Effectiveness of different pre-treatments in recovering pre-burial isotopic ratios of charred plants.

RATIONALE: Isotopic analysis of archaeological charred plant remains offers useful archaeological information. However, adequate sample pre-treatment protocols may be necessary to provide a contamination-free isotopic signal while limiting sample loss and achieving a high throughput. Under these constraints, research was undertaken to compare the performance of different pre-treatment protocols. METHODS: Charred archaeological plant material was selected for isotopic analysis (δ13 C and δ15 N values) by isotope ratio mass spectrometry from a variety of plant species, time periods and soil conditions. Preservation conditions and the effectiveness of cleaning protocols were assessed through Fourier transform infrared spectroscopy and X-ray fluorescence (XRF) spectrometry. An acid-base-acid protocol, successfully employed in radiocarbon dating, was used to define a contamination-free isotopic reference. Acid-base-acid isotopic measurements were compared with those obtained from untreated material and an acid-only protocol. RESULTS: The isotopic signals of untreated material and the acid-only protocol typically did not differ more than 1‰ from those of the acid-base-acid reference. There were no significant isotopic offsets between acid-base-acid and acid-only or untreated samples. Sample losses in the acid-base-acid protocol were on average 50 ± 17% (maximum = 98.4%). Elemental XRF measurements showed promising results in the detection of more contaminated samples albeit with a high rate of false positives. CONCLUSIONS: For the large range of preservation conditions described in the study, untreated charred plant samples, water cleaned of sediments, provide reliable stable isotope ratios of carbon and nitrogen. The use of pre-treatments may be necessary under different preservation conditions or more conservative measurement uncertainties should be reported.

High NaCl concentrations induce the nod genes of Rhizobium tropici CIAT899 in the absence of flavonoid inducers.

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.

Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs.

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.

A catalogue of molecular, physiological and symbiotic properties of soybean-nodulating rhizobial strains from different soybean cropping areas of China.

We have analysed 198 fast-growing soybean-nodulating rhizobial strains from four different regions of China for the following characteristics: generation time; number of plasmids; lipopolysaccharide (LPS), nodulation factors (LCOs) and PCR profiles; acidification of growth medium; capacity to grow at acid, neutral, and alkaline pH; growth on LC medium; growth at 28 and 37 degrees C; melanin production capacity; Congo red absorption and symbiotic characteristics. These unbiased analyses of a total subset of strains isolated from specific soybean-cropping areas (an approach which could be called "strainomics") can be used to answer various biological questions. We illustrate this by a comparison of the molecular characteristics of five strains with interesting symbiotic properties. From this comparison we conclude, for instance, that differences in the efficiency of nitrogen fixation or competitiveness for nodulation of these strains are not apparently related to differences in Nod factor structure.

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations.

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.

Rhizobium leguminosarum bv. trifolii produces lipo-chitin oligosaccharides with nodE-dependent highly unsaturated fatty acyl moieties. An electrospray ionization and collision-induced dissociation tandem mass spectrometric study.

The lipo-chitin oligosaccharides (LCO) or nodulation factors synthesized by Rhizobium leguminosarum bv. trifolii were analyzed using positive mode fast atom bombardment and positive and negative mode electrospray ionization mass spectrometry. From their mass spectrometric behavior it is clearly possible to distinguish between the [M + Na]+ pseudomolecular ion of the nodE-independent molecule IV(C18:1,Ac) and the [M + H]+ pseudomolecular ion of the nodE-dependent molecule IV(C20:4,Ac), although they both have the same mass value. The results unequivocally show that the bacterial strain investigated produces nodE-dependent LCOs with highly unsaturated fatty acyl moieties. We further demonstrate that the interpretation of the mass spectrometric data by Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Orgambide, G. G., Bradford, J. J., Smith, D. K., Hollingsworth, R. I., and Dazzo, F. B. (1995) J. Biol. Chem. 270, 20968) is incorrect and that their data do not contradict our hypothesis that the nodE gene determines the host specificity of R. leguminosarum bv. trifolii.

Bacteriocin small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors.

Small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants. The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone. The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(+)- and R-(-)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions. The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R. leguminosarum.

Nod factors produced by Rhizobium leguminosarum biovar viciae induce ethylene-related changes in root cortical cells of Vicia sativa ssp. nigra.

