypernodulating mutant) and Williams showed that root isoflavonoid concentration and content was more than twofold greater when the shoot genotype was NOD1-3. When grafted, NOD1-3 shoots also induced hypernodulation on roots of both Williams and NOD1-3, while Williams shoots induced normal nodulation on both root genotypes. This shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by the shoot. In contrast, the nonnodulating characteristic of the NN5 mutant was strictly root controlled, based on reciprocal grafts. Delayed inoculation (7 days after planting) resulted in greater nodule numbers on both NOD1-3 and Williams, compared with a seed inoculation treatment. The nodulation pattern of grafted plants was independent of whether the shoot portion was derived from inoculated seed or uninoculated seed, when grafted at day 7 onto seedling roots derived from inoculated seed. This observation, coupled with the fact that no difference existed in nodule number of NOD1-3 and Williams until after 9 days from seed inoculation, indicated that if isoflavonoids play a role in differential nodulation of the hypernodulating mutant and the wild type, the effect is on advanced stages of nodule ontogeny, possibly related to autoregulation, rather than on initial infection stages.

Effect of localized nitrate application on isoflavonoid concentration and nodulation in split-root systems of wild-type and nodulation-mutant soybean plants.

Although isoflavonoids are known to be inducers of nod genes in Bradyrhizobium japonicum, it was recently proposed that internal root levels of isoflavonoids may be important in nodule development on soybean (Glycine max [L.] Merr.). The hypernodulating soybean mutants were shown to accumulate higher root concentrations of isoflavonoid compounds (daidzein, genistein, and coumestrol) and to be more extensively nodulated than was the Williams parent when inoculated with B. japonicum. The hypernodulating mutants and the parent line, Williams, also showed decreased isoflavonoid concentrations and decreased nodule development if N was applied. The current study evaluated the effect of localized NO(3) (-) application on root isoflavonoid concentration and on nodulation in split-root systems of the Williams wild type and a hypernodulating mutant (NOD1-3). Nitrate application markedly decreased isoflavonoid concentrations in non-inoculated soybean roots. When roots were inoculated, nodule number, weight, and nitrogenase activity were markedly suppressed on the root-half receiving 5 millimolar NO(3) (-) compared with the other root-half receiving 0 millimolar NO(3) (-). High performance liquid chromatographic analyses of root extracts showed that the root-half receiving 5 millimolar NO(3) (-) was markedly lower in isoflavonoid concentrations in both soybean lines. This was partially due to the localized stimulatory effect of NO(3) (-) on root growth. The inoculated NOD1-3 mutant had higher isoflavonoid concentrations than did the Williams control in both the presence and absence of NO(3) (-). These results provide evidence that the site of N application primarily controls the site of nodulation inhibition, possibly through decreasing isoflavonoid levels. Although the effect of NO(3) (-) on nodule development and root isoflavonoid concentration was strongly localized, there was evidence that NO(3) (-) also resulted in a systemic effect on root isoflavonoids. The results are consistent with previous speculation that internal levels of root isoflavonoids may affect nodule development.

Effect of inoculation and nitrogen on isoflavonoid concentration in wild-type and nodulation-mutant soybean roots.

