Hydrogen Evolution from Alfalfa and Clover Nodules and Hydrogen Uptake by Free-Living Rhizobium meliloti

A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H₂ evolution in air, rates of H₂ evolution under Ar and O₂, and rates of C₂H₂ reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H₂. The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H₂ at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H₂ uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H₂ evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H₂-recycling system.     

Chemotaxis of Rhizobium meliloti towards Nodulation Gene-Inducing Compounds from Alfalfa Roots

Luteolin, a flavone present in seed exudates of alfalfa, induces nodulation genes (nod) in Rhizobium meliloti and also serves as a biochemically specific chemoattractant for the bacterium. The present work shows that R. meliloti RCR2011 is capable of very similar chemotactic responses towards 4′,7-dihydroxyflavone, 4′,7-Dihydroxyflavanone, and 4,4′-dihydroxy-2-methoxychalcone, the three principal nod gene inducers secreted by alfalfa roots. Chemotactic responses to the root-secreted nod inducers in capillary assays were usually two- to four-fold above background and, for the flavone and flavonone, occurred at concentrations lower than those required for half-maximal induction of the nodABC genes. Complementation experiments indicated that the lack of chemotactic responsiveness to luteolin seen in nodD1 and nodA mutants of R. meliloti was not due to mutations in the nod genes, as previously thought. Thus, while nod gene induction and flavonoid chemotaxis have the same biochemical specificity, these two functions appear to have independent receptors or transduction pathways. The wild-type strain was found to suffer selective, spontaneous loss of chemotaxis towards flavonoids during laboratory subculture.     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

Alteration of the Trifoliin A-Binding Capsule of Rhizobium trifolii 0403 by Enzymes Released from Clover Roots

The effect of white clover root exudate on capsules of Rhizobium trifolii 0403 was examined. The clover lectin trifoliin A was detected in root exudate of two clover varieties by indirect immunofluorescence with antibody against this lectin purified from clover seed. Trifoliin A bound uniformly to encapsulated, heat-fixed cells during 1 h of incubation with root exudate. After 4 to 8 h of incubation, trifoliin A was only bound to one pole of the cells. Transmission electron microscopy showed that the capsule itself was altered. The disorganization of the acidic polymers of the capsule began in the equatorial center of the rod-shaped cell and then progressed toward the poles at unequal rates. Trifoliin A could no longer be detected on heat-fixed cells after 12 h of incubation with root exudate. However, trifoliin A was detected in situ on one pole of cells grown for 4 days in the clover root environment of Fahraeus slide cultures. Inhibition studies with the hapten 2-deoxy-d-glucose showed that trifoliin A in root exudate had a higher affinity for one of the cell poles. Immunoelectrophoresis was used to monitor the alteration of the extracellular polysaccharides from R. trifolii 0403 by concentrated root exudate. These polysaccharides were converted into products which eventually lost their ability to immunoprecipitate with homologous antibody. This progressive loss of antigenic reactivity proceeded more rapidly with root exudate from seedlings grown under nitrogen-free conditions than with root exudate from plants grown with 15 mM KNO(3). The root exudate, depleted of trifoliin A by immunoaffinity chromatography, was still able to alter the capsule of R. trifolii 0403. Reconstitution experiments showed that the substance(s) in root exudate which induced this alteration of the capsule was of a high molecular weight, heat labile, trypsin sensitive, and antigenically unrelated to trifoliin A. A variety of glycosidase activities were also detected in the fraction depleted of trifoliin A. These results suggest that enzymes in clover root exudate alter the trifoliin A-binding capsule in a way which would favor polar attachment of R. trifolii to clover root hairs.     

Formation of Tumor-Like Structures on Legume Roots by Rhizobium

Tumor-like structures appeared on the roots of Medicago sativa, Alysicarpus vaginalis, and Trifolium pratense inoculated with a non-nodulating strain of Rhizobium trifolii or with irradiated cultures of either of two nodulating Rhizobium strains. The structures were composed of disorganized plant tissues which, on the basis of microscopic examination, were devoid of bacterial cells. Rhizobia which could nodulate legumes of one cross-inoculation group and which were able to induce formation of such tumor-like structures on plants of a second cross-inoculation group were isolated from extracts of these root growths. The apparent tumorogenic activity of some of the rhizobia, but not their nodulating capacity, was lost when the bacteria were transferred in laboratory media.     

