Induction Of Nitrate Reductase Activity Research Articles

Nitric oxide is involved in nano-titanium dioxide-induced activation of antioxidant defense system and accumulation of osmolytes under water-deficit stress in Vicia faba L.

Nano-titanium dioxide (nTiO2) has been reported to improve tolerance of plants against different environmental stresses by modulating various physiological and biochemical processes. Nitric oxide (NO) has been shown to act as an important stress signaling molecule during plant responses to abiotic stresses. The present work was planned to investigate the involvement of endogenous NO in nTiO2-induced activation of defense system of fava bean (Vicia faba L.) plants under water-deficit stress (WDS) conditions. Water-suffered plants showed increased concentration of hydrogen peroxide (H2O2) and superoxide (O2-) content coupled with increased electrolyte leakage and lipid peroxidation which adversely affected nitrate reductase (NR) activity, chlorophyll content and growth of the plants. However, application of 15mgL-1 nTiO2 to stressed plants significantly induced NR activity and synthesis of NO which elevated enzymatic and non-enzymatic defense system of the stressed plants and suppressed the generation of H2O2 and O2- content, leakage of electrolytes, and lipid peroxidation. Application of nTiO2, in association with NO, also enhanced the accumulation of osmolytes (proline and glycine betaine) that assisted the stressed plants in osmotic adjustment as witnessed by improved hydration level of the plants. Involvement of NO in nTiO2-induced activation of defense system was confirmed with NO scavenger cPTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] which caused recurrence of WDS.

Ectopic Expression of PII Induces Stomatal Closure in Lotus japonicus.

The PII protein in plants has been associated to many different tissue specialized roles concerning the Nitrogen assimilation pathways. We report here the further characterization of L. japonicus transgenic lines overexpressing the PII protein encoded by the LjGLB1 gene that is strongly expressed in the guard cells of Lotus plants. Consistently with a putative role played by PII in that specific cellular context we have observed an alteration of the patterns of stomatal movement in the overexpressing plants. An increased stomatal closure is measured in epidermal peels from detached leaves of normally watered overexpressing plants when compared to wild type plants and this effect was by-passed by Abscisic Acid application. The biochemical characterization of the transgenic lines indicates an increased rate of the Nitric Oxide biosynthetic route, associated to an induced Nitrate Reductase activity. The phenotypic characterization is completed by measures of the photosynthetic potential in plants grown under greenhouse conditions, which reveal a higher stress index of the PII overexpressing plants.

Rapid and slow modulation of nitrate reductase activity in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA110 impaired in nitrate reductase

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.

REGULATION OF NITRATE REDUCTASE ACTIVITY IN RICE (ORYZA SATIVA L.) BY GROWTH REGULATORS

The effect of three growth regulators, namely kinetin, 6 benzyl adenine, 2 chloro ethyl trimethyl ammonium chloride at three concentrations (10-6 M, 5 x 10-5 M 10-4 M) was studied on the catalytic activity of nitrate reductase in green and etiolated seedlings. A concentration of 5 x 10-5 M was optimal for all the growth regulators treatments. All the growth regulators stimulated nitrate reductase activity effectively at 5 x 10-5M concentration in both etiolated and green seedlings and had an additive effect when supplemented by NO-3 up to 140% to 160%. The 99.2% and 93.4% inhibition of nitrate reductase activity resulted in development of etiolated and green seedlings, respectively when treated with eukaryotic 80S ribosome protein synthesis inhibitor cycloheximide. Prokaryotic 70S inhibitor chloromphenicol did not have any effect on measured parameters. Actinomycin D, a RNA synthesis inhibitor also inhibited the enzyme activity as 80s inhibitors (Green 80%, etiolated 98%). One may suggest from this that both DNA and protein synthesis are involved in the induction of nitrate reductase activity. The differential effect of aminoacids was observed on enzyme activity in combination with growth regulators.

Isolation and characterization of a chlorate-resistant mutant (Clo- R) of the symbiotic cyanobacterium Nostoc ANTH: heterocyst formation and N(2)-fixation in the presence of nitrate, and evidence for separate nitrate and nitrite transport systems.

