The infectible cells of soybean roots appear to be located at any given time just above the zone of root elongation and just below the position of the smallest emergent root hairs. The location of infectible cells on the primary root at the time of inoculation was inferred from the position of subsequent nodule development, correcting for displacement of epidermal cells due to root elongation. Marks were made on the seedling growth pouches at the time of inoculation to indicate the position of the root tip and the zones of root hair development. Virtually all of the seedlings developed nodules on the primary root above the marks made at the root tips at the time of inoculation. None of the plants formed nodules on the root where mature root hairs were present at the time of inoculation. These results and profiles of nodulation frequency indicate that the location of infectible cells is developmentally restricted. When inoculations were delayed for intervals of I to 4 hours after marking the positions of the root tips. progressively fewer nodules were formed above the root tip marks, and the uppermost of these nodules were formed at progressively shorter distances above the marks. These results indicate that the infectibility of given host cells is a transient property that appears and then is lost within a few hours. The results also indicate that host responses leading to infection and nodulation are triggered or initiated in less than 2 hours after inoculation. The extent of nodulation above the root tip mark increased in proportion to the logarithm of the number of bacteria in the inoculum.The mechanisms by which soybean plants interact with Rhizobiumjaponicum to establish a symbiotic association have not been studied extensively (3). Bieberdorf (2) (9) modified to contain 0.5% mannitol, 0.5% sodium gluconate and 0.5% yeast extract. Every 2 to 3 weeks, single cell colonies from the stock cultures were used to inoculate 10 ml modified B5 medium (1), adjusted to pH 6.8 to 7.0, in 25-ml culture flasks. These starter cultures were maintained on a rotary shaker (100 rpm) at 25 C for 7 days (early stationary growth phase). The A620 nm of the starter culture was determined and a volume of the culture equivalent to 0.5 ml of an 1.0 A620 nm suspension was added to 50 ml fresh medium in a 125-ml culture flask. The remaining starter culture was stored in the refrigerator for use the following week. Inoculum cultures were grown for 3 days in the same manner as the starter cultures, harvested by centrifugation, washed once with sterile phosphate-buffered saline (pH 7.2) (1), and resuspended in onetenth strength phosphate-buffered saline. Suspensions used for inoculation were diluted to 0.08 A620 nm (I x 108 cells/ml) with sterile, half-strength N-free Jensen's plant growth medium (9) prior to use. Growth of Seedlings. Soybean seeds of cultivars Beeson and Williams were obtained from DeWine and Hamma Seed Co., Yellow Springs, OH. Harris seeds were a gift from Dr. K. Nadler, Michigan State University. Seeds (cv. Wil...
Rhizobium nod genes are essential for root hair deformation and cortical cell division, early stages in the development ofnitrogen-fixing root nodules. Nod-mutants are unable to initiate nodules on legume roots. We observed that N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid, compounds known to function as auxin transport inhibitors, induced nodule-like structures on alfalfa roots. The nodule-like structures (pseudonodules) were white, devoid of bacteria, and resembled nodules elicited by Rhizobium meliloti exopolysaccharide (exo) mutants at both the histological and molecular level. Two nodulin genes, ENOD2 and Nms-30, were expressed. RNA isolated from the nodule-like structures hybridized to pGmENOD2, a soybean early nodulin cDNA clone. RNA isolated from roots did not hybridize. We determined by in vitro translations of total RNA that the alfalfa nodulin transcript Nms-30 was also expressed in the nodule-like structures. The late expressed nodulin genes, such as the leghemoglobin genes, were not transcribed. Because N-(1-naphthyl)-phthalamic acid and 2,3,5-triiodobenzoic acid induce the development of nodules on alfalfa roots, we suggest that the auxin transport inhibitors nmimic the activity of compound(s) made upon the induction of the Rhizobium nod genes.The nodulation (nod) genes of Rhizobium play an essential role in the induction of nodules on the roots of leguminous plants. The (17,18). However, the internal structure of these cytokinin-induced pseudonodules differed from that of Rhizobium-induced nodules. Substituted benzoic acids, chemicals that presumably modify auxin levels in the plant (19,20), also induce nodule-like structures on legume roots (refs. 21 and 22; J.G.T., unpublished results).We found that the histology of the nodule-like structures induced by the auxin transport inhibitors N-(1-naphthyl)-phthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA)closely resembled that ofRhizobium-induced legume nodules.We studied these nodule-like outgrowths to determine their relation to root nodules initiated by Rhizobium. In
Plants naturally cycle amino acids across root cell plasma membranes, and any net efflux is termed exudation. The dominant ecological view is that microorganisms and roots passively compete for amino acids in the soil solution, yet the innate capacity of roots to recover amino acids present in ecologically relevant concentrations is unknown. We find that, in the absence of culturable microorganisms, the influx rates of 16 amino acids (each supplied at 2.5 μ m) exceed efflux rates by 5% to 545% in roots of alfalfa (Medicago sativa), Medicago truncatula, maize (Zea mays), and wheat (Triticum aestivum). Several microbial products, which are produced by common soil microorganisms such as Pseudomonas bacteria and Fusarium fungi, significantly enhanced the net efflux (i.e. exudation) of amino acids from roots of these four plant species. In alfalfa, treating roots with 200 μ m phenazine, 2,4-diacetylphloroglucinol, or zearalenone increased total net efflux of 16 amino acids 200% to 2,600% in 3 h. Data from 15N tests suggest that 2,4-diacetylphloroglucinol blocks amino acid uptake, whereas zearalenone enhances efflux. Thus, amino acid exudation under normal conditions is a phenomenon that probably reflects both active manipulation and passive uptake by microorganisms, as well as diffusion and adsorption to soil, all of which help overcome the innate capacity of plant roots to reabsorb amino acids. The importance of identifying potential enhancers of root exudation lies in understanding that such compounds may represent regulatory linkages between the larger soil food web and the internal carbon metabolism of the plant.
