Lateral fusion of membranes in bacteroid-containing cells of leguminous root nodules.
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Cell Membrane↗
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Invasion of Lotus japonicus root hairless 1 by Mesorhizobium loti involves the nodulation factor-dependent induction of root hairs.
In many legumes, including Lotus japonicus and Medicago truncatula, susceptible root hairs are the primary sites for the initial signal perception and physical contact between the host plant and the compatible nitrogen-fixing bacteria that leads to the initiation of root invasion and nodule organogenesis. However, diverse mechanisms of nodulation have been described in a variety of legume species that do not rely on root hairs. To clarify the significance of root hairs during the L. japonicus-Mesorhizobium loti symbiosis, we have isolated and performed a detailed analysis of four independent L. japonicus root hair developmental mutants. We show that although important for the efficient colonization of roots, the presence of wild-type root hairs is not required for the initiation of nodule primordia (NP) organogenesis and the colonization of the nodule structures. In the genetic background of the L. japonicus root hairless 1 mutant, the nodulation factor-dependent formation of NP provides the structural basis for alternative modes of invasion by M. loti. Surprisingly, one mode of root colonization involves nodulation factor-dependent induction of NP-associated cortical root hairs and epidermal root hairs, which, in turn, support bacterial invasion. In addition, entry of M. loti through cracks at the cortical surface of the NP is described. These novel mechanisms of nodule colonization by M. loti explain the fully functional, albeit significantly delayed, nodulation phenotype of the L. japonicus ROOT HAIRLESS mutant.
Base Sequence↗
Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules.
Biological nitrogen fixation involves the reduction of atmospheric N2 to ammonia by the bacterial enzyme nitrogenase. In legume-rhizobium symbioses, the nitrogenase-producing bacteria (bacteroids) are contained in the infected cells of root nodules within which they are enclosed by a plant membrane to form a structure known as the symbiosome. The plant provides reduced carbon to the bacteroids in exchange for fixed nitrogen, which is exported to the rest of the plant. This exchange is controlled by plant-synthesised transport proteins on the symbiosome membranes. This review summarises our current understanding of these transport processes, focusing on ammonia and amino acid transport.
Amino Acids↗
Identification and expression of a soybean nodule-enhanced PEP-carboxylase kinase gene (NE-PpcK) that shows striking up-/down-regulation in vivo.
Various isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post-translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC-kinase (PpcK), a novel, 'minimal but functional' Ser/Thr kinase. To date, this plant-specific kinase has been investigated in molecular terms exclusively in non-leguminous plants, such as Crassulacean-acid-metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up-regulated in mature nodules, the expression pattern of this transcript is distinct from that of a second soybean PpcK isogene (GmPpcK). The steady-state abundance of this former, nodule-enhanced transcript (NE-PpcK) is markedly influenced by photosynthate supply from the shoots. This latter up-/down-regulation of NE-PpcK transcript level occurs in vivo in concert with the corresponding changes in the nodule PpcK activity, the phosphorylation-state of PEPC, and the abundance of a previously identified, nodule-enhanced transcript (GmPEPC7) that encodes the target enzyme (NE-Ppc). Furthermore, genomic Southern analysis and inspection of the public database indicate that there are at least three distinct PpcK and Ppc isogenes in soybean. Collectively, these and recent findings with Arabidopsis implicate the existence of multiple PpcK-Ppc'expression-partners' in plants, exemplified by NE-PpcK and NE-Ppc in the soybean nodule.
Amino Acid Sequence↗
Signaling and host range variation in nodulation.
Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts, in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes, which determine host specificity, infection, and nodulation, are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins, which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in turn in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule-meristem formation.
Fabaceae↗
Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides.
The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40-1 peptide or different flavonoids. The results show that clover-nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot-inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O-acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot-inoculation, but only after delivery by microtargeting. Non-O-acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.
Journal Article↗
Progress in assisted natural remediation of an arsenic contaminated agricultural soil.