Nostoc ANTH is a filamentous, heterocystous cyanobacterium capable of N(2)-fixation in the absence of combined nitrogen. A chlorate-resistant mutant (Clo- R) of Nostoc ANTH was isolated that differentiates heterocysts and fixes N(2) in the presence of nitrate, but not in the presence of nitrite or ammonium. The mutant lacks nitrate uptake and thereby also lacks induction of nitrate reductase activity by nitrate. However, this mutant is able to transport and assimilate nitrite, indicating that there is a transport system for nitrite that is distinct from that for the nitrate. The lack of inhibitory effect of nitrate on N(2)-fixation was owing to lack of nitrate uptake and not to lack of enzymes for its assimilation (nitrate reductase and glutamine synthetase) or the lack of an ammonium transport system for retention of ammonia. The mutant has potential for use as a biofertilizer supplementing chemical nitrate fertilizer in rice fields, without N(2)-fixation being adversely affected.

Nitrate reductase activity in some subarctic species and UV influence in the foliage of Betula pendula Roth. seedlings

Nitrate reductase (NR) activity was studied in the foliage of five subarctic species: mature trees of European white birch (Betula pubescens Erch. S.S.), Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies L. Karst), Ericaceous shrub bilberry (Vaccinium myrtillus L.), naturally growing in a forest, and seed-grown silver birch (Betula pendula Roth.) seedlings in an ultraviolet (UV) exclusion field experiment at the Pallas-Ounastunturi National Park in Finnish Lapland (68 degrees N). Mean NR activity ranged from 0 in bilberry to 1477 (S.D. = 277.7) and 1910 (S.D. = 785.4) nmol g(-1) DW h(-1) in mature trees of European white birch and silver birch seedlings, respectively. Significant differences due to UV exclosure treatments were determined for the NR activity of silver birch seedlings (F = 3.62, P= 0.025*) after three growing seasons (191 days) of UV exclusion. The ambient and control silver birch seedlings had or tended to have higher NR activity than those grown under UV exclusion. No relationship was found between the foliage NR activity and total nitrogen content, which ranged from 0.61 to 1.35% per seedling. The present study suggests large differences in NR activity between the species and the induction of NR activity in silver birch seedlings due to ambient UV radiation.

Effect of phytohormones and tungsten on light-induced and nitrate-induced nitrate reductase activity of mustard cotyledons

Mustard (Brassica juncea Coss cv. T-59 ‘Varuna’) seedlings pretreated with gibberellic acid (GA) and kinetin (KiN) were grown in light. In vivo nitrate reductase (NR) activity was estimated and effect of tungsten on light-induced and NO3su−-induced NR activity was investigated. Different concentrations of GA did not show any effect on induction of light-induced NR; addition of nitrate promoted in vivo NR activity but no concentration effect of GA was evident. Light-induced NR was promoted by KiN and like in GA treatment, addition of nitrate increased NR activity. Addition of Na-tungstate inhibited NO3− induced NR while light-induced NR was not much affected in both GA and KiN treated seedlings. The two forms of NRs were further characterized by studying the decay kinetics using Na-tungstate. In light-induced NR, tungstate did not affect NR activity up to 11 h, while at later periods, a slight decay was observed. On the other hand, NO3−-induced NR activity increased up to 4 h and subsequently a rapid fall was observed. It was therefore apparent that light-induced NR had a very low turnover rate as compared to NO3−-induced NR. These results further support the earlier conclusion that in mustard seedlings two distinct types of NR enzyme exist and that nitrate requirement for NR induction is not absolute.

Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba

Nitrogen assimilation was studied in the deciduous, perennial climber Clematis vitalba. When solely supplied with NO3– in a hydroponic system, growth and N‐assimilation characteristics were similar to those reported for a range of other species. When solely supplied with NH4+, however, nitrate reductase (NR) activity dramatically increased in shoot tissue, and particularly leaf tissue, to up to three times the maximum level achieved in NO3– supplied plants. NO3– was not detected in plant material that had been solely supplied with NH4+, there was no NO3– contamination of the hydroponic system, and the NH4+‐induced activity did not occur in tobacco or barley grown under similar conditions. Western Blot analysis revealed that the induction of NR activity, either by NO3– or NH4+, was matched by NR and nitrite reductase protein synthesis, but this was not the case for the ammonium assimilation enzyme glutamine synthetase. Exposure of leaf disks to N revealed that NO3– assimilation was induced in leaves directly by NO3– and NH4+ but not glutamine. Our results suggest that the NH4+‐induced potential for NO3– assimilation occurs when externally sourced NH4+ is assimilated in the absence of any NO3– assimilation. These data show that the potential for nitrate assimilation in C. vitalba is induced by a nitrogenous compound in the absence of its substrate and suggest that NO3– assimilation in C. vitalba may have a significant role beyond the supply of reduced N for growth.