The isoflavones, daidzein and genistein, have been isolated and identified as the major inducers of nod genes of Bradyrhizobium japonicum. The common nod genes of rhizobia are in turn responsible for stimulating root hair curling and cortical root cell division, the earliest steps in the host response. This study evaluated whether there was a relationship between root isoflavonoid production and the hypernodulation phenotype of selected soybean (Glycine max [L.] Merr.) mutants. Three independently selected hypernodulating soybean mutants (NOD1-3, NOD2-4, and NOD3-7) and a nonnodulating mutant (NN5) were compared with the Williams parent for isoflavonoid concentrations. High performance liquid chromatographic analyses of soybean root extracts showed that all lines increased in daidzein, genistein, and coumestrol concentrations throughout the 12-day growth period after transplanting of both inoculated and noninoculated plants; transplanting and inoculation were done 6 days after planting. No significant differences were detected in the concentration of these compounds among the three noninoculated hypernodulating mutants and the Williams parent. In response to inoculation, the three hypernodulating mutants had higher isoflavonoid concentrations than did the Williams control at 9 to 12 days after inoculation when grown at 0 millimolar N level. However, the inoculated nonnodulating mutant also had higher isoflavonoid concentrations than did Williams. N application [urea, (NH(4))(2)SO(4) and NO(3) (-)] decreased the concentration of all three isoflavonoid compounds in all soybean lines. Application of NO(3) (-) was most inhibitory to isoflavonoid concentrations, and inhibition by NO(3) (-) was concentration dependent. These results are consistent with a conclusion that differential NO(3) (-) inhibition of nodulation may be partially due to changes in isoflavonoid levels, although the similar response of the nonnodulating mutant brings this conclusion into question. Alternatively, the nodulation control in the NN5 mutant may be due to factors totally unrelated to isoflavonoids, leaving open the possibility that isoflavonoids play a role in differential nodulation of lines genetically competent to nodulate.

Sequence specificity of the human mRNA N6-adenosine methylase in vitro.

N6-adenosine methylation is a frequent modification of mRNAs and their precursors, but little is known about the mechanism of the reaction or the function of the modification. To explore these questions, we developed conditions to examine N6-adenosine methylase activity in HeLa cell nuclear extracts. Transfer of the methyl group from S-[3H methyl]-adenosylmethionine to unlabeled random copolymer RNA substrates of varying ribonucleotide composition revealed a substrate specificity consistent with a previously deduced consensus sequence, Pu[G greater than A]AC[A/C/U]. 32-P labeled RNA substrates of defined sequence were used to examine the minimum sequence requirements for methylation. Each RNA was 20 nucleotides long, and contained either the core consensus sequence GGACU, or some variation of this sequence. RNAs containing GGACU, either in single or multiple copies, were good substrates for methylation, whereas RNAs containing single base substitutions within the GGACU sequence gave dramatically reduced methylation. These results demonstrate that the N6-adenosine methylase has a strict sequence specificity, and that there is no requirement for extended sequences or secondary structures for methylation. Recognition of this sequence does not require an RNA component, as micrococcal nuclease pretreatment of nuclear extracts actually increased methylation efficiency.

Nitrogen fixation of nodulation mutants of soybean as affected by nitrate.

It was previously reported that three soybean (Glycine max [L.] Merr.) nodulation mutants (NOD1-3, NOD2-4, and NOD3-7) were partially tolerant to nitrate when nitrate was supplied simultaneously with inoculation at the time of transplanting. The current study evaluated the effect of short-term nitrate treatment on nitrogenase activity (C(2)H(2) reduction per plant and per nodule weight) and on relative abundance of ureides when nitrate application was delayed until plants were 3 weeks old and nodules were fully developed. Nitrogenase activity of the mutants was similar to that of Williams after an initial 3-week growth period, prior to nitrate treatment. Application of 5 millimolar nitrate resulted in greater inhibition of nitrogenase activity in Williams than in the three mutants. NOD1-3 was most tolerant of nitrate among the mutants tested and showed the highest relative abundance of ureides. Although C(2)H(2) reduction activity per plant for NOD1-3 was higher than for Williams in the presence of nitrate, C(2)H(2) reduction activity per gram of nodules was lower for NOD1-3 than for Williams in the presence and absence of nitrate. Compared to Williams, NOD1-3 had higher nodule ureide concentration and had similar glutamine synthetase activity in nodule tissue, indicating its nodules have normal nitrogen assimilation pathways. Nitrate application resulted in ureide accumulation in nodule tissue as well as in all plant parts assayed. Unexpectedly, nitrate treatment also increased the rate of ureide degradative capacity of leaves in both NOD1-3 and Williams. The data confirmed that nitrogenase activity of the selected nodulation mutants was more, but still only partially, tolerant of nitrate compared with the Williams parent.

Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean.