Vicia sativa ssp. nigra plants develop the "Thick short root" (Tsr) phenotype when both (i) the roots are inoculated with the root nodule inducing bacterium Rhizobium leguminosarum biovar viciae, and (ii) the plants, including the roots, are grown in the light. Tsr roots have a reduced length, are locally twice as thick as normal roots and have an increased number of root hairs. Development of the Tsr phenotype is correlated with the presence of nod (nodulation) genes in the rhizobia. Nod factors (lipochitin oligosaccharides), products of these nod genes, can induce the Tsr phenotype in the absence of rhizobia. The Tsr phenotype can be mimicked by addition of the ethylene-releasing compound ethephon. Using several microscopical techniques, we compared roots showing the Tsr phenotype (Tsr roots) with normal roots and roots grown in the presence of the ethylene inhibitor aminoethoxyvinylglycine (AVG). The thickening of Tsr roots appeared to be caused by a swelling of the cortical cells, which corresponded with (i) a reorientation of the interphase cortical microtubules from a transverse to a longitudinal direction, (ii) general cell wall modifications, (iii) frequent absence of middle lamellae, and (iv) local maceration. The same changes could be induced by ethephon and were inhibited by AVG. This strongly suggests that the Tsr phenotype is caused by excessive ethylene production. The ethylene-related changes mentioned above are also seen during infection thread formation, but only very locally. Apparently, Vicia roots when grown in the light overrespond to Nod factors leading to overproduction of ethylene and to a non-local "ripening" process. These phenomena inhibit nodulation of the main root by preventing formation of pre-infected threads and by reducing formation of root nodule primordia. Local controlled production of ethylene, as induced by Nod factors, may, however, be an essential element of the nodulation process.

Uridine, a cell division factor in pea roots.

Nodulation (root nodule formation) in legume roots is initiated by the induction of cell divisions and formation of root nodule primordia in the plant root cortex, usually in front of the protoxylem ridges of the central root cylinder. We isolated a factor from the central cylinder (stele) of pea roots which enhances hormone-induced cell proliferation in root cortex explants at positions similar to those of nodule primordia. The factor was identified as uridine. Uridine may act as a morphogen in plant roots at picomolar concentrations.

Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2.

Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species. Here we show that strains of Rhizobium sp. GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs). Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine. The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone. The majority of the LCOs have an oligosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units. LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A. melanoxylon plants. We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A. cyanophylla, and A. melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.

Cell wall degradation during infection thread formation by the root nodule bacterium Rhizobium leguminosarum is a two-step process.

In the nitrogen-fixing root-nodule symbiosis of Rhizobium leguminosarum biovar viciae and its host plants pea and vetch, the bacteria enter one root cortical cell after another via a tip-growing structure, the infection thread. Rhizobial Nod (nodulation) factors induce the formation of preinfection thread structures (Van Brussel, A.A.N., R. Bakhuizen, P.C. van Spronsen, H.P. Spaink, T. Tak, B.J.J. Lugtenberg, J.W. Kijne, Science 257, 70-72 (1992)), but formation of infection threads requires the presence of bacterial cells. Passing of an infection thread from cell to cell requires local cell wall degradation. We compared at the ultrastructural level local cell wall changes in the outer root cortex of pea and vetch related to preinfection thread formation and infection thread formation, respectively. Cell wall modifications in the outer periclinal walls of root cortical cells induced by Nod factors appeared to be similar to those induced by rhizobia. These modifications take place opposite cytoplasmic bridges and are probably related to induction of tip growth. However, complete cell wall degradation was never observed in the absence of rhizobia. We propose a two-step cell wall degradation process for infection thread formation. The first step is a local cell wall modification by plant enzymes, induced by rhizobial Nod factors. The second step is complete cell wall degradation in the presence of rhizobia.

The structures and biological activities of the lipo-oligosaccharide nodulation signals produced by type I and II strains of Bradyrhizobium japonicum.

Bradyrhizobium japonicum produces lipo-oligosaccharide signal molecules that induce deformation of root hairs and meristematic activity on soybeans. B. japonicum USDA135 (a Type I strain) produces modified chitin pentasaccharide molecules with either a terminal N-C16:0- or N-C18:1-glucosamine with and without an O-acetyl group at C-6 and with 2-O-methylfucose linked to C-6 of the reducing N-acetylglucosamine. An additional molecule has N-C16:1-glucosamine and no O-acetyl group. All of these molecules cause root hair deformation on Vicia sativa and Glycine soja. The C18:1-containing molecules were tested and found to induce meristem formation on G. soja. USDA61 (a Type II strain) produces eight additional molecules. Five have a carbamoyl group on the terminal N-acylglucosamine. Six have chitin tetrasaccharide backbones. Three have a terminal N-acyl-N-methylglucosaminosyl residue. In four molecules, the reducing-end N-acetylglucosamine is glycosidically linked to glycerol and has a branching fucosyl, rather than a 2-O-methylfucosyl, residue. One molecule has a terminal N-acylglucosamine that has both acetyl and carbamoyl groups (one each).

Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium.

Root nodules of leguminous plants are symbiotic organs in which Rhizobium bacteria fix nitrogen. Their formation requires the induction of a nodule meristem and the formation of a tubular structure, the infection thread, through which the rhizobia reach the nodule primordium. In the Rhizobium host plants pea and vetch, pre-infection thread structures always preceded the formation of infection threads. These structures consisted of cytoplasmic bridges traversing the central vacuole of outer cortical root cells, aligned in radial rows. In vetch, the site of the infection thread was determined by the plant rather than by the invading rhizobia. Like nodule primordia, pre-infection thread structures could be induced in the absence of rhizobia provided that mitogenic lipo-oligosaccharides produced by Rhizobium leguminosarum biovar viciae were added to the plant. In this case, cells in the two outer cortical cell layers containing cytoplasmic bridges may have formed root hairs. A common morphogenetic pathway may be shared in the formation of root hairs and infection threads.

Activation of flavonoid biosynthesis in roots of Vicia sativa subsp. nigra plants by inoculation with Rhizobium leguminosarum biovar viciae.

Infective (nodulating) Rhizobium leguminosarum biovar viciae (R.l. viciae) bacteria release Nod factors which stimulate the release of nodulation gene-inducing flavanones and chalcones from roots of the host plant Vicia sativa subsp. nigra (K. Recourt et al., Plant Mol Biol 16: 841-852; H.P. Spaink et al., Nature 354: 125-130). The hypothesis that this release results from increased synthesis of flavonoids was tested by studying the effect of inoculation of V. sativa with infective and uninfective R.l. viciae bacteria on (i) activity of L-phenylalanine ammonia-lyase, (ii) level of chalcone synthase mRNA, and (iii) activity of (eriodictyol) methyltransferase in roots. Consistent with the hypothesis, each of these parameters was found to increase 1.5 to 2-fold upon inoculation with infective R.l. viciae bacteria relative to the situation for uninoculated roots and for roots inoculated with uninfective rhizobia.

Major flavonoids in uninoculated and inoculated roots of Vicia sativa subsp. nigra are four conjugates of the nodulation gene-inhibitor kaempferol.

Inoculation of Vicia sativa subsp. nigra (V. sativa) roots with Rhizobium leguminosarum biovar. viciae (R.l. viciae) bacteria substantially increases the ability of V. sativa to induce rhizobial nodulation (nod) genes. This increase is caused by the additional release of flavanones and chalcones which all induce the nod genes of R.l. viciae (K. Recourt et al., Plant Mol Biol 16: 841-852). In this paper, we describe the analyses of the flavonoids present in roots of V. sativa. Independent of inoculation with R.l. viciae, these roots contain four 3-O-glycosides of the flavonol kaempferol. These flavonoids appeared not capable of inducing the nod genes of R.l. viciae but instead are moderately active in inhibiting the activated state of those nod genes. Roots of 7-day-old V. sativa seedlings did not show any kaempferol-glycosidase activity consistent with the observation that kaempferol is not released upon inoculation with R.l. viciae. It is therefore most likely that inoculation with infective (nodulating) R.l. viciae bacteria results in de novo flavonoid biosynthesis and not in liberation of flavonoids from a pre-existing pool.

A novel highly unsaturated fatty acid moiety of lipo-oligosaccharide signals determines host specificity of Rhizobium.

In Rhizobium leguminosarum biovar viciae, the nodABC and nodFEL operons are involved in the production of lipo-oligosaccharide signals which mediate host specificity. The structure of these metabolites and those produced in nod mutants links the nodE and nodL genes to specific chemical features of the signal molecules. A nodE-determined, highly unsaturated fatty acid and a nodL-determined O-acetyl substituent are essential for the ability of the signals to induce nodule meristems on the host plant Vicia sativa.

Inoculation of Vicia sativa subsp. nigra roots with Rhizobium leguminosarum biovar viciae results in release of nod gene activating flavanones and chalcones.

Flavonoids released by roots of Vicia sativa subsp. nigra (V. sativa) activate nodulation genes of the homologous bacterium Rhizobium leguminosarum biovar viciae (R. l. viciae). Inoculation of V. sativa roots with infective R. l. viciae bacteria largely increases the nod gene-inducing ability of V. sativa root exudate (A.A.N. van Brussel et al., J Bact 172: 5394-5401). The present study showed that, in contrast to sterile roots and roots inoculated with R. l. viciae cured of its Sym plasmid, roots inoculated with R. l. viciae harboring its Sym plasmid released additional nod gene-inducing flavonoids. Using 1H-NMR, the structures of the major inducers released by inoculated roots, 6 flavanones and 2 chalcones, were elucidated. Roots extracts of (un)inoculated V. sativa contain 4 major non-inducing, most likely glycosylated, flavonoids. Therefore, the released flavonoids may either derive from the root flavonoids or inoculation with R. l. viciae activates de novo flavonoid biosynthesis.

