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.

Analysis of mRNA synthesis following induction of the Escherichia coli SOS system.

Escherichia coli responds to impairment of DNA synthesis by inducing a system of DNA repair known as the SOS response. Specific genes are derepressed through proteolytic cleavage of their repressor, the lexA gene product. Cleavage in vivo requires functional RecA protein in a role not yet understood. We used mRNA hybridization techniques to follow the rapid changes that occur with induction in cells with mutations in the recA operator or in the repressor cleavage site. These mutations allowed us to uncouple the induction of RecA protein synthesis from its role in inducing the other SOS functions. Following induction with ultraviolet light, we observed increased rates of mRNA synthesis from five SOS genes within five minutes, maximum expression ten to 20 minutes later and then a later decline to near the initial rates. The presence of a recA operator mutation did not significantly influence these kinetics, whereas induction was fully blocked by an additional mutation in the repressor cleavage site. These experiments are consistent with activation of RecA protein preceding repressor cleavage and derepression of SOS genes. The results also suggest that the timing and extent of induction of individual SOS genes may be different.

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.

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.

Canopy and Seasonal Profiles of Nitrate Reductase in Soybeans (Glycine max L. Merr.).

Nitrate reductase activity of soybeans (Glycine max L. Merr.) was evaluated in soil plots and outdoor hydroponic gravel culture systems throughout the growing season. Nitrate reductase profiles within the plant canopy were also established. Mean activity per gram fresh weight per hour of the entire plant canopy was highest in the seedling stage while total activity (activity per gram fresh weight per hour times the total leaf weight) reached a maximum when plants were in the full bloom to midpod fill stage. Nitrate reductase activity per gram fresh weight per hour was highest in the uppermost leaf just prior to full expansion and declined with leaf position lower in the canopy. Total nitrate reductase activity per leaf was also highest in the upper-most fully expanded leaf during early growth stages. Maximum total activity shifted to leaf positions lower in the plant canopy with later growth stages.Nitrate reductase activity of soybeans grown in hydroponic systems was significantly higher than activity of adjacent soil grown plants at later growth stages, which suggested that under normal field conditions the potential for nitrate utilization may not be realized. Nitrate reductase activity per gram fresh weight per hour and nitrate content were positively correlated over the growing season with plants grown in either soil or solution culture. Computations based upon the nitrate reductase assay of plants grown in hydroponics indicated that from 1.7 to 1.8 grams N could have been supplied to the plant via the nitrate reductase process. The harvested seed contained 1.1 to 1.2 grams N per plant. Thus, based on previous estimates of approximately 32% of the final N distribution being in the vegetative plant parts, the estimated input of reduced nitrogen via the enzyme assay was in agreement with the actual N accumulation.The amount of calculated N(2)-fixation by nodules per season with plants grown in hydroponics was less than 2% of the computed nitrate reduced via leaf nitrate reductase. Thus, the level of nitrate in the nutrient solution appeared to be quite inhibitory to N(2)-fixation.

Evolution of Nitrogen Oxide(s) during In Vivo Nitrate Reductase Assay of Soybean Leaves.

Studies were conducted to quantitate the evolution of nitrogen oxides (NO((x))) from soybean [Glycine max (L.) Merr.] leaves during in vivo nitrate reductase (NR) assays with aerobic and anaerobic gas purging. Anaerobic gas purging (N(2) and argon) consistently resulted in greater NO((x)) evolution than did aerobic gas purging (air and O(2)). The evolution of NO((x)) was dependent on gas flow rate and on NO(2) (-) formation in the assay medium; although a threshold level of NO(2) (-) appeared to exist beyond which the rate of NO((x)) evolution did not increase further.The loss of NO((x)) from in vivo NR assays under gas purging explains partially, but not stoichiometrically, the decrease in NO(2) (-) accumulation in in vivo NR assay medium with young soybean leaves. The lack of stoichiometry between NO((x)) evolution and apparent NO(2) (-) loss suggests that other mechanisms are also involved in loss of NO(2) (-) or inhibition of formation of NO(2) (-) during anaerobic and aerobic incubation conditions imposed on the in vivo NR assay of soybean. The mechanism of NO((x)) evolution under the assay conditions imposed and the relevance of this phenomenon to intact plants remains unclear.

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.

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.

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.

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.

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.

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.

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.

Identification of the lexA gene product of Escherichia coli K-12.