Since NO(3) (-) availability in the rooting medium seriously limits symbiotic N(2) fixation by soybean (Glycine max [L.] Merr.), studies were initiated to select nodulation mutants which were more tolerant to NO(3) (-) and were adapted to the Midwest area of the United States. Three independent mutants were selected in the M(2) generation from ethyl methanesulfonate or N-nitroso-N-methylurea mutagenized Williams seed. All three mutants (designated NOD1-3, NOD2-4, and NOD3-7) were more extensively nodulated (427 to 770 nodules plant(-1)) than the Williams parent (187 nodules plant(-1)) under zero-N growth conditions. This provided evidence that the mutational event(s) affected autoregulatory control of nodulation. Moreover, all three mutants were partially tolerant to NO(3) (-); each retained greater acetylene reduction activity when grown hydroponically with 15 millimolar NO(3) (-) than did Williams at 1.5 millimolar NO(3) (-). The NO(3) (-) tolerance did not appear to be related to an altered ability to take up or metabolize NO(3) (-), based on solution NO(3) (-) depletion and on in vivo nitrate reductase assays. Enhanced nodulation appeared to be controlled by the host plant, being consistent across four Bradyrhizobium japonicum strains tested. In general, the mutant lines produced less dry weight than the control, with root dry weights being more affected than shoot dry weights. The nodulation trait has been stable through the M(5) generation in all three mutants.

The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean.

A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO((x))) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO((x)) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent K(m) for nitrite of 0.49 millimolar, and an apparent K(m) for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO((x)) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH(2)) and reduced methyl viologen (MV) can serve as electron donors for NO((x)) evolution activity. The NADPH-, FMNH(2)-, and reduced MV-NO((x)) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH(2) and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO((x)). NADPH eluted both NR and NO((x)) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean.

A method for the separation and partial purification of the three forms of nitrate reductase present in wild-type soybean leaves.

A rapid and simple purification method was used to separate and purify nitrate reductases (NR) from Williams soybean leaves. Blue Sepharose columns were sequentially eluted with 50 millimolar NADPH and 50 millimolar NADH, thus separating NAD(P)H:NR from NADH:NRs. Subsequent purification of the collected peaks on a fast protein liquid chromatography-Mono Q column enabled separation of two NADH:NRs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the subunit relative molecular mass for all three NR forms (constitutive NAD(P)H:NR [pH 6.5], EC 1.6.6.2; constitutive NADH:NR [pH 6.5], EC not assigned; and inducible NADH:NR [pH 7.5], EC 1.6.6.1) was approximately 107 to 109 kilodaltons. All three NRs showed similar spectra with absorption maxima at 413 and 273 nanometers in the oxidized state, and with the characteristics of a cytochrome b type heme upon reduction with NADH (absorption maxima at 556, 527, and 424 nanometers). The technique developed provides an improved separation of the three NR forms from soybean leaves. The similarity of the NRs with regard to their cytochrome b(556) type heme content and in relative molecular mass indicated that other differences must exist to account for the different kinetic and physical properties previously reported.

Nitric Oxide and Nitrous Oxide Production by Soybean and Winged Bean during the in Vivo Nitrate Reductase Assay.

This study was conducted to determine by gas chromatography (GC) and mass spectrometry (MS) the identity and the quantity of volatile N products produced during the helium-purged in vivo NR assay of soybean (Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identification and quantitation was collected by cryogenic trapping of volatile compounds carried in the He-purge gas stream. As opposed to an earlier report, acetaldehyde oxime production was not detected by our GC method, and acetaldehyde oxime was shown to be much more soluble in water than the compound(s) evolved from soybean leaflets. Nitric oxide (NO) and nitrous oxide (N(2)O) were identified by GC and GC/MS as the main N products formed. NO and N(2)O produced from soybean leaflets were both labeled with (15)N when (15)N-nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Other compounds co-trapped with NO and N(2)O were identified as air (N(2), O(2)), CO(2), methanol, acetaldehyde, and ethanol. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N(2)O (13.9 and 0.37 micromole per gram fresh weight per 30 minutes, respectively) than did the soybean cv Williams (1.67 and 0.09 micromole per gram fresh weight per 30 minutes, respectively). In both species NO production was dominant. In contrast, with similar assays, NO and N(2)O were not evolved from leaves of the nr(1) soybean mutant which lacks the constitutive NR enzymes. In addition to soybean cv Williams, six other Glycine sp. examined evolved significant quantities of NO((x)) (NO and NO(2)). Other species including Neonotonia wightii (Arn.) Lackey comb. nov., Pueraria montana (Lour.) Merr., and Pueraria thunbergiana Benth. evolved lower levels of NO((x)).

