Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules.

Nitrogen-fixing bacteroids are degraded during nodule senescence. This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules. Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp. population in the soil. Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.     

The initiation,development and structure of root nodules inElaeagnus angustifolia L. (Elaeagnaceae)

Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species,Elaeagnus angustifolia L. (Elaeagnaceae).Two pure culturedFrankia strains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such asAlnus orMyrica the mode of infection ofElaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomyceteFrankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizingFrankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizingFrankia. No random division of root cortical cells, indicative of prenodule formation was observed inElaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancingFrankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.     

Effects of Boron on Rhizobium-Legume Cell-Surface Interactions and Nodule Development

Boron (B) is an essential micronutrient for the development of nitrogen-fixing root nodules in pea (Pisum sativum). By using monoclonal antibodies that recognize specific glycoconjugate components implicated in legume root-nodule development, we investigated the effects of low B on the formation of infection threads and the colonization of pea nodules by Rhizobium leguminosarum bv viciae. In B-deficient nodules the proportion of infected host cells was much lower than in nodules from plants supplied with normal quantities of B. Moreover, the host cells often developed enlarged and abnormally shaped infection threads that frequently burst, releasing bacteria into damaged host cells. There was also an over-production of plant matrix material in which the rhizobial cells were embedded during their progression through the infection thread. Furthermore, in a series of in vitro binding studies, we demonstrated that the presence of B can change the affinity with which the bacterial cell surface interacts with the peribacteroid membrane glycocalyx relative to its interaction with intercellular plant matrix glycoprotein. From these observations we suggest that B plays an important role in mediating cell-surface interactions that lead to endocytosis of rhizobia by host cells and hence to the correct establishment of the symbiosis between pea and Rhizobium.     

Development of Stem Nodules in a Tropical Forage Legume, Aeschynomene afraspera

Rhizobial infection occurred on the stem of Aeschynomene afrasperaat the site of emergence of adventitious root primordia. Rhizobiainvaded cortical cells at the base of the root primordium. Thefirst infected cell enlarged and collapsed after rhizobia hadmultiplied in large numbers. At this time, a meristematic zonewas initiated some distance beneath the first infected cell.Rhizobial penetration into the deeper cortex was by progressivecollapse of infected cells towards the meristematic zone; rhizobiaentering the cortical cells by invagination of the host cellwall. At the entry point, rhizobia were embedded in digitatecell wall material. These infection structures were restricted,always originating from the cell wall of an adjacent infectedcell. Soon after infection, the cell collapsed progressivelyforming infection strand-like structures which developed upto the meristematic zone. When infection had reached the meristematiczone, invaded host cells ceased to collapse but divided repeatedlyto form the nodule. Key words: Aeschynomene afraspera, stem nodulation     

Rhizobia can induce nodules in white clover by "hijacking" mature cortical cells activated during lateral root development

We examined a range of responses of root cortical cells to Rhizobium sp. inoculation to investigate why rhizobia preferentially nodulate legume roots in the zone of emerging root hairs, but generally fail to nodulate the mature root. We tested whether the inability to form nodules in the mature root is due to a lack of plant flavonoids to induce the bacterial genes required for nodulation or a failure of mature cortical cells to respond to Rhizobium spp. When rhizobia were inoculated in the zone of emerging root hairs, changes in beta-glucuronidase (GUS) expression from an auxin-responsive promoter (GH3), expression from three chalcone synthase promoters, and the accumulation of specific flavonoid compounds occurred in cortical cells prior to nodule formation. Rhizobia failed to induce these responses when inoculated in the mature root, even when co-inoculated with nod gene-inducing flavonoids. However, mature root hairs remained responsive to rhizobia and could support infection thread formation. This suggests that a deficiency in signal transduction is the reason for nodulation failure in the mature root. However, nodules could be initiated in the mature root at sites of lateral root emergence. A comparison between lateral root and nodule formation showed that similar patterns of GH3:gusA expression, chalcone synthase gene expression, and accumulation of a particular flavonoid compound occurred in the cortical cells involved in both processes. The results suggest that rhizobia can "hijack" cortical cells next to lateral root emergence sites because some of the early responses required for nodule formation have already been activated by the plant in those cells.     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

