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Alfalfa Controls Nodulation during the Onset of Rhizobium-induced Cortical Cell Division
Abstract
The formation of first nodules inhibits subsequent nodulation in younger regions of alfalfa (Medicago sativa L.) roots by a feedback regulatory mechanism that controls nodule number systemically (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Following inoculation with wild-type Rhizobium meliloti, almost all infections associated with cortical cell division developed into mature nodules. While the distribution of Rhizobium- induced cell divisions closely paralleled the distribution of first emergent nodules, only 9 to 15% of total cell division foci failed to become functional nodules. Nodule formation was restricted to the primary root when plants were inoculated before lateral root emergence. Excision of these primary root nodules allowed nodules to reappear in lateral roots clustered around the location of the root tip at the time of nodule removal. Apparently, this region regained susceptibility to infection within the first hours after excision of primary nodules and suppression of nodulation was restored a day later probably due to the development of new infection foci. Our results suggest that alfalfa controls nodulation during the onset of cell division in the root cortex and not during infection development as in soybean.
... Plants have evolved molecular pathways to control the number of formed nodules. Autoregulation of nodulation (AON) responds to rhizobial infection to maintain an adequate number of nodules; this pathway works systematically throughout the shoot [27][28][29][30]. The genes involved in the AON process are LjHAR (Hypernodulation And Aberrant Root)/GmNARK (Nodule Autoregulation Receptor Kinase)/MtSUNN (Super Numeric Nodules), homologous to the Arabidopsis CLAVATA1 (CLV1) gene, which is a gene involved in the AON process and meristem maintenance. ...
The legume family includes approximately 19,300 species across three large subfamilies, of which Papilionoideae stands out with 13,800 species. Lentils were one of the first crops to be domesticated by humans, approximately 11,000 BP. They are diploid legumes that belong to the Papilionoidea subfamily and are of agricultural importance because of their resistance to drought and the fact that they grow in soil with a pH range of 5.5–9; therefore, they are cultivated in various types of soil, and so they have an important role in sustainable food and feed systems in many countries. In addition to their agricultural importance, lentils are a rich source of protein, carbohydrates, fiber, vitamins, and minerals. They are key to human nutrition since they are an alternative to animal proteins, decreasing meat consumption. Another characteristic of legumes, including lentils, is their ability to form nodules, which gives them a growth advantage in nitrogen-deficient soils because they enable the plant to fix atmospheric nitrogen, thus contributing nitrogen to the soil and facilitating the nutrition of other plants during intercropping. Lentils have also been applied for protection against various human diseases, as well as for phytoremediation, and they also have been applied as environmental bioindicators to identify cytotoxicity. This review addresses the importance of lentils in agriculture and human health.
... Nitrate also regulates the development of nodules, lateral root organs in legumes, where symbiotic bacteria rhizobia carry out nitrogen fixation (Carroll et al. 1985;Streeter and Wong 1988;Saito et al. 2014;Roy et al. 2020). The nodule number is negatively regulated by a systemic pathway known as autoregulation of nodulation (AON) to avoid excessive nodule formation (Nutman 1952;Caetano-Anollés and Gresshoff 1991). In the AON, the CLV1 orthologues in legumes, such as HYPERNODU-LATION ABERRANT ROOT FORMATION 1 (HAR1) in L. japonicus (Wopereis et al. 2000;Krusell et al. 2002;Nishimura et al. 2002), SUPER NUMERIC NODULES (SUNN) in Medicago truncatula (Schnabel et al. 2005), NODULE AUTOREGULATION RECEPTOR KINASE (NARK) in Glycine max (Searle et al. 2003), in Pisum sativum (Krusell et al. 2002), receive root-derived signals in the shoots. ...
Main conclusion
The local and long-distance signaling pathways mediated by the leucine-rich repeat receptor kinase HAR1 suppress root branching and promote primary root length in response to nitrate supply.