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase.

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M(2) seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH(2)-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c(1)NR), constitutive NADH:NR (c(2)NR), and nitrate-inducible NR (iNR).

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : II. Partial Activity, Inhibitor, and Complementation Analyses.

Soybean (Glycine max [L.] Merr.) leaves have been shown to contain three forms of nitrate reductase (NR). Two of the forms, which are present in leaves of wild-type (cv. Williams) plants grown in the absence of NO(3) (-), are termed constitutive and designated c(1)NR and c(2)NR. The third form, which is present in NO(3) (-)-grown mutant (nr(1)) plants lacking the constitutive forms, is termed inducible and designated iNR. Samples of c(1)NR, c(2)NR, and iNR obtained from appropriately treated plants were analyzed for the presence of partial activities, response to inhibitors, and ability to complement a barley NR which lacks the molybdenum cofactor (MoCo) but is otherwise active.The three forms were similar to most assimilatory NR enzymes in that they (a) exhibited NADH-cytochrome c reductase, reduced flavin mononucleotide-NR, and reduced methyl viologen-NR partial activities; (b) were inhibited by p-hydroxymercuribenzoate at the site of initial electron transport through each enzyme; (c) were more inhibited by CN(-) in their reduced enzyme state as compared with their oxidized state; and (d) complemented a MoCo-defective NR (e.g. contained cofactors with characteristics similar to the MoCo found in barley NR and commercial xanthine oxidase). However, among themselves, they showed dissimilarities in their response to treatment with HCO(3) (-) and CN(-), and in their absolute ability to complement the barley NR. The site of effect for these treatments was the terminal cofactor-containing portion of each enzyme. This indicated that, although a terminal cofactor (presumably a MoCo) was present in each form, structural or conformational differences existed in the terminal cofactor-protein complex of each form.

Effect of multiple factor source-sink manipulation on nitrogen and carbon assimilation by soybean.

The objectives of this study were to determine the effect of light enhancement and hastened reproductive development on nitrogen and dry matter accumulation by field-grown soybean (Glycine max [L.] Merr.). The impacts of photosynthate supply and reproductive development on change in the season-long profiles of in vivo leaf nitrate reductase (NR) activity and root nodule acetylene reduction (AR) activity were evaluated.Light enhancement resulted in significant increases in dry matter accumulation, root nodule fresh weight and AR activity. Seed yield was increased in both light enhanced treatments in 1978 and in one in 1979.Hastened flowering and seed development was accomplished through photoperiod manipulation within a single genotype. Seasonal decline in leaf NR activity was most rapid in plants entering reproductive development early. An early increase in root nodule fresh weight and AR activity was also observed in response to this treatment and was followed similarly by early decline.The addition of high levels of soil-applied nitrogen increased leaf NR activity and delayed late season decline in NR activity for both control and early reproductive plants. Nitrate supply was therefore implicated as limiting to leaf NR activity during the decline associated with flowering and early seed development. A limited additional increase in leaf NR activity was observed in response to light enhancement plus soil-applied nitrogen. As no significant increase in leaf NR activity was observed in response to light enhancement alone, leaf nitrate supply was further implicated as more limiting to leaf NR activity than was photosynthate supply during flowering and early seed development.

Leaf Nitrate Reductase, d-Ribulose-1,5-bisphosphate Carboxylase, and Root Nodule Development of Genetic Male-Sterile and Fertile Soybean Isolines.