A contaminated soil was collected in a field adjacent to a derelict As((III)) smelter in Reppel (Bocholt, Belgium). A single soil treatment (% by soil weight) based on either iron grit (SS, 1%), beringite (B, 5%), or iron grit (1%) + beringite (5%) (BSS) was applied. Untreated and treated Reppel soils and a control soil were placed in lysimeters inside a greenhouse and cropped annually. The efficiency of soil treatments in decreasing As and metals in exposure sources and restoring soil functions was assessed 6 years after the treatments commenced. Decreases in extractable Cd, Mn, Zn and As occurred in the BSS soil. Only BSS treatment reduced both As and metal concentrations in leachates. BSS treatment produced best growth of lettuce and cabbage, the highest shoot and pod yields for dwarf bean, the lowest As, Cd and Zn concentrations in plant tissues, and partly restored Rhizobium nodulation on bean roots. The epigeic earthworm (Dendrobaena octaedra) could only survive in the BSS soil. Depurated living worms from the BSS soil had Cd concentration similar to those in control worms, but higher As, Ca, Fe, and Zn concentrations. Based on physiologically based extraction test (PBET), As bioaccessibility was reduced from 12% (untreated soil) to 7.4% (BSS) and 3% (SS), but only the SS treatment decreased the bioaccessibility of Cd (-30%) and Pb (-35%). The range of chemical and biological indicators suggested that BSS amendment was the most effective treatment for restoration of normal soil functions 6 years after initial treatment of the Reppel soil.
Agriculture↗
The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.
Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.
Amino Acid Sequence↗
Localized Changes in Flavonoid Biosynthesis in Roots of Lotus pedunculatus after Infection by Rhizobium loti.
Two-dimensional paper chromatography in four solvent systems, high-sensitivity spray reagents, and UV absorption spectroscopy were used to separate and characterize flavonoids and isoflavonoids in roots and root nodules of 20-d-old Lotus pedunculatus Cav. Seedlings were grown either under sterile conditions or after inoculation with Fix(+) or Fix(-) strains of Rhizobium loti. Flavonoids rather than isoflavonoids predominated in all tissues. Flavonoid profiles in sterile and denodulated root tissues were remarkably similar, both qualitatively and quantitatively. At least 14 partially purified flavonoid aglycones and conjugates were found in root extracts; denodulated root tissues contained no compounds that were not also present in sterile roots. Fix(+) rhizobia were responsible for major postinfection shifts in plant flavonoid biosynthesis at the sites of nodule morphogenesis. Polymeric flavolans were absent from Fix(+) nodules but present in all root tissues and in Fix(-) nodules. Catechin was detected only in Fix(+) nodules.
Journal Article↗
Subcellular location of delta-pyrroline-5-carboxylate reductase in root/nodule and leaf of soybean.
The expression of Delta(1)-pyrroline-5-carboxylate reductase (P5CR) gene was found to be higher in soybean root nodules than in leaves and roots, and its expression in roots appeared to be osmoregulated (AJ Delauney, DPS Verma [1990] Mol Gen Genet 221: 299-305). P5CR was purified to homogeneity as a monomeric protein of 29 kilodaltons by overexpression of a soybean P5CR cDNA clone in Escherichia coli. The pH optimum of the purified P5CR was altered by increasing the salt concentration, and maximum enzyme activity was attainable at a lower pH under high salt (0.2-1 molar NaCl). Kinetic studies of the purified enzyme suggested that nicotinamide adenine dinucleotide phosphate(+) inhibited P5CR activity, whereas nicotinamide adenine dinucleotide(+) did not. Subcellular fractionation and antibodies raised against purified soybean P5CR were used to investigate location of the enzyme in different parts of soybean as well as in leaves of transgenic tobacco plants synthesizing soybean P5CR. P5CR activity was present in cytoplasm of soybean roots and nodules as well as in leaves, but in leaves, about 15% of the activity was detected in the plastid fraction. The location of P5CR was further confirmed by western blot assay of the proteins from cytosol and plastid fractions of different parts of the plant. Expression of soybean nodule cytosolic P5CR in transgenic tobacco under the control of cauliflower mosaic virus 35S promoter led to the accumulation of this protein exclusively in the cytoplasm, suggesting that the chloroplastic activity may be due to the presence of a plastid form of the enzyme. The different locations of P5CR in root and leaf suggested that proline may be synthesized in different subcellular compartments in root and leaf. Proline concentration was not significantly increased in transgenic plants exhibiting high level P5CR activity, indicating that reduction of P5C is not a rate-limiting step in proline production.