In-situ localization of chalcone synthase mRNA in pea root nodule development

In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it is hypothesized that the Rhizobium Nod factor induces cell division in the root cortex by stimulating the production of flavonoids that function as auxin transport inhibitors. In nodules CHS mRNA is predominantly present in a region at the apex of the nodule consisting of meristematic and cortical cells. These cells are not infected by Rhizobium. Therefore it is postulated that CHS plays a role in nodule development rather than in a defence response. In roots CHS mRNA is located at a similar position as in nodules, suggesting that CHS has the same function in both root and nodule development. When nodules are formed by mutants of Rhizobium leguminosarum bv. viciae that are unable to secrete β(1-2) glucan and to synthesize the O-antigen containing LPS I, CHS genes are also expressed in regions of the nodule that are infected by Rhizobium. It is postulated that the impaired development of nodules formed by these mutants is due to an induction of a plant defence response.     

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.     

Stem Nodulation in Legumes: Diversity,Mechanisms, and Unusual Characteristics

Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A few aquatic legumes, however, can form nodules on their stem at dormant root primordia. The stem-nodulating legumes described so far are all members of the genera Aeschynomene, Sesbania, Neptunia, and Discolobium. Their rhizobial symbionts belong to four genera already described: Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. This review summarizes our current knowledge on most aspects of stem nodulation in legumes, the infection process and nodule development, the characterization and unusual features of the associated bacteria, and the molecular genetics of nodulation. Potential use as green manure in lowland rice of these stem-nodulating legumes, giving them agronomical importance, is also discussed.     

Root-to-shoot primordium conversion on Sesbania rostrata Brem. stem explants

The morphogenetic responses of cultured stem explants of Sesbaniarostrata Brem. from various positions along the stem axis wereanalysed after treatment with four growth regulators (BAP, NAA,kinetin, and GAJ. Internodal explants formed adventitious shootbuds when cultured on a Murashige and Skoog basal medium withoutadded growth regulators. Histological studies of regenerated shoot buds revealed thatapproximately 30% of the buds resulted from the conversion ofa preformed root primordium (characteristic of this species)into a shoot bud without a callogenesis phase. Each bud whichoriginated from a single root primordium grew into a leafy shoot.Preformed root primordia of stem explants of Sesbania rostratamay constitute an excellent model for physiological researchon plant differentiation. Key words: Organogenesis, adventitious bud, preformed root primordium, conversion, Sesbania rostrata     

Rhizobial communication with rice roots: Induction of phenotypic changes,mode of invasion and extent of colonization

Legume-rhizobial interactions culminate in the formation of structures known as nodules. In this specialized niche, rhizobia are insulated from microbial competition and fix nitrogen which becomes directly available to the legume plant. It has been a long-standing goal in the field of biological nitrogen fixation to extend the nitrogen-fixing symbiosis to non-nodulated cereal plants, such as rice. To achieve this goal, extensive knowledge of the legume-rhizobia symbioses should help in formulating strategies for developing potential rice-rhizobia symbioses or endophytic interactions. As a first step to assess opportunities for developing a rice-rhizobia symbiosis, we evaluated certain aspects of rice-rhizobia associations to determine the extent of predisposition of rice roots for forming an intimate association with rhizobia. Our studies indicate that: a. Rice root exudates do not activate the expression of nodulation genes such as nodY of Bradyrhizobium japonicum USDA110, nodA of R. leguminosarum bv. trifolii, or nodSU of Rhizobium. sp. NGR234; b. Neither viable wild-type rhizobia, nor purified chitolipooligosaccharide (CLOS) Nod factors elicit root hair deformation or true nodule formation in rice; c. Rhizobia-produced indole-3-acetic acid, but neither trans-zeatin nor CLOS Nod factors, seem to promote the formation of thick, short lateral roots in rice; d. Rhizobia develop neither the symbiont-specific pattern of root hair attachment nor extensive cellulose microfibril production on the rice root epidermis; e. A primary mode of rhizobial invasion of rice roots is through cracks in the epidermis and fissures created during emergence of lateral roots; f. This infection process is nod-gene independent, nonspecific, and does not involve the formation of infection threads; g. Endophytic colonization observed so far is restricted to intercellular spaces or within host cells undergoing lysis. h. The cortical sclerenchymatous layer containing tightly packed, thick walled fibers appears to be a significant barrier that restricts rhizobial invasion into deeper layers of the root cortex. Therefore, we conclude that the molecular and cell biology of the Rhizobium-rice association differs in many respects from the biology underlying the development of root nodules in the Rhizobium-legume symbiosis.     

