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PubMed2005

Evidence for carbon flux shortage and strong carbon/nitrogen interactions in pea nodules at early stages of water stress.

Symbiotic N2 fixation in legume nodules declines under a wide range of environmental stresses. A high correlation between N2 fixation decline and sucrose synthase (SS; EC 2.4.1.13) activity down-regulation has been reported, although it has still to be elucidated whether a causal relationship between SS activity down-regulation and N2 fixation decline can be established. In order to study the likely C/N interactions within nodules and the effects on N2 fixation, pea plants (Pisum sativum L. cv. Sugar snap) were subjected to progressive water stress by withholding irrigation. Under these conditions, nodule SS activity declined concomitantly with apparent nitrogenase activity. The levels of UDP-glucose, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate decreased in water-stressed nodules compared with unstressed nodules. Drought also had a marked effect on nodule concentrations of malate, succinate, and alpha-ketoglutarate. Moreover, a general decline in nodule adenylate content was detected. NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42) was the only enzyme whose activity increased as a result of water deficit, compensating for a possible C/N imbalance and/or supplying NADPH in circumstances that the pentose phosphate pathway was impaired, as suggested by the decline in glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) activity. The overall results show the occurrence of strong C/N interactions in nodules subjected to water stress and support a likely limitation of carbon flux that might be involved in the decline of N2 fixation under drought.

Carbon↗

PubMed2005

Promoter trapping in Lotus japonicus reveals novel root and nodule GUS expression domains.

Agrobacterium-based transformation was used to introduce a promoter-less glucuronidase uidA gene (beta-glucuronidase; GUS) into Lotus japonicus. Transgenic plants were screened for GUS activation at different stages after inoculation with its symbiont, Mesorhizobium loti. Functional GUS fusion frequencies ranged from about 2 to 5% of the total number of transgenic lines. These lines provide excellent histological markers for tissue ontogeny analysis. Some of the activations generated GUS expression patterns that correspond to well-known tissue types, such as lateral root and nodule primordia, root tips and developing nodules (line CHEETAH). Others generated GUS activation associated with predictable but previously unknown (i) tissue types, such as the vascular bundle of the nodule (line VASCO); or (ii) expression domains, such as pericycle, nodule primordia, nodule and flower connective/vascular tissue (line FATA MORGANA) or inner root cortex cells in the vicinity of a curled root hair, nodule primordia and nodule cortex (line TIMPA). Putative members of two gene superfamilies, EH (Esp homolog) and AAA ATPase (ATPase associated with various cellular activities), were located next to the CHEETAH and VASCO insertions, respectively, and a nodulin gene, LjENOD40-2, was located next to the FATA MORGANA insertion. We utilized promoter GUS fusions to investigate the genetic regulation of LjENOD40-2 and FATA MORGANA GUS. The LjENOD40-2 promoter defined a novel expression domain and the FATA MORGANA nodule expression was reiterated by the 2 kb sequence upstream of the T-DNA insertion.

Base Sequence↗

PubMed2006

Phosphoenolpyruvate carboxylase plays a crucial role in limiting nitrogen fixation in Lotus japonicus nodules.

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is believed to play a significant role in supporting nitrogen fixation via anaplerotic CO2 fixation for recycling carbon in nodules. Using the antisense technique, we decreased the expression levels of the nodule-enhanced PEPC gene (Ljpepc1) in a determinate legume plant (Lotus japonicus) in order to look at the influence of the symbiotic phenotype and biochemical parameters. Three independent transgenic L. japonicus plants (designated as Asppc1, Asppc2 and Asppc3) were prepared using a Ljpepc1 DNA fragment which is under the control of the cauliflower mosaic virus 35S promoter. Extensive suppression of the Ljpepc1 transcript in nodules of Asppc plants (T3 homologous plants) was confirmed by RNA gel blot, Western blot and enzyme activity assays. In nodules of Asppc plants, PEPC activity was reduced to about 10% of that of non-transformants and the plants showed typical nitrogen-deficient symptoms without a supply of nitrogen nutrient, and returned to normal growth when nitrate was supplied at 2.5 mM. The acetylene reduction activity per fresh weight of nodules of these Asppc plants decreased by 29% at 35 dai (days after infection). Various enzyme activities and metabolite levels were surveyed using Asppc plants at 35 dai. Significant reduction of sucrose synthase and asparagine aminotransferase activities was observed in Asppc nodules. In addition, sucrose, succinate, asparagine, aspartate and glutamate contents also decreased in Asppc nodules. The data are discussed in terms of a role for PEPC in the carbon/nitrogen metabolic flux in nodules.