A biovar-specific signal of Rhizobium leguminosarum bv. viciae induces increased nodulation gene-inducing activity in root exudate of Vicia sativa subsp. nigra.

Flavonoids in root exudate of leguminous plants activate the transcription of Rhizobium genes involved in the formation of root nodules (nod genes). We report that inoculation with the homologous symbiont R. leguminosarum bv. viciae results in an increased nod gene-inducing activity (Ini) in root exudate of V. sativa subsp. nigra, whereas inoculation with heterologous Rhizobium strains results in exudates with nod gene-inducing activity comparable to that of uninfected plants. Ini can be demonstrated by using either of the isogenic indicator strains containing an inducible nod promoter fused to the Escherichia coli lacZ reporter gene and the regulatory nodD gene of R. leguminosarum bv. viciae, R. leguminosarum bv. trifolii, or R. meliloti. The presence of genes nodDABCEL of R. leguminosarum bv. viciae appeared to be essential for induction of Ini. Mutation of the genes nodI and nodJ causes a delay of Ini, whereas gene nodF appears to be required for both the timely appearance and the maximum level of Ini activity. The nodE gene is responsible for the biovar specificity of induction of Ini by Rhizobium spp. Ini is caused by a soluble heat-stable factor of rhizobial origin. This Rhizobium-produced Ini factor has an apparent molecular weight between 1,000 and 10,000 and does not originate from flavonoid precursors.

Analysis of the major inducers of the Rhizobium nodA promoter from Vicia sativa root exudate and their activity with different nodD genes.

Root exudate of Vicia sativa contains 7 inducers for the nodA promoter of Rhizobium leguminosarum biovar viciae. Six of these inducers are flavanones. One inducer was identified as 3,5,7,3'-tetrahydroxy-4'-methoxyflavanone, and a second inducer most likely is 7,3'-dihydroxy-4'-methoxyflavanone. The inducing activity of these compounds and the other inducers depends on the nodD gene present in the test strains, which originated either from R. leguminosarum biovars viciae or trifolii, or from R. meliloti. Three inducers are 'common', three others almost exclusively induce the nodA promoter in the presence of the R. leguminosarum biovar viciae nodD gene, and the last one is active with the noD genes of either R. leguminosarum biovar viciae or that of R. meliloti. Testing of a large number of flavonoids revealed two classes of structural features required for inducing ability: (i) features required for induction in general, and (ii), features restricting the inducing ability to (a) specific nodD gene(s). These features are discussed in relation to current models of the process of nodD-mediated transcription activation of the inducible nod genes.

Accumulation of a nod gene inducer, the flavonoid naringenin, in the cytoplasmic membrane of Rhizobium leguminosarum biovar viciae is caused by the pH-dependent hydrophobicity of naringenin.

Most Sym plasmid-localized nodulation genes of Rhizobium leguminosarum bv. viciae are only expressed upon activation of the NodD protein by plant flavonoids, e.g., naringenin (S. A. J. Zaat, C. A. Wijffelman, H. P. Spaink, A. A. N. van Brussel, and B. J. J. Lugtenberg, J. Bacteriol, 169:198-204, 1987). As part of a study on the mechanism of NodD protein activation, the mechanism of uptake and the intracellular fate of [3H]naringenin were studied. Naringenin was accumulated by Rhizobium cells without apparent metabolic conversion to an 80-fold-higher concentration in a process which did not require any of the other Sym plasmid-localized nod genes. Naringenin accumulation was nonsaturable, highly reversible, and not inhibited by the presence of other flavonoids or the metabolic inhibitors potassium cyanide, sodium azide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenylhydrazone. These data indicate an accumulation mechanism without high affinity sites which does not use cellular energy. In vitro, naringenin has high affinity for the cytoplasmic membrane. This binding was pH dependent, very high at pH 5.7 and not present anymore at pH 9.7. A similar pH dependency was found for the affinity of naringenin for the olive oil fraction of a biphasic olive oil-water system. pH-dependent changes in the UV spectrum indicate ionization of naringenin at high pH to a negatively charged form. Since it has recently been shown that the nodD gene product is located in the cytoplasmic membrane (H. R. M. Schlaman, H. P. Spaink, R. J. H. Okker, and B. J. J. Lugtenberg, J. Bacteriol., in press), our data are consistent with a model in which the un-ionized form of naringenin accumulates in the cytoplasmic membrane and activates, in a metabolically unaltered form, the NodD protein.