Lithium inhibits phytochrome-stimulated nitrate reductase activity and transcript level in etiolated maize leaves

Our earlier work indicated an involvement of signals generated via polyphosphoinositide turnover in phytochrome (Pfr) mediated light stimulation of nitrate reductase (NR) activity. We now show that lithium, an inhibitor in the cycling of inositol phosphates, inhibits red-light stimulated nitrate induced NR activity. The effect of lithium was more pronounced if it was given immediately after red light irradiation than if supplied1 h after. The inhibition of NR activity by lithium was partially reversed by myo-inositol and totally reversed by calcium. Red light stimulated NR transcript level was also inhibited by lithium, however, calcium could only partially reverse this effect. In the present system we show that lithium decreased the level of phosphoinositide-bis-phosphate as well as other phosphoinositides in red light irradiated tissues. These results further suggests an involvement of calcium and signals produced via turnover of polyphosphoinositides in signal transduction in Pfr regulation of NR transcript level and NR activity.

Soluble and membrane-bound nitrate reductase from Bradyrhizobium japonicum bacteroids

Nitrate reductase (NR) activity was detected in the cytosol and membranes from bacteroids of Bradyrhizobium japonicum strain L-236 isolated from nodules of soybean ( Glycine max, Merr., cv. Williams) plants that were only N 2-dependent. The molecular mass ( M) of the NR from each source was estimated by gel filtration to be about 76 kDa. Treatment of plants with 20 mM KNO 3 induced NR activity in the bacteroid cytosol, but did not affect rates of activity in the membranes. Furthermore, the M of the cytosolic enzyme was 160 kDa, while that of the NR from the membranes was 76 kDa. When bacteroids that had been isolated from nodules of plants not receiving nitrate were incubated microaerobically (2 % O 2 v/v) in both the absence and presence of nitrate, the M of the membrane-bound NR enzyme was calculated to be 230 kDa; in bacteroids incubated in the absence of nitrate, the M of the soluble enzyme was 76 kDa, whereas that of bacteroids incubated with nitrate was about 160 kDa. Incubation of bacteroids under aerobic conditions with nitrate induced NR activity in the soluble fraction, and the M of the enzyme was 160 kDa; in the absence of nitrate the M of the soluble NR was 76 kDa. No NR activity was found in membranes from aerobically incubated bacteroids.

NO2‐induced nitrate reductase activity in needles of Norway spruce (Picea abies) under laboratory and field conditions

The induction of activity of the enzyme nitrate reductase (NR, EC 1.6.6.1, 1.6.6.2) in needles of Norway spruce (Picea abies[L.] Karst.) by nitrogen dioxide (NO2) was studied under laboratory and field conditions. In fumigation chambers an increase in nitrate reductase activity (NRA) was detected 4 h after the start of the NO2 treatment. During the first 2 days with 100 µg NO2 m−3, NRA reached a constant level and did not change during the following 4 days. At the same level of NO2, NRA was lower in needles from trees grown on NPK‐fertilized soil than on non‐fertilized soil. After the transfer of spruce trees from fertilized soil to NPK‐rich nutrient solution, NRA was transiently increased. This effect was assigned to root injuries causing nitrate transport to the shoot and subsequent induction of NRA. Neither trees on fertilized soil nor trees transferred to NPK‐poor nutrient solution had increased NRA unless NO2 was provided. The NO2 gradient in the vicinity of a highway was used to test the long‐term effect of elevated levels of NO2 on needle NRA of potted and field‐grown spruce trees. Compared with less polluted sites, permanently increased NRAs were detected when NO2 concentrations were above 20 µg m−3. Controls of field measurements some 10 years after the introduction of catalytic converters in cars showed no significant change neither in NO2 levels nor in the decreasing NRA of spruce needles with the distance from the highway.

Stable Transformation of Chlorella: Rescue of Nitrate Reductase-Deficient Mutants with the Nitrate Reductase Gene

Unicellular green algae, like Chlorella, offer a potentially useful system for the expression of heterologous proteins. However, the development of Chlorella as a bioreactor has been delayed owing to the lack of a stable transformation technique. Here we report on the use of micro-projectile bombardment to introduce the nitrate reductase (NR) gene from Chlorella vulgaris into NR-deficient Chlorella sorokiniana mutants, resulting in stable transformants. The stable transformants were able to grow on nitrate medium after repeated passages between selective and nonselective medium and exhibited inducible nitrate reductase activity comparable to that of wild-type cells. Southern analysis suggests homologous recombination occurs with insertion of the wild type gene into the mutated gene and that the genes of the two Chlorellaspecies used are very similar. Specific RNase protection assays, selecting for a poorly conserved region of the gene, identified the presence of the C. vulgaris NR transcript only in the transformed C. sorkiniana mutant and not in the mutant.