Acetylene↗

PubMed2006

Identification of a Sed5-like SNARE gene LjSYP32-1 that contributes to nodule tissue formation of Lotus japonicus.

We identified a Sed5-like clone LjSYP32-1 which contributes to nodule tissue formation and plant growth in Lotus japonicus. In the L. japonicus expressed sequence tag (EST) clone databases of Kazusa DNA Research Institute, another syntaxin-related clone (LjSYP32-2) was also detected, and the nucleotide and amino acid sequences of these two clone are very similar to each other. Real-time PCR and promoter analysis indicated that expression of LjSYP32-1 was dominant compared with LjSYP32-2 in the various plant organs. Promoter analysis and in situ hybridization revealed that LjSYP32-1 was expressed significantly in the inner cortex cell layer surrounding the infected zone of young nodules and in the meristem area of developing lateral root. To explore the function and physiological role of LjSYP32-1 in nodules and other plant organs, stable transformation lines of L. japonicus expressing either sense or antisense LjSYP32-1 were prepared. The antisense plants showed a significantly retarded plant growth phenotype, suggesting a role for LjSYP32-1 in supporting plant growth. In the same transgenic lines, the plants were capable of forming nodules, but the acetylene reduction activity was reduced by around 50% per plant. The nodules were much smaller and some nodules were fused to each other by sharing the inner cortex. The rate of occurrence of such irregular nodules was twice that observed in wild-type plants. The data suggest that LjSYP32-1 contributes to the support of plant growth and normal nodule tissue differentiation.

Amino Acid Sequence↗

PubMed2006

RNAi knock-down of ENOD40s leads to significant suppression of nodule formation in Lotus japonicus.

ENOD40 is one of the most intriguing early nodulin genes that is known to be induced very early in response to interaction of legume plants with symbiotic Rhizobium bacteria, but its function in the nodulation process is still not known. Lotus japonicus has two ENOD40 genes: LjENOD40-1 is abundantly induced in very early stages of bacterial infection or Nod factor application, whereas LjENOD40-2 is abundantly expressed only in mature nodules. We generated transgenic lines of L. japonicus with an RNAi (RNA interference) construct that expresses hairpin double-stranded RNA for LjENOD40-1 to induce sequence-specific RNA silencing. In the transgenic plants, expression of both LjENOD40-1 and -2 was significantly reduced, and no accumulation of ENOD40 transcripts was detected upon Mesorhizobium loti inoculation. The transgenic plants exhibited very poor nodulation (only 0-2 nodules per plant) and could not grow well without additional nitrogen supply. Analysis of segregation in the T(2) progeny indicated that the suppression of nodulation is perfectly linked with the presence of the transgene. Microscopic observation of the infection process using lacZ-labeled M. loti, together with expression analysis of infection-related nodulin genes, demonstrated that ENOD40 knock-down did not inhibit the initiation of the bacterial infection process. In contrast, nodule primordium initiation and subsequent nodule development were significantly suppressed in the transgenic plants. These results clearly indicate that ENOD40 is required for nodule initiation and subsequent organogenesis, but is not involved in early infection events.

Base Sequence↗

PubMed1998

Identification of novel putative regulatory genes induced during alfalfa nodule development with a cold-plaque screening procedure.

Until now very few plant genes with possible regulatory functions during nodule development have been isolated. We have used a modified cold-plaque screening method to identify new transcripts expressed at low levels that are induced during nodulation. Several clones were isolated and characterized by their mRNA expression patterns during nodule development and in spontaneous nodules. Sequence homology with known genes of other organisms indicated that transcripts corresponded to (i) "basic" genes probably required during the growth of the nodule organ (e.g., structural proteins), (ii) genes related to the metabolic adaptations taking place during nodule morphogenesis and function (e.g., carbonic anhydrase), and (iii) genes containing regulatory motifs and/or homologies (three clones out of the 20 identified). The latter genes encode a zinc-finger-containing protein, a putative protein kinase, and a Wilm's tumor (WT) suppressor homologue, respectively. Expression of the kinase and WT suppressor homologues was induced early in nodulation, although the latter was activated transiently. Accumulation of the Zn-finger gene transcripts was detected at a later stage of development and seems to be regulated in a complex manner. Hence, using a cold-plaque screening procedure, we could identify genes that may play regulatory roles in the signal transduction pathways activated during nodule development.

Amino Acid Sequence↗

PubMed1998

The Bradyrhizobium japonicum noeD gene: a negatively acting, genotype-specific nodulation gene for soybean.