Journal Article↗
Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase.
Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C(4) PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C(4) plant species. Neither antiserum reacted strongly with any C(3) leaf proteins. The molecular weight of the PEPC polypeptide from C(4) leaves and legume nodules appears to be similar.
Journal Article↗
Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants.
Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated [Landsmann et al. (1986). Nature 324, 166-168; Bogusz et al. (1988). Nature 331, 178-180]. The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus. Both promoters directed root-specific expression in transgenic tobacco. When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene. The T. tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots. The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes. We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes [Stougaard et al. (1987). EMBO J. 6, 3565-3569].
Base Sequence↗
Expression of cysteine protease genes in pea nodule development and senescence.
Coding sequences for two cysteine proteases were amplified from cDNA derived from pea nodule mRNA using primers based on conserved regions of plant cysteine proteases. One of the amplified cDNA sequences corresponded to a previously described cysteine protease gene, Cyp15a, expressed in pea shoots in response to dehydration (J.T. Jones and J.E. Mullet, Plant Mol. Biol. 28:1055-1065, 1995). Inside the pea root nodule, in situ hybridization revealed that this gene is expressed strongly in the apical region and more weakly in the uninfected cortex and in the central infected tissue where nitrogen fixation takes place. The complete sequence of the cDNA corresponding to the other gene, PsCyp1, was obtained. Expression of this gene, which was studied both on RNA blots and in situ, showed good correlation with the onset of nodule senescence. In situ hybridization studies revealed that PsCyp1 was expressed in senescent infected tissue at the base of the nodule. This signal was just detectable in normal symbiotically wild-type nodules but was much stronger in the early senescing nodules formed by a symbiotically defective mutant of Rhizobium leguminosarum.
Amino Acid Sequence↗
Nodule parenchyma-specific expression of the sesbania rostrata early nodulin gene SrEnod2 is mediated by its 3' untranslated region
The early nodulin Enod2 gene encodes a putative hydroxyproline-rich cell wall protein and is expressed exclusively in the nodule parenchyma cell layer. The latter finding suggests that the Enod2 protein may contribute to the special morphological features of the nodule parenchyma and to the creation of an oxygen diffusion barrier. The Enod2 gene of the stem-nodulating legume Sesbania rostrata (SrEnod2) is induced specifically in roots by the plant hormone cytokinin, and this induction occurs at a post-transcriptional level. Here, we characterize the cis determinant(s) in the SrEnod2 locus responsible for nodule parenchyma-specific expression and show that the 3' untranslated region (UTR) of the SrEnod2 gene is both required and sufficient for directing chimeric reporter gene expression in the nodule parenchyma of transgenic Lotus corniculatus plants. Moreover, we show that the SrEnod2 3' UTR does not act as a tissue-specific enhancer element. By conducting a detailed deletion analysis of the 5' and 3' SrEnod2 regions, we delimited the minimal promoter of the SrEnod2 gene, and it appears that the 5' flanking sequences are not essential for nodule parenchyma-specific expression. This finding is in contrast with the report that the 5' upstream region of the soybean Enod2 gene directs nodule parenchyma-specific expression, indicating that different mechanisms may be involved in regulating the expression of these two genes. We definitively demonstrate that the cis element(s) for tissue-specific expression is located within the 3' UTR of a plant nuclear gene.
Journal Article↗
Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L.