Abstract
The root morphology of higher plants changes plastically to effectively absorb nutrients and water from the soil. In particular, legumes develop root organ nodules, in which symbiotic rhizobia fix atmospheric nitrogen in nitrogen-poor environments. The number of nodules formed in roots is negatively regulated by a long-distance signaling pathway that travels through shoots called autoregulation of nodulation (AON). In the model plant Lotus japonicus, defects in AON genes, such as a leucine-rich repeat receptor kinase HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1), an orthologue of CLAVATA1, and the F-box protein TOO MUCH LOVE (TML), induce the formation of an excess number of nodules. The loss-of-function mutant of HAR1 exhibits a short and bushy root phenotype in the absence of rhizobia. We show that the har1 mutant exhibits high nitrate sensitivity during root development. The uninfected har1 mutant significantly increased lateral root number and reduced primary root length in the presence of 3 mM nitrate, compared with the wild-type and tml mutant. Grafting experiments indicated that local and long-distance signaling pathways via root- and shoot-acting HAR1 additively regulated root morphology under the moderate nitrate concentrations. These findings allow us to propose that HAR1-mediated signaling pathways control the root system architecture by suppressing lateral root branching and promoting primary root elongation in response to nitrate availability.
... Hence, we present a model in which down-regulation of NF perception would inhibit further nodulation events from the initiation stage. Because in L. japonicus, nodule primordia and rhizobial infections might both be targets of AON systemic signals (Suzaki et al., 2012;Tsikou et al., 2018), and because depending on the legume species, such as soybean and Medicago sativa (alfalfa), an AON effect on infections or on cortical cell divisions had been reported, respectively (Mathews et al., 1989;Caetano-Anollés and Gresshoff, 1991), the AON pathway might inhibit different nodulation steps, even depending on legume species. ...
The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed.
... To achieve this, legumes have evolved a number of molecular pathways enabling them to control nodulation in response to different growing conditions. The autoregulation of nodulation (AON) pathway acts in response to rhizobial infection to maintain an optimal number of nodules and functions systemically through the shoot (Caetano-Anollés & Gresshoff, 1991aGresshoff, , 1991bDelves et al., 1986;Ferguson et al., 2010;Kosslak & Bohlool, 1984;Reid, Ferguson, Hayashi, Lin, & Gresshoff, 2011). The nitrogen regulation pathway acts locally in the roots, and may involve systemic components in some species, to inhibit nodulation in nitrogen-rich growing conditions, helping the plant to conserve resources (Lim, Lee, Lee, & Hwang, 2014;. ...
Global demand to increase food production and simultaneously reduce synthetic nitrogen‐fertiliser inputs in agriculture are underpinning the need to intensify the use of legume crops. The symbiotic relationship that legume plants establish with nitrogen‐fixing rhizobia bacteria is central to their advantage. This plant‐microbe interaction results in newly developed root organs, called nodules, where the rhizobia convert atmospheric nitrogen gas into forms of nitrogen the plant can use. However, the process of developing and maintaining nodules is resource‐intensive; hence, the plant tightly controls the number of nodules forming. A variety of molecular mechanisms are used to regulate nodule numbers under both favourable and stressful growing conditions, enabling the plant to conserve resources and optimise development in response to a range of circumstances. Using genetic and genomic approaches, many components acting in the regulation of nodulation have now been identified. Discovering and functionally characterising these components can provide genetic targets and polymorphic markers that aid in the selection of superior legume cultivars and rhizobia strains that benefit agricultural sustainability and food security. This review addresses recent findings in nodulation control, presents detailed models of the molecular mechanisms driving these processes and identifies gaps in these processes that are not yet fully explained.
... Limited nitrogen supply to leaves was shown to reduce light-saturated leaf photosynthesis in regrowth crops subjected to frequent defoliations (Teixeira et al., 2008). In the seedling phase, nitrogen supply to leaves could be limited by the absence of root reserve pools (Avice et al., 2003) or nitrate (NO 3 -) availability from the still forming Rhizobium meliloti root nodules (Baysdorfer and Bassham, 1985;Caetano-Anolles and Gresshoff, 1991) that fix atmospheric nitrogen (Vance et al., 1979). This initial period of low RUE shoot , further indicated by the projection of negative y-intercepts in seedlings ( Figure 5), overlaps with the juvenile period of development ( Figure 7) when seedling crops remain vegetative for longer than regrowth crops. ...