Bradyrhizobium japonicum strain USDA 110 is restricted for nodulation by soybean genotype PI 417566. We previously reported the identification of a USDA 110 Tn5 mutant, strain D4.2-5, that had the ability to overcome nodulation restriction conditioned by PI 417566 (S. M. Lohrke, J. H. Orf, E. Martínez-Romero, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:2378-2383, 1995). In this study, we report the cloning and characterization of the negatively acting DNA region mutated in strain D4.2-5 that is involved in the genotype-specific nodulation of soybean. The Tn5 integration site was localized to a 5.2-kb EcoRI fragment isolated from wild-type USDA 110 genomic DNA. Saturation Tn5 mutagenesis of this 5.2-kb region and DNA homogenitization studies indicated that a 0.9-kb DNA region was involved in the genotype-specific nodulation of PI 417566. A single open reading frame (ORF) of 474 nucleotides, encoding a predicted protein of 158 amino acids, was identified within this region by DNA sequencing. This ORF was named noeD. Computer comparisons with available data bases revealed no significant similarities between the noeD DNA or predicted amino acid sequence and any known genes or their products. However, comparisons done with the region upstream of noeD revealed a high degree of similarity (about 76% similarity and 62% identity) to the N-terminal regions of the Rhizobium leguminosarum bv. viciae and R. meliloti nodM genes, which have been postulated to encode a glucosamine synthase. Southern hybridization analysis indicated that noeD is not closely linked to the main or auxiliary nodulation gene clusters in B. japonicum and that both nodulation-restricted and -unrestricted B. japonicum serogroup 110 strains contain a noeD homolog. High-performance liquid chromatography and fast atom bombardment-mass spectrometry analyses of the lipo-chitin oligosaccharide (LCO) nodulation signals produced by an noeD mutant showed a higher level of acetylation than that found with wild-type USDA 110. These results suggest that specific LCO signal molecules may be one of the factors influencing nodulation specificity in this symbiotic system.

Amino Acid Sequence↗

PubMed2000

Construction of transgenic pea lines with modified expression of diamine oxidase and modified nodulation responses with exogenous putrescine.

Diamine oxidase (DAO) might influence pea nodule development either by regulating the peroxide-driven cross-linking of glycoproteins in the infection thread matrix or by modifying the metabolism of diamines and polyamines in host cells. Transformed lines of pea (Pisum sativum) with the coding sequence for DAO (PSAO-1) in sense orientation behind a tissue-specific promoter (pENOD12A) showed strong co-suppression of DAO activity in extracts from nodules and epicotyls, whereas the antisense constructs were relatively unaffected. No difference in nodule number was observed between transformed lines and controls, suggesting that DAO does not normally have an essential role in nodule initiation. However, lines showing co-suppression of DAO were less sensitive to the inhibitory effects of exogenous putrescine and less active in the cross-linking of matrix glycoprotein, indicating that putrescine-derived products of DAO activity could retard nodule development. Inoculation of co-suppressed lines with Rhizobium strain B661 (a lipopolysaccharide-defective mutant) resulted in more extreme impairment of nodule development and nitrogen fixation capacity, relative to lines with normal levels of DAO, which suggests that DAO may serve to reduce the endogenous level of inhibitory diamines or polyamines in nodules under physiological stress. We conclude that the most critical role of DAO in pea nodule development is apparently in the regulation of diamine levels in host tissues.

Amine Oxidase (Copper-Containing)↗

PubMed2001

Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa reveal abundance of iron-containing isozymes, posttranslational regulation, and compensation of isozyme activities.

The composition of antioxidant enzymes, especially superoxide dismutase (SOD), was studied in one nontransgenic and three transgenic lines of nodulated alfalfa plants. Transgenic lines overproduced MnSOD in the mitochondria of nodules and leaves (line 1-10), MnSOD in the chloroplasts (line 4-6), and FeSOD in the chloroplasts (line 10-7). In nodules of line 10-7, the absence of transgene-encoded FeSOD activity was due to a lack of mRNA, whereas in nodules of line 4-6 the absence of transgene-encoded MnSOD activity was due to enzyme inactivation or degradation. Transgenic alfalfa showed a novel compensatory effect in the activities of MnSOD (mitochondrial) and FeSOD (plastidic) in the leaves, which was not caused by changes in the mRNA levels. These findings imply that SOD activity in plant tissues and organelles is regulated, at least partially, at the posttranslational level. All four lines had low CuZnSOD activities and an abundant FeSOD isozyme, especially in nodules, indicating that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. This was confirmed by the detection of FeSOD cDNAs and proteins in nodules of other legumes such as cowpea, pea, and soybean. The cDNA encoding alfalfa nodule FeSOD was characterized and the deduced protein found to contain a plastid transit peptide. A comparison of sequences and other properties reveals that there are two types of FeSODs in nodules.

