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Comparison of nodule induction in legume and actinorhizal symbioses: the induction of actinorhizal nodules does not involve ENOD40.

Two types of root nodule symbioses are known for higher plants, legume and actinorhizal symbioses. In legume symbioses, bacterial signal factors induce the expression of ENOD40 genes. We isolated an ENOD40 promoter from an actinorhizal plant, Casuarina glauca, and compared its expression pattern in a legume (Lotus japonicus) and an actinorhizal plant (Allocasuarina verticillata) with that of an ENOD40 promoter from the legume soybean (GmENOD40-2). In the actinorhizal Allocasuarina sp., CgENOD40-GUS and GmENOD40-2-GUS showed similar expression patterns in both vegetative and symbiotic development, and neither promoter was active during nodule induction. The nonsymbiotic expression pattern of CgENOD40-GUS in the legume genus Lotus resembled the nonsymbiotic expression patterns of legume ENOD40 genes; however, in contrast to GmENOD40-2-GUS, CgENOD40-GUS was not active during nodule induction. The fact that only legume, not actinorhizal, ENOD40 genes are induced during legume nodule induction can be linked to the phloem unloading mechanisms established in the zones of nodule induction in the roots of both types of host plants.

Base Sequence↗

Genetic regulation of nitrogen fixation in Rhizobium meliloti.

The soil bacterium Rhizobium meliloti fixes dinitrogen when associated with root nodules formed on its plant host, Medicago sativa (alfalfa). The expression of most of the known genes required for nitrogen fixation (nif and fix genes), including the structural genes for nitrogenase, is induced in response to a decrease in oxygen concentration. Induction of nif and fix gene expression by low oxygen is physiologically relevant because a low-oxygen environment is maintained in root nodules to prevent inactivation of the highly oxygen-sensitive nitrogenase enzyme. The genes responsible for sensing and transducing the low oxygen signal, fixL and fixJ, encode proteins (FixL and FixJ, respectively) that are homologous to a large family of bacterial proteins involved in signal transduction, the two component regulatory system proteins. The two components consist of a sensor protein, to which FixL is homologous, and a response regulator protein, to which FixJ is homologous. The sensor protein respond to an activating signal by autophosphorylating and then transferring the phosphate to its cognate response regulator protein. The phosphorylated response regulator, which is often a transcriptional activator, is then able to activate its target. A cascade model of nif and fix gene regulation in R. meliloti has been proposed, whereby FixL acts as an oxygen sensor as the initial event in the cascade and transmits this information to FixJ. FixJ, which possesses a putative helix-turn-helix DNA-binding motif, then activates transcription of the nifA and fixK genes. The nifA and fixK gene products, are transcriptional activators of at least 14 other nif and fix genes.

Bacterial Proteins↗

Silencing the flavonoid pathway in Medicago truncatula inhibits root nodule formation and prevents auxin transport regulation by rhizobia.

Legumes form symbioses with rhizobia, which initiate the development of a new plant organ, the nodule. Flavonoids have long been hypothesized to regulate nodule development through their action as auxin transport inhibitors, but genetic proof has been missing. To test this hypothesis, we used RNA interference to silence chalcone synthase (CHS), the enzyme that catalyzes the first committed step of the flavonoid pathway, in Medicago truncatula. Agrobacterium rhizogenes transformation was used to create hairy roots that showed strongly reduced CHS transcript levels and reduced levels of flavonoids in silenced roots. Flavonoid-deficient roots were unable to initiate nodules, even though normal root hair curling was observed. Nodule formation and flavonoid accumulation could be rescued by supplementation of plants with the precursor flavonoids naringenin and liquiritigenin. The flavonoid-deficient roots showed increased auxin transport compared with control roots. Inoculation with rhizobia reduced auxin transport in control roots after 24 h, similar to the action of the auxin transport inhibitor N-(1-naphthyl)phthalamic acid (NPA). Rhizobia were unable to reduce auxin transport in flavonoid-deficient roots, even though NPA inhibited auxin transport. Our results present genetic evidence that root flavonoids are necessary for nodule initiation in M. truncatula and suggest that they act as auxin transport regulators.