The objectives of this study were to determine the effect of pod and seed development on leaf chlorophyll concentration, and on activities of leaf ribulose bisphosphate carboxylase, leaf nitrate reductase, and root nodule acetylene reduction in field-grown soybean (Glycine max [L.] Merr.). Two genetic male-sterile lines and their fertile counterparts (Williams and Clark 63) were compared in both 1978 and 1979. Two additional lines (Wells x Beeson and Wells x Corsoy) were compared in 1979.The expression of male-sterile character was nearly complete as very little outcrossing due to insect pollinators was observed. Male-sterile plants showed a delayed late season decline in leaf chlorophyll content and ribulose bisphosphate carboxylase activity when compared with fertile plants. A slight delay in the loss of in vivo leaf nitrate reductase activity was also observed for male-sterile plants. Root nodule fresh weight and acetylene reduction activity declined slightly more rapidly for fertile lines than for male-sterile lines in both years with differences significant on the last two to three sampling dates as leaf loss occurred in the control plants.Seed development was found to increase slightly, the rate of decline of metabolic activity in fertile lines compared with that of male-sterile lines. However, pod development was not an a priori requirement for leaf and root nodule senescence. Male-sterile plants also lost photosynthetic and nitrogen metabolic competence, but at a slower rate. These results support the concept that pod and seed development does not signal monocarpic senescence per se but rather affects the rate at which senescence occurs after flowering.

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties.

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr(1)-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c(1)NR, and the form eluted with NADH was designated c(2)NR. Nitrate-grown nr(1) mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c(1)NR and c(2)NR had similar pH optima of 6.5, sedimentation behavior (s(20,w) of 5.5-6.0), and electrophoretic mobility. However, c(1)NR was more active with NADPH than with NADH, while c(2)NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c(1)NR) and 0.19 millimolar (c(2)NR). The iNR from the mutant had a pH optimum of 7.5, s(20,w) of 7.6, and was less mobile on polyacrylamide gels than c(1)NR and c(2)NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

Uptake and reduction of [N]nitrate by intact soybean plants in the dark.

Experiments were conducted to determine if nitrate ((15)N-labeled) was taken up and assimilated by intact soybean (Glycine max [L.] Merr. cv Williams) plants during extended periods of dark. Nitrate was taken up by soybean roots throughout a 12-hour dark period. The (15)N-labeled nitrogen was also translocated to the plant shoots, but at a slower rate than the rate of accumulation in the roots. Much of the nitrogen ((15)N-labeled) was present in a nonreduced form, although considerable soluble-reduced nitrogen ((15)N-labeled) accumulated throughout the dark period. The (15)N-labeled, soluble-reduced nitrogen fraction accounted for nearly 30% of the total (15)N found in plant roots and more than 63% of the total (15)N found in plant tops after 12 hours of dark. This provided evidence that intact soybean plants take up and metabolize significant quantities of nitrate to reduced N forms in the dark.In addition to nitrate influx during the dark, it was shown that there was a concomitant loss of (15)N-labeled nitrogen compounds from previously (15)N-labeled plants to a natural abundance (15)N nutrient solution. Thus, evidence was obtained which indicated that light was not directly essential for flux and reduction of nitrate by intact soybean plants.

Physiology of the SOS response: kinetics of lexA and recA transcriptional activity following induction.

The products of the lexA and recA genes play central roles in the regulation of the Escherichia coli SOS response. We have measured the rate of mRNA synthesis from each gene at intervals following various inducing treatments in order to obtain a more precise timing of the induction process. Further, we provide quantitative evidence for kinetics of decay from fully induced levels of mRNA synthesis to basal levels as the cells shut down the SOS response which are in agreement with previously published data on the expression of specific SOS functions. The induction kinetics of lexA and recA gene expression are parallel except for nalidixic acid (NAL) treatment, with the actual levels of lexA mRNA synthesis being about 10-fold lower than that of recA. Reestablishment of repression from RecA commenced over 30 min earlier than from lexA. These results are fully consistent with the model that the functions result from the increased gene expression.