Amino Acid Sequence↗

PubMed2001

Cell biological changes of outer cortical root cells in early determinate nodulation.

In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex. This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant. We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants. Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides [LCOs]) on this phenomenon, and sensitivity of the nodulation process to ethylene. Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L. japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L. japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylene-biosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L. japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L. japonicus.

Ethylenes↗

PubMed2002

Expression of genes encoding late nodulins characterized by a putative signal peptide and conserved cysteine residues is reduced in ineffective pea nodules.

Five nodulin genes, PsN1, PsN6, PsN314, PsN335, and PsN466, with reduced expression in ineffective nodules on the pea (Pisum sativum) mutant E135 (sym13) were characterized. They encode small polypeptides containing a putative signal peptide and conserved cysteine residues and show homology to the nodulins PsENOD3/14 and PsNOD6. For each gene, multiple bands were detected by genomic Southern analysis. Northern analysis showed that all five genes were expressed exclusively in nodules and that their temporal expression patterns were similar to that of the leghemoglobin (Lb) gene during nodule development. Their transcripts were localized predominantly from the interzone II-III to the distal part of nitrogen-fixing zone III in effective nodules, resembling the Lb gene. However, transcripts in ineffective E135 nodules were localized in narrower regions than those in the effective nodules. These results indicate that these nodulins are abundant in pea nodules and that their successive expression during nodule development is associated with nitrogen-fixing activity.

Amino Acid Sequence↗

PubMedDate not supplied

Small nodule detection in cirrhotic livers: evaluation with US, spiral CT, and MRI and correlation with pathologic examination of explanted liver.

PURPOSE: The purpose of this work was to evaluate the detection and characterization of nodules > or = 8 mm and small hepatocellular carcinomas (HCCs) in liver cirrhosis. METHOD: Pathologic examination and results of US, helical CT, and dynamic MRI with gadolinium were compared after orthotopic liver transplantation (OLT) of 43 cirrhotic patients. Nodules were classified as macroregenerative nodules (MRNs), borderline nodules (BNs), and HCC. RESULTS: Pathologic examination classified 69 nodules: 50 MRNs, 6 BNs, and 13 HCCs. Sensitivities of MRN, BN, and HCC detection were, respectively, for US imaging 2% (1/50), 33.3% (2/6), and 46.2% (6/13); for helical CT 2% (1/50), 50% (3/6), and 53.8% (7/13); and for MRI 42% (21/50), 50% (3/6), and 76.9% (10/13). MRI detected 21 MRNs. They presented on T1/T2-weighted images as hyperintense/hypointense (n = 8), hyperintense/isointense (n = 7), hypointense/hypointense (n = 4), hypointense/isointense (n = 1), and hypointense depicted only on echo planar imaging (n = 1). The three detected BNs were hyperintense/hypointense nodules. The 10 detected HCCs appeared hyperintense/isointense (n = 7), hyperintense/hypointense (n = 2), and hypointense/isointense (n = 1). None of the MRNs but eight HCCs and one BN were enhanced after gadolinium injection. CONCLUSION: Contrast-enhanced MRI is the most sensitive technique for detecting liver nodules. No MR signal intensity pattern characteristic of small HCCs enables differentiation from benign nodules, however. Gadolinium enhancement is the most sensitive and specific characteristic of HCC.

Adult↗

PubMed2002

Fat augmentation following microsurgical removal of the vocal nodules.

OBJECTIVES: Autogenous fat augmentation has been used as a treatment for glottic insufficiency. However, no information is available on the effectiveness of fat injection in patients with vocal nodules or recurrent vocal nodules after surgery. STUDY DESIGN: The retrospective study reviews the efficiency of fat injection after surgery in patients with vocal nodules (n = 18) and recurrent vocal nodules (n = 5). METHODS: The perceptual acoustic, phonatory function, and video laryngostroboscopic data were evaluated before and after surgery in 23 patients. RESULTS: Mean follow-up time was 7.5 months. Nineteen patients had excellent results. Two patients had improvement, and no change was observed in two patients. Phonatory function showed significant improvement in shimmer, harmonic-to-noiseratio (P <.05), maximum phonation time, grade, roughness, and breathiness (P <.001). Video laryngostroboscopic rating showed significant improvement in linearity of the vocal fold edge, amplitude of vocal fold vibration, and excursion of the mucosal wave (P <.001). Less improvement was observed in recurrent vocal nodules than in nonrecurrent vocal nodules. CONCLUSIONS: Fat injection is an effective autogenous implant and may be considered as an option in management of patients with vocal nodules after surgery. Recurrence of nodules is a problem, but the procedure may be repeated.