Acyltransferases↗

Carbohydrate supply and n(2) fixation in soybean : the effect of varied daylength and stem girdling.

When arrival of shoot supplied carbohydrate to the nodulated root system of soybean was interrupted by stem girdling, stem chilling, or leaf removal, nodule carbohydrate pools were utilized, and a marked decline in the rates of CO(2) and H(2) evolution was observed within approximately 30 minutes of treatment. Nodule excision studies demonstrated that the decline in nodulated root respiration was associated with nodule rather than root metabolism, since within 3.5 hours of treatment, nodules respired at less than 10% of the initial rates. Apparently, a continuous supply of carbohydrate from the shoot is required to support nodule, but not root, function. Depletion of nodular carbohydrate pools was sufficient to account for the (diminishing) nodule respiration of girdled plants. Of starch and soluble sugar pools within the whole plant, only leaf starch exhibited a diurnal variation which was sufficient to account for the respiratory carbon loss of nodules over an 8 hour night. Under 16 hour nights, or in continuous dark, first the leaf starch pools were depleted, and then nodule starch reserves declined concomitant with a decrease in the rates of CO(2) and H(2) evolution from the nodules. Nodule soluble sugar levels were maintained in dark treated plants but declined in girdled plants. The depletion of starch in root nodules is an indicator of carbohydrate limitation of nodule function.

Journal Article↗

The response of the phosphate uptake system and the organic acid exudation system to phosphate starvation in Sesbania rostrata.

It is well known that the P(i) uptake system via the high-affinity P(i) transporter and the organic acid exudation system via PEPC are enhanced in the roots of P(i)-starved plants. In this paper, we compared the expression of these two systems in Sesbania rostrata, a leguminous plant, on whose roots and stems it forms nodules. When S. rostrata plants were transferred to a 0 microM P(i) nutrient solution, the expression of both the high-affinity P(i) transporter and PEPC was enhanced within 2 d. The enhancement of the expression of the high-affinity P(i) transporter genes and the PEPC gene coordinated with the increases in the P(i) uptake rate and the PEPC activity, respectively. This suggests that the expression of the high-affinity P(i) transporters and PEPC is regulated in part at the transcript level. Furthermore, we examined which of the environmental or the endogenous P(i) level regulates the expression of these two systems. The P(i) content in the 6-day-old plants decreased to a lower level than that in the 15-day-old plants when grown in a 30 microM P(i) solution. At that time, the expression of the high-affinity P(i) transporters and PEPC was enhanced only in the 6-day-old plants. Moreover, the P(i) content in plants forming many nodules on their stems decreased. The expression of the high-affinity P(i) transporters and PEPC was then enhanced in the nodulated plants. These facts suggest that the expression of these two systems may be regulated by the P(i) content in the plants, not by the P(i) concentration in the soil.

Fabaceae↗

A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning.

In the establishment of the legume-rhizobial symbiosis, bacterial lipochitooligosaccharide signaling molecules termed Nod factors activate the formation of a novel root organ, the nodule. Nod factors elicit several responses in plant root hair cells, including oscillations in cytoplasmic calcium levels (termed calcium spiking) and alterations in root hair growth. A number of plant mutants with defects in the Nod factor signaling pathway have been identified. One such Medicago truncatula mutant, dmi3, exhibits calcium spiking and root hair swelling in response to Nod factor, but fails to initiate symbiotic gene expression or cell divisions for nodule formation. On the basis of these data, it is thought that the dmi3 mutant perceives Nod factor but fails to transduce the signal downstream of calcium spiking. Additionally, the dmi3 mutant is defective in the symbiosis with mycorrhizal fungi, indicating the importance of the encoded protein in multiple symbioses. We report the identification of the DMI3 gene, using a gene cloning method based on transcript abundance. We show that transcript-based cloning is a valid approach for cloning genes in barley, indicating the value of this technology in crop plants. DMI3 encodes a calcium/calmodulin-dependent protein kinase. Mutants in pea sym9 have phenotypes similar to dmi3 and have alterations in this gene. The DMI3 class of proteins is well conserved among plants that interact with mycorrhizal fungi, but it is less conserved in Arabidopsis thaliana, which does not participate in the mycorrhizal symbiosis.