ABSTRACT'To whom reprint requests should be addressed.Commonwealth Scientific and Industrial Research Organization, Australia.General procedures for maintenance of stock cultures, initiation of starter cultures, and preparation of inocula have been described (3). R meliloti strain 102F71 and R. trifolii strain TA1 were grown on modified B5 medium supplemented with 0.5 ,ug of biotin/L (3). All cultures were harvested in mid-exponential growth phase for preparation of inocula (3.5 days for Rhizobium spp. 32H1 and 3G4b4 and 36 h for R. meliloti 102F71 and R. trifolii TAI).Growth of Plants. Cowpea seeds (Vigna sinensis [L.] Endl. cv. California Black Eye) were obtained from W. Atlee Burpee Co., Warminster, PA. Seeds were surface-disinfected and grown in plastic growth pouches (Scientific Products, Evanston, IL) as described earlier for soybean (3). Plants were inoculated by adding 0.5 ml of inoculum (A620 nm = 0.08) in drops onto the lower portion of each root.Seeds of alfalfa (Medicago sativa L. cvs. Moapa and Vernal) were obtained from Dewine and Hamma Seed Company, Yellow Springs, OH. Seeds were surface-sterilized as described (3) and soaked for 2 h in sterile H20. Ten to 12 swollen seeds were transferred directly to each growth pouch. Plants were inoculated 48 h after transfer by adding 100 ,ul of inoculum (A620nm = 0.08) onto each root.Changes in the position ofepidermal cells on cowpea and alfalfa roots, relative to the RT4 and SERH marks (Fig. 1) made on the pouches, were measured as described for soybean (3).Seeds ofpeanut (Arachis hypogaea L. cv. Spanish) were obtained from W. Atlee Burpee Co., surface-sterilized for 5 min, and germinated as described (3). Healthy seedlings were transferred to growth pouches on the fourth day after transfer to plates and inoculated with 0.5 ml of inoculum (A620 = 0.08) when they possessed a few emergent lateral roots. Due to the large seed size and slow rate of growth, peanut plants were not well suited for growing in pouches.White clover seeds (Trifolium repens L. cv. Regal Ladino) were obtained from Cal-West Seed Company, Woodland, CA. Seeds were surface-sterilized for 10 min with 5% sodium hypochlorite containing a drop of Tween 20 (Sigma Chemical Co.), washed, and then soaked in sterile H20 for 24 h in the cold (7). Swollen seeds were transferred to plates containing sterile half-strength Nfree Jensen's medium (10) with 0.5 g agar/L. Germinated seeds were transferred 24 h later to growth pouches wetted with 8 ml of the N-free Jensen's medium. Plants were inoculated 48 h after transfer to pouches with 100 id inoculum (A620nm = 0.08).In some cases, clover seedlings were grown on agar plates. The seeds were surface-sterilized, soaked in cold water, and germinated on Petri plates containing Fahraeus soft agar (10), 0.5 g agar/L.
Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium (3H-SBL) and repurified by affinity chromatography. FITC-SBL was found to bind to living ceUls of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with Dgalactose or N-acetyl-D-galactosamine.The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of ceUs in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The Intimate and specific symbiotic associations between leguminous plants and bacteria of the genus Rhizobium provide most of the biologically fixed nitrogen available for agriculture. The rhizobia enter root hairs of the host plant in a structure called the infection thread (4). The infection thread, which is believed to be a tubular, inward growing invagination of the host cell wall, carries the bacterial symbiont into the cortex of the root (11). There the bacteria enter the cytoplasm of host cell, surrounded by an envelope of host cell plasma membrane (12).Both the bacteria and cortical cells of the host proliferate to form a root nodule where nitrogen fixation takes place.The specificity of the Rhizobium-legume symbiosis is manifested by the failure of soil microorganisms other than rhizobia to gain effective entry into the plant by induction of infection thread structures, and also by host range specificity between members of the genus Rhizobium and the family Leguminosae. Dazzo and Hubbell (6), in a study of the symbiotic association between Rhizobium trifolii and white clover, have found that clover roots and R. trifolii possess a common antigen. This common antigen was shown to be present on the cell surfaces of infective strains of R. trifolii, but absent, inaccessible, or present in reduced quantities on noninfective strains. These authors isolated capsular polysaccharide material from infective R. trifolii which possessed cross-reactive antigenicity. They also provided evidence for the presence of a lectin in clover seed extracts capable of binding to isolated capsular antigen and to infective-but not the noninfective-strains of rhizobia. These results led Dazzo and Hubbell (6) to propose that the clover lectin provides a bridge between common antigen structures for the preferential adsorption of infective strains of R. trifolii to the root surface of the host.Wolpert and Albersheim (20) isolated lectins from four different legumes and obtained lipopolysaccharide preparations from four strains of Rhizobium, each capable of nodulating one of the legume species. The authors reported that the rhizobial lipopolysaccharides interacted with the host lectins, but not with the lectins ...
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