Adipose Tissue↗

PubMed1985

Characterization of the anomalous infection and nodulation of subterranean clover roots by Rhizobium leguminosarum 1020.

Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.

Autoradiography↗

PubMed2002

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↗

PubMed2003

Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry.

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo (15)N nuclear magnetic resonance (NMR) by exposing detached nodules to (15)N(2) via a perfusion medium, while recording a time course of spectra. In vivo (31)P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and (15)N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by (15)N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo (15)N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed (15)N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo (15)N chemical shifts were observed. Extensive (15)N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn (15)N amino signal was absent in in vivo (15)N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized (15)N gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.

Amino Acids↗

PubMed1993

Phosphorylation of Soybean (Glycine max L.) Nodule Phosphoenolpyruvate Carboxylase in Vitro Decreases Sensitivity to Inhibition by L-Malate.

Phosphoenolpyruvate carboxylase (PEPC) from soybean (Glycine max L.Merr.) nodules was purified 187-fold to a final specific activity of 56 units mg-1 of protein. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed one major polypeptide band, with a molecular mass of 110 kD, after the final purification step. Two-dimensional PAGE resolved four isoelectric forms of the purified enzyme. Antibodies raised against the purified enzyme immunoprecipitated PEPC activity from a desalted nodule extract. Two cross-reacting bands were obtained when protein immunoblots of crude nodule extracts subjected to SDS-PAGE were probed with the antiserum. One of these corresponded to the 110-kD subunit of PEPC, and the other had a molecular mass of about 60 kD. PEPC was shown to be activated in a time-dependent manner when desalted soybean nodule extracts were preincubated with Mg.ATP in vitro. Activation was observed when PEPC was assayed at pH 7 in the absence of glycerol but not at pH 8 in the presence of glycerol. When o.5 mM L-malate was included in the assay, activation was much more pronounced than without malate. Maximal activation was 30% in the absence of L-malate and 200% in its presence. The L-malate concentrations producing 50% inhibition of PEPC activity were o.35 and 1.24 mM, respectively, before and after preincubation with Mg.ATP. The antiserum against soybean nodule PEPC was used to immunoprecipitate PEPC from a desalted nodule extract that had been preincubated with Mg.[[gamma]-32P]ATP. The immunoprecipitate was then subjected to SDS-PAGE, followed by autoradiography. The autoradiograph revealed intense labeling of the 110-kD subunit of PEPC following preincubation with [[gamma]-32P]ATP. The data suggest that soybean nodule PEPC becomes phosphorylated by an endogenous protein kinase, resulting in decreased sensitivity of the enzyme to inhibition by L-malate in vitro. The results are discussed in relation to the proposed functions of PEPC in legume nodules.

Journal Article↗

PubMed2004

Adenylate gradients and Ar:O(2) effects on legume nodules: I. Mathematical models.

Mathematical models were developed to test the likelihood that large cytosolic adenylate concentration gradients exist across the bacteria-infected cells of legume nodules. Previous studies hypothesized that this may be the case to account for the unusually low adenylate energy charge (AEC; 0.65) measured in the plant fraction of metabolically active nodules (M.M. Kuzma, H. Winter, P. Storer, I. Oresnik, C.A. Atkins, D.B. Layzell [1999] Plant Physiol 119: 399-407). Simulations coupled leghemoglobin-facilitated O(2) diffusion into the infected cell, through bacteroid nitrogenase activity, with the ATP demand for transport and ammonia assimilation in the plant fraction of ureide- and amide-producing nodules. Although large cytosolic adenylate gradients were predicted to exist in both nodule types, amide nodules were predicted to have steeper AEC gradients (0.82-0.52) than ureide nodules (0.82-0.61). The differences were attributed to an additional ATP demand for Asn synthesis in the amide nodule. Simulations for nodules transferred to an Ar:O(2) atmosphere predicted a major reduction in the magnitude of adenylate gradients and an increase in the AEC of the plant fraction. Results were consistent with a number of experimental studies and were used to propose an experimental test of the models.

Adenosine Monophosphate↗