Amino Acid Sequence↗

A strong constitutive positive element is essential for the ammonium-regulated expression of a soybean gene encoding cytosolic glutamine synthetase.

In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean gene GS15 encoding cytosolic glutamine synthetase, a series of 5' promoter deletions were fused to the GUS reporter gene. To allow the detection of positive and negative regulatory elements, a series of 3' deletions were fused to a -90 CaMV 35S promoter fragment placed upstream of the GUS gene. Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation. Both spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the promoter distal region is indispensable for the ammonium-regulated expression of GS15. Interestingly, this SCPE was able to direct constitutive expression in both a legume and non-legume background to a level similar to that driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression. This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nodule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-specific elements. The presence of positive, negative and organ-specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA for trans-acting factor accessibility.

Base Sequence↗

Effect of light, dark, and temperature on root nodule activity (acetylene reduction) of soybeans.

The objectives of this study were (a): to define the effects of light, dark, and temperature on nodule activity (acetylene reduction), and (b) to establish the contributions of reserve carbohydrate and recent photosynthate to the support of nodule function. An in situ assay of nodule activity was developed for use with intact, hydroponically grown soybeans (Glycine max [L.] Merr. cv. Calland).Nodule activity of 35-day-old plants grown in controlled environment chambers decreased during a 10-hour dark period at 18 C, compared with activity during the preceding and subsequent 14-hour light periods at 27 C. In contrast, plants that were maintained at a constant 27 C did not vary in nodule activity during diurnally varying dark and light exposure. Nodules of plants exposed to diurnal 18 and 27 in 24-hour continuous dark were less active at 18 C than at 27 C. At constant 27 C, nodule activity was sustained throughout the 24-hour dark period. Thus, nodule activity was independent of short term dark periods but dependent on temperature; nodule activity was decreased at the lower temperature.Temperature also affected the nodule activity of plants maintained in the light. Exposure of shoots and roots of intact plants to the lower temperature (5 hours at 18 C) during the light period resulted in a marked decrease in nodule activity, compared with that of plants maintained at 27 C. Exposure of only the shoot portion to 18 C (roots were maintained at 27 C) resulted in a similar decrease in nodule activity.Nodules of plants exposed to 10 days of diurnally variable dark, light, and temperature had high activity in the light at 27 C and low activity in the dark at 18 C. Nodule activity of plants at a constant 27 C was not affected by diurnally variable dark and light exposure throughout the 10-day period, although activity generally increased with time due to increased nodule mass. At a constant 27 C, nodules of intact plants in continuous dark sustained activity through 72 hours before declining to zero by 7 days. At diurnally varying 18 and 27 C in continuous dark, peak diurnal nodule activity was sustained through 5 days, and then declined to zero by 8 days. Analyses of the carbohydrate content of tissue harvested from this study suggested use of reserve photosynthate (primarily of shoot origin) in support of nodule activity in the absence of concurrent photosynthesis.

Journal Article↗

In vivo and in vitro phosphorylation of membrane and soluble forms of soybean nodule sucrose synthase.

Sucrose synthase (SS) is a known phosphoserine (SerP)-containing enzyme in a variety of plant "sink" organs, including legume root nodules, where it is phosphorylated primarily at Ser-11. Using immunofluorescence confocal microscopy, we documented that part of the total SS (nodulin-100) pool in mature soybean (Glycine max) nodules is apparently associated with the plasma membrane in situ, and we report that this association is very "tight," as evidenced by a variety of chemical and enzymatic pretreatments of the isolated microsomal fraction. To investigate the in situ and in planta phosphorylation state of the membrane (m) and soluble (s) forms of nodule SS, three complementary approaches were used. First, excised nodules were radiolabeled in situ with [(32)P]Pi for subsequent analysis of phosphorylated m- and s-SS; second, immunopurified s- and m-SS were used as substrate in "on-bead" assays of phosphorylation by nodule Ca(2+)-dependent protein kinase; and third, SS-Ser-11(P) phosphopeptide-specific antibodies were developed and used. The collective results provide convincing evidence that microsomal nodulin-100 is phosphorylated in mature nodules, and that it is hypophosphorylated relative to s-SS (on an equivalent SS protein basis) in attached, unstressed nodules. Moreover, the immunological data and related phosphopeptide mapping analyses indicate that a homologous N-terminal seryl-phosphorylation domain and site reside in microsomal nodulin-100. We also observed that mild, short-term inorganic nitrogen and salt stresses have a significant negative impact on the content and N-terminal phosphorylation state of nodule m- and s-SS, with the former being the more sensitive of the two SS forms.