The Escherichia coli lexA gene encodes a product important in induction of the recA gene and the expression of various cellular functions, including mutagenesis and prophage induction. As a start in a biochemical analysis of the lexA function, a family of lambda transducing phages carrying lexA+, lexA3, lexA3 spr-54, and lexA3 spr-55 alleles of the lexA gene was isolated and characterized. Polypeptides synthesized by these phages were examined. lambdalexA+ made a distinctive protein 24 kilodaltons (kd) in size. Lambda lexA3, which encodes an active mutant form of the protein dominant to wild-type function, made a slightly larger protein 25 kd in size. The latter protein was shown to be the mutant lexA3 gene product by the fact that lambda lexA3 spr-55, which carries an amber mutation in lexA3, made the 25-kd protein in hosts with an amber suppressor but not in a suppressor-free host. In hosts carrying a multicopy lexA3 plasmid, neither the 25-kd nor the 24-kd protein was made. This result suggests that lexA is autoregulated and that expression of the 24-kd protein made by lambda lexA+ is subject to the same controls. This and other evidence argues that the 24-kd protein is the product of the wild-type lexA+ gene.

Nitrogen metabolism of soybeans: I. Effect of tungstate on nitrate utilization, nodulation, and growth.

The effects of N source (6 mm nitrogen as NO(3) (-) or urea) and tungstate (0, 100, 200, 300, and 400 mum Na(2) WO(4)) on nitrate metabolism, nodulation, and growth of soybean (Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO(3) (-) content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO(3) (-)-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO(3) (-)-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 mum tungstate levels, acetylene reduction activity of nodulated roots of NO(3) (-)-grown plants exceeded the activity of comparable urea-grown plants.Addition of tungstate to the nutrient solution decreased plant growth, regardless of the N source, although the effect was more pronounced with NO(3) (-) nutrition. The increased nodulation and decreased nitrate reductase activity noted with plants grown in the presence of tungstate and a high (6 mm) external supply of NO(3) (-) suggests that NO(3) (-) does not directly inhibit nodulation but rather affects nodulation indirectly through subsequent metabolism of NO(3) (-).

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.

Nitrogen assimilation and protein synthesis in wheat seedlings as affected by mineral nutrition. I. Macronutrients.

Deficiencies of each macronutrient (N, P, K, Ca. Mg, S, and Fe) decreased the specific activity of nitrate reductase from Triticum aestivum L. seedlings. Nitrate content was decreased by N, P, K, Ca, and Mg deficiencies and unaffected by S and Fe deficiencies. Glutamic acid dehydrogenase activity was decreased by N, P, and S deficiencies, unchanged by K deficiency, and increased by Ca, Mg, and Fe deficiencies. Glutamine synthetase activity closely paralleled nitrate reductase activity and was decreased by deficiencies of N, P, K, Ca, Mg, and S. Glutamic-oxaloacetic transaminase was not sensitive to macronutrient deficiencies. High (14)C-leucine incorporation into tissue sections of N-, P-, K-, Ca-, and S-deficient seedlings did not appear indicative of protein synthesis rates in intact seedlings. Nutritional deficiencies apparently depleted endogenous amino acid pools and caused less inhibition of exogenous (14)C-leucine incorporation into protein.

Nitrogen Assimilation and Protein Synthesis in Wheat Seedlings as Affected by Mineral Nutrition. II. Micronutrients.

Activity of nitrate reductase from Triticum aestivum L. seedlings was decreased by deficiencies of molybdenum, zinc, and chlorine. Nitrate accumulated in molybdenum-deficient seedlings, declined in zinc-deficient seedlings, and was unaffected by the other micronutrient treatments. Glutamic acid dehydrogenase activity was decreased by deficiency of molybdenum, the only nutrient that affected the enzyme. Glutamine synthetase activity was decreased only by copper deficiency, and glutamic-oxaloacetic transaminase was not affected by any micronutrient deficiencies. Incorporation of (14)C-leucine into protein by wheat seedlings was increased by molybdenum deficiency, apparently because of decreased inhibition from endogenous amino acids, and was decreased by copper deficiency. Protein content was not affected significantly by the micronutrient treatments.

Evidence for a role of calcium in nitrate assimilation in wheat seedlings.

Severely Ca-deficient Triticum aestivum L. seedlings accumulated high levels of nitrite and moderate levels of nitrate and organic nitrogen, but contained unaltered levels of hydroxylamine. Nitrite accumulation was not related to molybdenum deficiency, or altered cellular pH. Nitrate reductase was decreased by Ca deficiency, apparently by repression of enzyme synthesis from accumulated nitrite and not by inhibition of enzyme activity. Nitrite reductase and NADP diaphorase activities were not affected by Ca deficiency, and Ca did not restore activity to nitrite reductase inactivated by cyanide. The results indicated that the role of Ca is in intracellular transport of nitrite and not in induction or activity of enzymes.