根瘤菌多相分类的研究进展

根瘤菌可以入侵豆科植物形成根瘤或茎瘤,并可固定空气中的氮为其宿主植物提供必需的氮素营养。根瘤菌的分类系统可分为早期根瘤菌的分类系统和现代根瘤菌的分类系统两个阶段。根瘤菌的多相分类技术包括表型的分群方法和遗传型的分群方法两大类。     

根瘤菌结瘤因子的结构和功能

结瘤因子是根瘤菌分泌的寡糖,它作为外在信号,诱发宿主植物根部各种生理反应。引起根毛变形,诱导皮层细胞分裂,形成根瘤原基,作者主要就这一早期结瘤过程中结瘤因子的结构和功能作一综述。     

Regulation of nitrogen fixation by Rhizobia. Export of fixed N2 as NH+4.

The metabolic fate of gaseous nitrogen (15N2) fixed by free-living cultures of Rhizobia (root nodule bacteria) induced for their N2-fixation system was followed. A majority of the fixed 15N2 was found to be exported into the cell supernatant. For example, as much as 94% of the 15N2 fixed by Rhizobium japonicum (soybean symbiont) was recovered as 15NH+4 from the cell supernatant following alkaline diffusion. Several species of root nodule bacteria also exported large quantities of NH+4 from L-histidine. Evidence is presented that overproduction and export of NH+4 by free-living Rhizobia may be closely linked to the control of several key enzymes of NH+4 assimilation. For instance, NH+4 was found to repress glutamine synthetase whereas L-glutamate repressed glutamate synthase. Assimilation of NH+4 as nitrogen source for growth of Rhizobia was inhibited by glutamate. The mechanism of regulation of NH+4 production by root nodule bacteria is discussed.     

Early nodulin gene expression during Nod factor-induced processes in Vicia sativa

Rhizobium leguminosarum bv. viciae -secreted Nod factors are able to induce root hair deformation, the formation of nodule primordia and the expression of early nodulin genes in Vicia sativa (vetch). To obtain more insight into the mode of action of Nod factors the expression of early nodulin genes was followed during Nod factor-induced root hair deformation and nodule primordium formation. The results of these studies suggested that the expression of VsENOD5 and VsENOD12 is not required for root hair deformation. In the Nod factor-induced primordia both VsENOD12 and VsENOD40 are expressed in a spatially controlled manner similar to that found in Rhizobium -induced nodule primordia. In contrast, VsENOD5 expression has never been observed in Nod factor-induced primordia, showing that the induction of VsENOD5 and VsENOD12 expression are not coupled. VsENOD5 expression is induced in the root epidermis by Nod factors and in Rhizobium -induced nodule primordia only in cells infected by the bacteria, suggesting that the Nod factor does not reach the inner cortical cells.     

Abortion of infection during the Rhizobium meliloti—alfalfa symbiotic interaction is accompanied by a hypersensitive reaction

Nodulation, the organogenetic process resulting from the symbiotic interaction between Rhizobium and legumes, is under the feedback control of the plant. However, the autoregulatory mechanisms controlling root nodule formation are poorly understood. In this paper it is shown that alfalfa can react to infection by its symbiont Rhizobium meliloti by eliciting a defence mechanism similar to the hypersensitive reaction (HR) observed in incompatible plant-pathogen interactions. After the first nodule primordia have been induced, an increasing proportion of infection threads abort in a single or a few root cortical cells in which both symbionts simultaneously undergo necrosis. Autofluorescent, cytochemical and immunolocalization assays revealed that phenolic compounds and proteins associated with defence mechanisms in plants have accumulated in the necrotic cells. These results lead to the proposition that the elicitation of a HR is part of the mechanism by which the plant controls infection and, therefore, regulates nodulation.     

Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation.