Ammonium Chloride↗

The LATD gene of Medicago truncatula is required for both nodule and root development.

The evolutionary origins of legume root nodules are largely unknown. We have identified a gene, LATD, of the model legume Medicago truncatula, that is required for both nodule and root development, suggesting that these two developmental processes may share a common evolutionary origin. The latd mutant plants initiate nodule formation but do not complete it, resulting in immature, non-nitrogen-fixing nodules. Similarly, lateral roots initiate, but remain short stumps. The primary root, which initially appears to be wild type, gradually ceases growth and forms an abnormal tip that resembles that of the mutant lateral roots. Infection by the rhizobial partner, Sinorhizobium meliloti, can occur, although infection is rarely completed. Once inside latd mutant nodules, S. meliloti fails to express rhizobial genes associated with the developmental transition from free-living bacterium to endosymbiont, such as bacA and nex38. The infecting rhizobia also fail to express nifH and fix nitrogen. Thus, both plant and bacterial development are blocked in latd mutant roots. Based on the latd mutant phenotype, we propose that the wild-type function of the LATD gene is to maintain root meristems. The strong requirement of both nodules and lateral roots for wild-type LATD gene function supports lateral roots as a possible evolutionary origin for legume nodules.

Chromosome Mapping↗

Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses.

Most land plants can form a root symbiosis with arbuscular mycorrhizal (AM) fungi for assimilation of inorganic phosphate from the soil. In contrast, the nitrogen-fixing root nodule symbiosis is almost completely restricted to the legumes. The finding that the two symbioses share common signaling components in legumes suggests that the evolutionarily younger nitrogen-fixing symbiosis has recruited functions from the more ancient AM symbiosis. The recent advances in cloning of the genes required for nodulation and AM symbioses from the two model legumes, Medicago truncatula and Lotus japonicus, provide a unique opportunity to address biological questions pertaining to the evolution of root symbioses in plants. Here, we report that nearly all cloned legume genes required for nodulation and AM symbioses have their putative orthologs in nonlegumes. The orthologous relationship can be clearly defined on the basis of both sequence similarity and microsyntenic relationship. The results presented here serve as a prelude to the comparative analysis of orthologous gene function between legumes and nonlegumes and facilitate our understanding of how gene functions and signaling pathways have evolved to generate species- or family-specific phenotypes.

DNA, Complementary↗

Sodium stimulation of uptake hydrogenase activity in symbiotic Rhizobium.

Initial observations showed a 100% increase in H(2)-uptake (Hup) activity of Rhizobium leguminosarum strain 3855 in pea root nodules (Pisum sativum L. cv Alaska) on plants growing in a baked clay substrate relative to those growing in vermiculite, and an investigation of nutrient factors responsible for the phenomenon was initiated. Significantly greater Hup activity was first measured in the clay-grown plants 24 days after germination, and higher activity was maintained relative to the vermiculite treatment until experiments were terminated at day 32. The increase in Hup activity was associated with a decrease in H(2) evolution for plants with comparable rates of acetylene reduction. Analyses of the clay showed that it contained more Na(+) (29 versus 9 milligrams per kilogram) and less K(+) (6 versus 74 milligrams per kilogram) than the vermiculite. Analyses of plants, however, showed a large increase in Na(+) concentration of clay-grown plants with a much smaller reduction in K(+) concentration. In tests with the same organisms in a hydroponic system with controlled pH, 40 millimolar NaCl increased Hup activity more than 100% over plants grown in solutions lacking NaCl. Plants with increased Hup activity, however, did not have greater net carbon or total nitrogen assimilation. KCl treatments from 5 to 80 millimolar produced slight increased in Hup activity at 10 millimolar KCl, and tests with other salts in the hydroponic system indicated that only Na(+) strongly promoted Hup activity. Treating vermiculite with 50 millimolar NaCl increased Na(+) concentration in pea plant tissue and greatly promoted Hup activity of root nodules in a manner analogous to the original observation with the clay rooting medium. A wider generality of the phenomenon was suggested by demonstrating that exogenous Na(+) increased Hup activity of other R. leguminosarum strains and promoted Hup activity of R. meliloti strain B300 in alfalfa (Medicago sativa L.).