Legume nodules have specialized transport functions for the exchange of carbon and nitrogen compounds between bacteroids and root cells. Plasma membrane-type (vanadate-sensitive) H+-ATPase energizes secondary active transporters in plant cells and it could drive exchanges across peribacteroidal and plasmatic membranes. A nodule cDNA corresponding to a major isoform of Phaseolus vulgaris H+-ATPase (designated BHA1) has been cloned. BHA1 is a functional proton pump because after removal of its inhibitory domain and can complement a yeast mutant unable to synthesize a H+-ATPase. BHA1 is not nodule-specific, since it is also expressed in roots of uninfected plants. It belongs to the subfamily of plasma membrane H+-ATPases defined by the Arabidopsis AHA1, AHA2 and AHA3 genes and the tobacco PMA4 and corn MHA2 genes. In situ hybridization in nodule sections indicates high expression of BHA1 limited to uninfected cells. These results were confirmed by immunocytochemistry. The relatively low expression of plasma membrane-type H+-ATPase in Rhizobium-infected cells put a note of caution on the origin of the vanadate-sensitive ATPase described in preparations of peribacteroidal membranes. Also, our results indicate that active transport in symbiotic nodules is most intense at the plasma membrane of uninfected cells and support a specialized role of uninfected tissue for nitrogen transport.
Amino Acid Sequence↗
Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii.
The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifolii exoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction.
Carbohydrate Sequence↗
Identification of ethylene-mediated protein changes during nodulation in Medicago truncatula using proteome analysis.
Ethylene has been hypothesised to be a regulator of root nodule development in legumes, but its molecular mechanisms of action remain unclear. The skl mutant is an ethylene-insensitive legume mutant showing a hypernodulation phenotype when inoculated with its symbiont Sinorhizobium meliloti. We used the skl mutant to study the ethylene-mediated protein changes during nodule development in Medicago truncatula. We compared the root proteome of the skl mutant to its wild-type in response to the ethylene precursor aminocyclopropane carboxylic acid (ACC) to study ethylene-mediated protein expression in root tissues. We then compared the proteome of skl roots to its wild-type after Sinorhizobium inoculation to identify differentially displayed proteins during nodule development at 1 and 3 days post inoculation (dpi). Six proteins (pprg-2, Kunitz proteinase inhibitor, and ACC oxidase isoforms) were down-regulated in skl roots, while three protein spots were up-regulated (trypsin inhibitor, albumin 2, and CPRD49). ACC induced stress-related proteins in wild-type roots, such as pprg-2, ACC oxidase, proteinase inhibitor, ascorbate peroxidase, and heat-shock proteins. However, the expression of stress-related proteins such as pprg-2, Kunitz proteinase inhibitor, and ACC oxidase, was down-regulated in inoculated skl roots. We hypothesize that during early nodule development, the plant induces ethylene-mediated stress responses to limit nodule numbers. When a mutant defective in ethylene signaling, such as skl, is inoculated with rhizobia, the plant stress response is reduced, resulting in increased nodule numbers.
Amino Acid Sequence↗
A 2-O-methylfucose moiety is present in the lipo-oligosaccharide nodulation signal of Bradyrhizobium japonicum.
Bradyrhizobium japonicum is a soil bacterium that forms nitrogen-fixing nodules on the roots of the agronomically important legume soybean. Microscopic observation of plant roots showed that butanol extract of B. japonicum strain USDA110 cultures induced for nod gene expression elicited root hair deformation, an early event in the nodulation process. The metabolite produced by B. japonicum responsible for root hair deformation activity was purified. Chemical analysis of the compound revealed it to be a pentasaccharide of N-acetylglucosamine modified by a C18:1 fatty acyl chain at the nonreducing end. In these respects, the B. japonicum metabolite is similar to the lipo-oligosaccharide signals described from Rhizobium species. However, the B. japonicum compound is unique in that an additional sugar, 2-O-methylfucose, is linked to the reducing end. Comparative analysis of the B. japonicum Nod metabolite and those characterized from Rhizobium species suggests that the presence of the fucosyl residue plays an important role in the specificity of the B. japonicum-soybean symbiosis. The availability of the purified B. japonicum nodulation signal should greatly facilitate further studies of soybean nodulation.
Carbohydrate Sequence↗