Regulation of the expression of ferredoxin-glutamate synthase in barley.

We have investigated the regulation of ferredoxin-glutamate synthase (Fd-GOGAT) in leaves of barley (Hordeum vulgare L. cv. Maris Mink) at the mRNA, protein and enzyme activity levels. Studies of the changes in Fd-GOGAT during plant development showed that the activity in shoots increases rapidly after germination to reach a maximum (on a fresh-weight basis) at day 10 and then declines markedly to less than 50% of the maximal activity by day 30, this decline being correlated with an equivalent loss of Fd-GOGAT protein. Growing the plants in darkness reduced the maximum activity attained in the shoots, but did not affect the overall pattern of the changes or their timing. The activity of Fd-GOGAT increased two- to three-fold within 48 h when etiolated leaves were exposed to light, and Northern blots indicated that the induction occurred at the mRNA level. However, whilst a carbon source could at least partially substitute for light in the induction of nitrate reductase activity, no induction of Fd-GOGAT activity was seen when etiolated leaves were treated with either sucrose or glucose. Interestingly, the levels of Fd-GOGAT mRNA and activity remained high up to a period of 16 h or 72 h darkness, respectively. Compared with plants grown in N-free medium, light-grown plants supplied with nitrate had almost two-fold higher Fd-GOGAT activities and increased Fd-GOGAT mRNA levels, but nitrate had no effect on the abundance of the enzyme or its mRNA in etiolated plants, indicating that light is required for nitrate induction of barley Fd-GOGAT.

Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH4+ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8‐day‐old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO3+ in the presence of 0, 1 or 10 mM NH4+, Exogenous NH4+ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO3+, In contrast, the induction of NiRA was inhibited by NH4+ at all NO3+ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH4+ Pre‐treatment with NH4+ had no effect on the subsequent induction of NRA in the absence of additional NH4+ whereas the induction of NiRA in NH4+‐pretreated roots was inhibited in the absence of NH4+ At 10 mM NO3+ L‐methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH4+ was present. In contrast, the induction of NiRA was inhibited by L‐methionine sulfoximine irrespective of NH4+ supply. During the postinduction phase, exogenous NH4+ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH3+ whereas, NiRA was unaffected by NH4+ at either substrate concentration. The results indicate that exogenous NH4+ regulates the induction of NRA in roots by limiting the availability of NO3+. Conversely, it has a direct effect, independent of the availability of NO3+, on the induction of NiRA. The lack of an NH4+ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.

Nitrate Reductase Activity is Endogenously Induced by Zeatin Riboside in Habituated Suspension Cultured Chenopodium rubrum Cells

Summary The natural cytokinins IP, IPA, Z, ZR, DHZ and DHZR of suspension cultured photoautotrophic and phytohormone-independent cells of Chenopodium rubrum were examined for their ability to induce nitrate reductase (EC 1.6.6.1) activity in resting cells of the batch culture. Each of the applied cytokinins was readily taken up from the medium and nitrate reductase activity was rapidly enhanced by all of them, ZR being a little more effective than the others. Nitrate reductase activity was also enhanced by adding ammonium to the cell culture in the G o phase. Subsequently, the endogenous level of ZR increased transitorily up to 2.5-fold, peaking about 4 h after addition of ammonium. The levels of the other endogenous cytokinins remained more or les unchanged. Supply of cytokinins to the stationary cell culture did not result in resumption of cell division. Induction of nitrate reductase activity by both ZR and ammonium could be inhibited with cycloheximide, indicating that protein de-novo -synthesis was involved in the enhancement of the enzyme activity. The time-span between the rise of the cytokinin level and the increase of nitrate reductase activity was about 2 h, irrespective of whether induction was triggered with ZR or ammonium nitrate. From these results, ZR was considered to be the endogenous cytokinin species that is involved in the induction of nitrate reductase in Chenopodium rubrum .