Journal Article↗

Role of methylotrophy during symbiosis between Methylobacterium nodulans and Crotalaria podocarpa.

Some rare leguminous plants of the genus Crotalaria are specifically nodulated by the methylotrophic bacterium Methylobacterium nodulans. In this study, the expression and role of bacterial methylotrophy were investigated during symbiosis between M. nodulans, strain ORS 2060T, and its host legume, Crotalaria podocarpa. Using lacZ fusion to the mxaF gene, we showed that the methylotroph genes are expressed in the root nodules, suggesting methylotrophic activity during symbiosis. In addition, loss of the bacterial methylotrophic function significantly affected plant development. Indeed, inoculation of M. nodulans nonmethylotroph mutants in C. podocarpa decreased the total root nodule number per plant up to 60%, decreased the whole-plant nitrogen fixation capacity up to 42%, and reduced the total dry plant biomass up to 46% compared with the wild-type strain. In contrast, inoculation of the legume C. podocarpa with nonmethylotrophic mutants complemented with functional mxa genes restored the symbiotic wild phenotype. These results demonstrate the key role of methylotrophy during symbiosis between M. nodulans and C. podocarpa.

Crotalaria↗

Burkholderia mimosarum sp. nov., isolated from root nodules of Mimosa spp. from Taiwan and South America.

Fourteen strains were isolated from nitrogen-fixing nodules on the roots of plants of the genus Mimosa growing in Taiwan, Brazil and Venezuela. On the basis of 16S rRNA gene sequence similarities, all of the strains were previously shown to be closely related to each other and to belong to the genus Burkholderia. A polyphasic approach, including DNA-DNA reassociation, whole-cell protein analysis, fatty acid methyl ester analysis and extensive biochemical characterization, was used to clarify the taxonomic position of these strains: all 14 strains were classified as representing a novel species, for which the name Burkholderia mimosarum sp. nov. is proposed. The type strain, PAS44(T) (=LMG 23256(T) =BCRC 17516(T)), was isolated from Mimosa pigra nodules in Taiwan.

Bacterial Proteins↗

[Nitrate assimilation activity of yellow lupine root nodules ?].

Low nitrate assimilation activity of the root nodules was demonstrated by assaying the nitrate reductase, glutamate synthase, glutamate dehydrogenase, and asparagine synthase activities, as well as the kinetics of 14C-labeled saccharose incorporation in the amino acids and amides of the cortex and the bacteroid-containing root nodule zones. Irrespective of the exogenous nitrogen concentration (0, 11.2, or 25 mM NO3-), nitrate concentration in the nodules was low as compared to the plant roots, leaves, and stems. This allowed us to propose the presence of structural and/or metabolic barriers in the nodules limiting nitrate accessibility and assimilation.

Amino Acids↗

Gene expression of Medicago sativa inoculated with Sinorhizobium meliloti as modulated by the xenobiotics cadmium and fluoranthene.