Effect of Meloidogyne incognita on Reproduction of Pratylenchus penetrans in Red Clover and Alfalfa

Roots of seedlings of red clover and alfalfa growing on 10⁻¹ Hoagland and Arnon solution agar were inoculated with various combinations of Meloidogyne incognita and Pratylenchus penetrans. Egg-laying by P. penetrans decreased as the number of nematodes, the ratio of entrant M. incognita to entrant P. penetrans, and the priority of invasion of roots by M. incognita increased. Embryogeny and hatching of eggs of P. penetrans, and development of larvae of M. incognita, were not affected. In red clover, the greatest red uction occurred when there were 65 entrant nematodes, the ratio of M. incognita:P. penetrans was 4:1 and M. incognita was inoculated four days prior to P. penetrans. In alfalfa, the less-favorable host for both nematodes, the greatest reduction occurred when there were 45 entrant nematodes, the ratio of M. incognita:P. penetrans was 2:1, and M. incognita was inoculated 4 days prior to P. penetrans.     

Comparison of DNA Polymerase of Rhizobium meliloti and Alfalfa Bacteroids

DNA dependent-DNA polymerase activity was established and partially purified from extracts of cultured Rhizobium meliloti, F-28, and nodule bacteroids (R. meliloti, F-28) of alfalfa plants (Medicago sativa). Polymerase activity in the partially purified fractions showed characteristic dependence on Mg²⁺, DNA, and a full complement of deoxyribonucleoside triphosphates. DNase activity, preference of “activated” double strand DNA, and inhibition by p-chloromercuribenzoate and MnCl₂ were responses common to both systems. The two systems however did exhibit some differences in pH, Mg²⁺, and primer optima. Polymerase activity in crude extracts of the cultured bacteria was more stable and had 10- to 18-fold greater specific activity than the bacteroid extracts. Preliminary measurements of specific DNA polymerase activity in crude extracts of cultured Rhizobium japonicum were not significantly higher than that in the crude extracts of soybean nodule bacteroids. A possible correlation between DNA synthesis and the successful establishment of rhizobia-legume symbiosis is discussed.     

Scanning Electron Microscopy of Rhizobium trifolii Infection Sites on Root Hairs of White Clover

White clover root hairs which were inoculated with Rhizobium trifolii 4S (infectious strain) contained infection threads which were observed by light microscopy and scanning electron microscopy. Three morphological types of root hairs retaining infection threads were recognized. The bacteria were strongly attached between the surfaces of two plant cell walls as follows: between surfaces of a root hair tip curled back on itself, between a protuberance from a root hair and its cell surface, or between two root hair tips clinging together. An anatomical analysis documented the attachment site of the infection thread sheath from the inside of the root hair cell.     

A Predicted Binding Site for Cholesterol on the GABAA Receptor

Modulation of the GABA type A receptor (GABA_AR) function by cholesterol and other steroids is documented at the functional level, yet its structural basis is largely unknown. Current data on structurally related modulators suggest that cholesterol binds to subunit interfaces between transmembrane domains of the GABA_AR. We construct homology models of a human GABA_AR based on the structure of the glutamate-gated chloride channel GluCl of Caenorhabditis elegans. The models show the possibility of previously unreported disulfide bridges linking the M1 and M3 transmembrane helices in the α and γ subunits. We discuss the biological relevance of such disulfide bridges. Using our models, we investigate cholesterol binding to intersubunit cavities of the GABA_AR transmembrane domain. We find that very similar binding modes are predicted independently by three approaches: analogy with ivermectin in the GluCl crystal structure, automated docking by AutoDock, and spontaneous rebinding events in unbiased molecular dynamics simulations. Taken together, the models and atomistic simulations suggest a somewhat flexible binding mode, with several possible orientations. Finally, we explore the possibility that cholesterol promotes pore opening through a wedge mechanism.     

小麦根系接受缺磷信号的部位（简报）

用局部供磷法研究小麦根系接受外界缺磷信号位点的结果表明,体内不缺磷的植株可以接受缺磷磷信号的刺激,根系局部缺磷有利于小麦的生长发育,小麦根的任何部位均可接受环境缺磷信号。     

Attachment of Rhizobium to Legume Roots as the Basis for Specific Interactions

Experiments were conducted to elucidate the basis of the observation that different strains of Rhizobium infect particular legumes. Rhizobia specific for a variety of legumes were grown with ¹³PO^2?₄ and exposed to pea roots (Pisum sativum L.), R. leguminosarum 128C53, which nodulates pea, did not attach to the roots in greater numbers than those strains of rhizobia incapable of infecting pea roots. A complex of R. leguminosarum 128C53 conjugated to a fluorochrome-labeled antibody exhibited a striking attachment to the tips of pea root hairs, where infection normally occurs, but this fluorescent complex also bound to the root hairs of Canavalia en siformis DC., Lupinus polyphyllus Lindl., Trifolium pratense L., and Medicago sativa L., which are not infected by this bacterium. A reproducible, quantitative technique developed for studying interactions between fluorochrome-labeled lectins and rhizobia revealed no relationship between lectin-Rhizobium interactions and the capacity to infect a plant. The data are interpreted as suggesting that simple attachment of Rhizobium to a legume root is not the basis of host-symbiont specificity in this system.     

Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots

Introduction of the pea (Pisum sativum L.) lectin (PSL) gene into white clover (Trifolium repens L.) hairy roots facilitates nodulation by the nitrogen-fixing bacterium Rhizobium leguminosarum biovar viciae, which normally nodulates pea and not white clover (C.L. Diaz, L.S. Melchers, P.J.J. Hooykaas, B.J.J. Lugtenberg, and J.W. Kijne [1989] Nature 338: 579-581). Here, we show that PSL is functionally expressed in transgenic white clover hairy roots transformed with the PSL gene. PSL could be isolated from these roots by affinity chromatography. Immunoanalysis of PSL showed the presence of polypeptides corresponding to the PSL precursor and its [beta] subunits. In addition, we developed a highly sensitive localization technique based on specific binding of a glycan moiety of rat IgE to PSL. Similar to the situation in pea roots, PSL appeared to be localized on the external cell surface of elongated epidermal cells and on the tips of emerging and growing root hairs of transgenic white clover hairy roots. PSL was not observed on normal white clover roots and on hairy roots without the PSL gene. These results show that (a) in transgenic white clover hairy roots, PSL is correctly processed and targeted to root cells susceptible to rhizobial infection, and (b) like in pea roots, PSL is surface bound with at least one of its two sugar-binding sites available for (rhizobial) ligands.     

Association of Rhizobium Strains with Roots of Trifolium repens

TWO TECHNIQUES WERE USED TO ASSESS THE BINDING OF RHIZOBIA TO CLOVER ROOTS: indirect counting after radiolabeling the bacteria and direct counting by using phase-contrast microscopy. Microscopic observations revealed a large variability in the number of bacteria associated with individual root hairs. This variability made unbiased counting by microscopy difficult. Systematic examination of all visible root hairs and "blind" counting of coded strains and treatments were adopted to minimize observer bias. The validity of the radiolabeling method was also examined in some detail. The reproducibility of results from this method was satisfactory. However, drawbacks of this method included its lack of sensitivity and its failure to distinguish between bacteria attached to mature root hairs, emerging root hairs, and undifferentiated epidermal cells. The method also failed to distinguish between individual bacteria and any aggregates that may be present. The ability of a number of chosen mutant strains of Rhizobium trifolii and their corresponding parent strains, as well as a number of nonhomologous strains, to bind to clover roots was assessed by using both of these methods. Our results gave no indication of specificity of R. trifolii binding to clover roots. 2-Deoxy-d-glucose did not appear to have a major inhibitory effect on the attachment of rhizobia to the host root, which suggests that lectin cross-bridging is not an obligatory step in the initiation of infection even though it may occur under some conditions. The presence or absence of the symbiotic plasmid was not correlated with bacterial adherence to the host plant root. Since host specificity functions are carried on this plasmid, our results suggest that binding of rhizobia to the legume root is not the basis of host specificity.     

Growth and Movement of Spot Inoculated Rhizobium meliloti on the Root Surface of Alfalfa

Inoculum droplets of approximately 10 nanoliter volume and containing about 10 Rhizobium meliloti cells were placed onto the root surface of alfalfa seedlings in plastic growth pouches at either the root tip, the position of the smallest emergent root hairs, or at a site midway between these points. The droplets were initially confined to an area of about 0.2 square millimeter at the point of application. By 48 and 96 hours after inoculation, the inoculum bacteria and their progeny were distributed over several centimeters of the root between the initial site of deposition and the growing root tip, reaching densities of 10³ to 10⁴ bacteria per centimeter near the site of initial deposition and decreasing exponentially from that point toward the root tip. Graphite particles deposited on the root surface close to the growing tip were similarly distributed along the root length by 48 and 96 hours, suggesting that passive displacement by root cell elongation was primarily responsible for the spread of bacteria. A nonmotile mutant of R. meliloti colonized alfalfa roots to the same extent as the wild type and was usually distributed in the same manner, indicating that bacterial motility contributed little under these conditions to long distance spread of the bacteria. However, when applied in low numbers, R. meliloti mutants defective in motility or chemotaxis were considerably less efficient in initiating nodules near the point of inoculation than the wild type. This implies that motility and/or chemotaxis contribute significantly to local exploration for suitable infection sites. Almost all nodules on the primary root formed within a few millimeters of the spot-inoculation site, indicating that, under our experimental conditions, movement and multiplication of R. meliloti on the root surface were not sufficient to maintain an adequate population in the infectible region of the root during root growth.