Rhizobia induce the formation of root nodules on the roots of leguminous plants. In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia. It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle. Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex. Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads. It has been proposed that the cytoplasmic bridges are related to phragmosomes. By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase. Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.     

Characteristics of rhizobia nodulating beans in the central region of Minnesota

Until recently, beans (Phaseolus vulgaris L.) grown in Minnesota were rarely inoculated. Because of this, we hypothesized that bean rhizobia collected in Minnesota would either share characteristics identifiable with Rhizobium etli of Mesoamerican or Andean origin, introduced into the region as seed-borne contaminants, or be indigenous rhizobia from prairie species, such as Dalea spp. The latter organisms have been shown to nodulate and fix N2 with Phaseolus vulgaris. Rhizobia recovered from the Staples, Verndale, and Park Rapids areas of Minnesota were grouped according to the results of BOXA1R-PCR fingerprint analysis into 5 groups, with only one of these having banding patterns similar to 2 of 4 R. etli reference strains. When representative isolates were subject to fatty acid - methyl ester analysis and 16S rRNA gene sequence analysis, the results obtained differed. 16S rRNA gene sequences of half the organisms tested were most similar to Rhizobium leguminosarum. Rhizobia from Dalea spp., an important legume in the prairie ecosystem, did not play a significant role as the microsymbiont of beans in this area. This appears to be due to the longer time needed for them to initiate infection in Phaseolus vulgaris. Strains of Rhizobium tropici IIB, including UMR1899, proved tolerant to streptomycin and captan, which are commonly applied as seed treatments for beans. Local rhizobia appeared to have very limited tolerance to these compounds.     

欧美杂种山杨微扦插不定根发生过程的解剖学研究

采用石蜡切片技术,以欧美杂种山杨插穗基部茎段为实验材料,连续解剖观察插穗不定根发生发育过程,分析根原基发生部位与扦插生根的关系。结果显示:欧美杂种山杨插穗不定根的发生过程分为4个时期,为根原基诱导期,不定根起始期、表达期和伸长生长期。根原基诱导期维管形成层产生具有分生组织特点的薄壁细胞;不定根起始期,维管形成层及附近的薄壁细胞脱分化,形成不定根原基发端细胞;不定根表达期,根原基发端细胞不断分裂成具有方向性的根原基,根原基穿过韧皮射线和皮层,向皮孔方向发展;不定根伸长生长期,根原基从皮孔伸出,其内部的维管系统开始发育,形成不定根。研究认为,欧美杂种山杨为皮部诱导生根类型,不定根原基起源于维管形成层区,起源部位单一,扦插难生根。     

The seminal root primordia in barley and the participation of their non-meristematic cells in root construction

In addition to the primary seminal primordium, the so-called secondary seminal root primordia are also initiated in a barley embryo. The primary root primordium is developmentally most advanced. It is formed by root meristem covered with the root cap, and by a histologically determined region with completed cell division. On germination, the restoration of growth processes begins in this non-meristematic region of root primordium by cell elongation, with the exception of the zone adjacent to the scutellar node, the cells of which do not elongate but continue differentiating. In the root primordia initiated later, the zone with completed cell division is relatively shorter, in the youngest primordia the non-meristematic cells may be lacking. The root meristem is reactivated after the primary root primordium has broken through the sheath-like coleorrhiza and emerges from the caryopsis as the primary root. The character of root meristem indicates a reduced water content at the embryonic development of root primordium. With progressing growth the root apex becomes thinner, the meristematic region becomes longer, and the differences in the extent of cell division between individual cell types increase. — The primary root base is formed of cells pre-existing in the seminal root primordium. Upon desiccation of caryopsis in maturation, and subsequent quiescent period, their development was temporarily broken, proceeding with the onset of germination. The length of this postembryonically non-dividing basal zone is different in individual cell types. The column of central metaxylem characteristic of the smallest number of cell cycles, has, under the given conditions, a mean length of about 22 mm, whereas the pericycle, as the tissue with most prolonged cell division, has a mean length of about 6 mm. In the seminal root primordia initiated later the non-dividing areas are relatively shorter. The basal region of seminal roots thus differs in its ontogenesis from the increase which is formed “de novo” by the action of root meristem upon seed germination.