Alfalfa plants (Medicago sativa cv. Europe) inoculated with Sinorhizobium meliloti 2011 (formerly Rhizobium meliloti, de Lajudie et al., 1994) were cultivated for 14 days under standardized growth conditions in mineral medium with addition of the heavy metal cadmium or the polycyclic aromatic hydrocarbon fluoranthene. These xenobiotics significantly reduced the numbers of root nodules before any visible damage to the plant could be detected. EC10, EC50, and EC90 (effective concentrations reducing nodulation, shoot and root fresh weight by 10, 50, or 90% compared to the control without pollutant) were calculated. EC50 for cadmium ranged from 5.8 microM (nodulation) to more than 20 microM (root fresh weight). Testing fluoranthene resulted in an EC50 of 2.5 microg cm(-2) for nodulation, and EC50 values of more than 35 microg cm(-2) for shoot and root biomass production, indicating that the effect parameter nodulation is 10-fold more sensitive than shoot and root fresh weight. With mRNA differential display techniques the effects of both xenobiotics on gene expression in alfalfa root systems were studied. 37 differentially displayed transcripts were detected. Two of them, called DDMs1 and DDMs2, were confirmed by northern hybridization to be down-regulated in the presence of the xenobiotics. The expression of transcript DDMs1 was enhanced in alfalfa control plants inoculated with rhizobia, the transcript level was increased 2.5-3-fold compared to non-inoculated plants. This positive effect of nodulation was suppressed, partly by 35 microg cm(-2) fluoranthene and totally by 20 microM cadmium. The decrease in DDMs1 transcription was highly affected by the cadmium concentration with an EC50 of 5.9 microM. Compared to nodulation, almost identical EC10, EC50, and EC90 values were found for DDMs1 expression. Sequence analysis of DDMs1 revealed a significant overall homology (50% identity) to a hypothetical protein from Arabidopsis thaliana with high similarity to a copper transporting ATPase. High levels of transcript DDMs2 were observed in control plants with a 50% decrease in the xenobiotic-treated plants. DDMs2 gave a strong homology (82% identity) to the cytoplasmatic 60S ribosomal protein L18 from Arabidopsis thaliana.

Amino Acid Sequence↗

Oligonucleotide Probes That Hybridize with rRNA as a Tool To Study Frankia Strains in Root Nodules.

Oligonucleotide probes that hybridize with specific sequences in variable regions of the 16S rRNA of the nitrogen-fixing actinomycete Frankia were used for the identification of Frankia strains in nodules. Frankia cells were released from plant tissue by grinding glutaraldehyde-fixed root nodules in guanidine hydrochloride solution. rRNA was obtained after sonication, precipitation with ethanol, and purification by phenolchloroform extraction. Degradation of rRNA, evident in Northern blots, did not affect hybridization with the oligonucleotides. Nodules of about 1 mg (fresh weight) provided sufficient rRNA for reliable detection of the Frankia strain. The utility of this rRNA extraction method was tested in a competition experiment between two effective Frankia strains on cloned Alnus glutinosa plants.

Journal Article↗

Effect of changes in shoot carbon-exchange rate on soybean root nodule activity.

The effect of short- and long-term changes in shoot carbon-exchange rate (CER) on soybean (Glycine max [L.] Merr.) root nodule activity was assessed to determine whether increases in photosynthate production produce a direct enhancement of symbiotic N(2) fixation. Shoot CER, root + nodule respiration, and apparent N(2) fixation (acetylene reduction) were measured on intact soybean plants grown at 700 microeinsteins per meter per second, with constant root temperature and a 14/10-hour light/dark cycle. There was no diurnal variation of root + nodule respiration or apparent N(2) fixation in plants assayed weekly from 14 to 43 days after planting. However, if plants remained in darkness following their normal dark period, a significant decline in apparent N(2) fixation was measured within 4 hours, and decreasing CO(2) concentration from 320 to 90 microliters CO(2) per liter produced diurnal changes in root nodule activity. Increasing shoot CER by 87, 84, and 76% in 2-, 3-, and 4-week-old plants, respectively, by raising the CO(2) concentration around the shoot from 320 to 1,000 microliters CO(2) per liter, had no effect on root + nodule respiration or acetylene-reduction rates during the first 10 hours of the increased CER treatment. When the CO(2)-enrichment treatment was extended in 3-week-old plants, the only measured parameter that differed significantly after 3 days was shoot CER. After 5 days of continuous CO(2) enrichment, root + nodule respiration and acetylene reduction increased, but such changes reflected an increase in root nodule mass rather than greater specific root nodule activity. The results show that on a 24-hour basis the process of symbiotic N(2) fixation in soybean plants grown under controlled environmental conditions functioned at maximum capacity and was not limited by shoot CER. Whether N(2)-fixation capacity was limited by photosynthate movement to root nodules or by saturation of metabolic processes in root nodules is not known.

Journal Article↗