A hypothesis of plant susceptibility to atmospheric pollution based on intrinsic nitrogen metabolism: Why acidity really is the problem

Several hypotheses have been put forward to explain the causes of damage to plants by atmospheric pollution. None however, fully explain why some species are more prone to damage than others. In the literature, certain higher plants are more commonly reported as suffering damage from atmospheric pollution. These include climax species such as conifers, some broadleaf species such as oak and beech, and ericaceous species. Pioneer species such as highly nitrophillous woody species and ruderal herbaceous species are seldom reported in connection with damage from atmospheric pollutants. A screening of 18 species (woody and herbaceous) for 5 physiological and metabolic parameters and subsequent regression analysis of the data, showed that plants could be grouped according to their tolerance to acidity. There was a marked positive correlation between leaf nitrate reductase (NR) activity and the buffering capacity index (BCI) of the leaf Foliar mistings and soil applications of 3 mol m -3 KNO 3 to 6 assorted plant species also revealed that pioneer species, can induce NR activity when NO 3 - is applied directly to the foliage or soil. However, climax species have a much lower ability to increase NR activity in response to foliar and soil NO 3 - inputs. Leaf NO 3 + assimilation carries with it the direct generation of OH - , involves greater uptake of base cations and confers greater physiological buffering capability through the close interaction of carbon and nitrogen acquisition in the leaf. The hypothesis states that leaf nitrate assimilators are physiologically more competent at buffering acidic inputs whether directly as H + , or indirectly through uptake of acidic pollutants.

Nitrate reduction in cotyledons of Cicer arietinum L.: regulatory role of cytokinins

Application of different concentrations of kinetin to chick-pea seeds inhibits the nitrate reductase ativity detected in cotyledons when the seeds are sown without a hormone. However, kinetin concentrations between 10 −6 and 10 −5 M, applied to excised cotyledons are able to induce nitrate reductase activity, which also becomes manifest in an active metabolism of nitrate and nitrite in the cotyledons. The development of nitrate reductase activity in excised cotyledons is due to synthesis of the enzyme, except when induction is carried out by applying low concentrations of cytokinins. In this case, apart from synthesis, an inactive nitrate reductase becomes activated.

Shoot nitrate reductase activities of field‐layer species in different forest types

Deposition of protons, nitrate and sulphate have caused a decrease in soil pH and base saturation in southern Scandinavia. Floral changes have been suggested to be due to this deposition. This study was made to test if differences in nitrate assimilation capability between plants could be one of the reasons behind reported changes in the flora. Current and maximally induced nitrate reductase activity (C‐NRA and MI‐NRA, respectively) were measured in four beech forest field‐layer species, which have been reported to behave differently with respect to acid rain. The study was conducted at four sites of different soil quality (pH). There were considerable differences between the species as regards both C‐NRA and MI‐NRA. For two of the studied species, Galium odoratum and Stellaria nemorum, the gap between C‐ and MI‐NRA was relatively small but for Mercurialis perennis and Oxalis acetosella, this gap was larger. A small difference between C‐ and MI‐NRA should indicate that a larger portion of the available resources for nitrate reduction is already used. The concentrations of soluble carbohydrates were correlated with the MI‐NRA after leafing of beech, while before leafing no such effect could be detected. It can be concluded that the light environment has a considerable effect on the nitrate nutrition of field‐layer plants. Apparently, some species receive as much nitrate as they can reduce. Fertiliser experiments are needed to test if higher addition rates are harmful to them.

Studies on the root control of non-nodulation and plant growth of non-nodulating mutants and a supernodulating mutant of soybean ( Glycine max (L.) Merr.)

Ethyl methanesulphonate mutagensis of soybean cv. Bragg led to the isolation of three non-nodulating mutants: nod49, nod772 and nod139, as well as the supernodulation mutant nts382. Mutants nod49 and nod772 are allelic to naturally-occuring mutation rj 1 while nod139 is non-allelic and represents a newly found gene conditioning non-nodulation in soybean. Using the standard reciprocal wedge grafting technique it was shown earlier that non-nodulation in mutant nod49 is root controlled and supernodulation in mutant nts382 is shoot controlled. Non-nodulation in mutants nod772 and nod139 (the newly found non-nodulation gene in soybean) is also root controlled. In addition, scions of nts382 failed to alter the nodulation phenotype when grafted onto stocks of the non-nodulating lines. Lateral roots developing from the scion of nts382 and Bragg exhibited supernodulation and the wild-type pattern of nodulation, respectively, indicating that the lesion(s) conditioning non-nodulation only affect the root in a localized manner. Physiological studies conducted on the mutants indicate that they are not altered in the assimilation of nitrogen and carbon when grown on nitrate, and a study of their nitrogen contents indicate that the anomaly in the non-nodulation mutants is clearly specific for the nodulation process. This was confirmed by assaying nitrate reductase (NR) under a range of conditions; it was observed that the non-nodulation mutants expressed inducible NR and constitutive NR activity in a manner similar to the wild type.