This study compared physiological responses of fully irrigated seedling and regrowth lucerne crops (Medicago sativa L.) grown under similar environmental field conditions. Measurements occurred for 2–4 years after sowing on 24 October, 15 November, 05 December and 27 December 2000 at Lincoln, Canterbury, New Zealand. Irrespective of the date of sowing, on average lucerne accumulated less shoot dry matter (DM) in the seedling year (11±0.44tha−1) than during the regrowth year (18±0.76tha−1). Slower shoot-growth rates in seedlings were explained by less intercepted light and reduced efficiency in conversion of light to biomass. Specifically, seedlings had a longer phyllochron (47±2.3°Cd leaf−1) and slower leaf area expansion rate (0.009m2m−2°Cd−1) than regrowth crops (35±1.8°Cd leaf−1 and 0.016m−2m−2°Cd, respectively). There were no differences in canopy architecture with a common extinction coefficient of 0.93. The radiation use efficiency (RUE) for shoot production (RUEshoot) was 1.2±0.16g DM MJ−1 of intercepted photosynthetically active radiation (PARi) in seedlings and 1.9±0.24g DM MJ−1 PARi in regrowth crops. Reproductive development was slower in seedling than regrowth crops due to an apparent juvenile period ranging from 240 to 530°Cd in seedlings. For both seedling and regrowth phases, the thermal time accumulation to reach 50% buds visible (Tt0-bv) and 50% open flowers (Tt0-fl) increased as photoperiod shortened in autumn. The minimum Tt0-bv, or the thermal-time duration of the basic vegetative period (TtBVP), was estimated at 270±48°Cd at photoperiods >14h for regrowth crops. The theoretical threshold below which reproductive development is projected to cease, or the base photoperiod (Ppbase), was estimated at a common 6.9h for seedling and regrowth crops. The transition from buds visible to open flowers (Ttbv-fl) was mainly controlled by air temperature and ranged from 161°Cd for seedlings to 274°Cd for regrowth crops. These results can be used as guidelines to develop differential management strategies for seedling and regrowth crops and improve the parameterization of lucerne simulation models.
... Three basic categories of mutant phenotypes have been identified: (i) non-nodulation; (ii) ineffective nodulation and/or early senescence; and (iii) hypernodulation/nitrate tolerance. Most of the phenotypes described thus far have been shown to be controlled by single recessive genetic elements, yet dominant inheritance of the mutation underlying an unusual fourth class of symbiotic traits, Nar (nodulation in the absence of rhizobia), has also been reported (Truchet et al. 1989;Caetano-Anolles and Gresshoff 1991a;Caetano-Anolles et al. 1993). The isolation and characterization of different classes of symbiotic mutants have provided a solid basis for the development of novel concepts in symbiotic nitrogen fixation. ...
A detailed microscopical analysis of the morphological features that distinguish different developmental stages of nodule organogenesis in wild-type Lotus japonicus ecotype Gifu B-129-S9 plants was performed, to provide the necessary framework for the evaluation of altered phenotypes of L. japonicus symbiotic mutants. Subsequently, chemical ethyl methanesulfonate (EMS) mutagenesis of L. japonicus was carried out. The analysis of approximately 3,000 M1 plants and their progeny yielded 20 stable L. japonicus symbiotic variants, consisting of at least 14 different symbiosis-associated loci or complementation groups. Moreover, a mutation affecting L. japonicus root development was identified that also conferred a hypernodulation response when a line carrying the corresponding allele (LjEMS102) was inoculated with rhizobia. The phenotype of the LjEMS102 line was characterized by the presence of nodule structures covering almost the entire root length (Nod++), and by a concomitant inhibition of both root and stem growth. A mutation in a single nuclear gene was shown to be responsible for both root and symbiotic phenotypes observed in the L. japonicus LjEMS102 line, suggesting that (a) common mechanism(s) regulating root development and nodule formation exists in legumes.
... In our semiquantitative assay, transgenic plants were scored positive for GUS staining in the cortex on the basis of a clear and unambiguous signal located at or below the region of the root that is normally susceptible to nodulation at the moment of bacterial inoculation. This susceptible region lies between the root tip and the zone of root hair emergence (Bhuvaneswari et al. 1981;Caetano-Anollés and Gresshoff 1991), and the use of growth pouches enabled us to monitor root growth following either S. meliloti inoculation or Nod factor addition and thus to position GUS expression in relation to the susceptible zone. This approach eliminated the potential risk of confusing Rhizobium/Nod factor responses with the development of spontaneous nodule primordia in the older region of the root (see Pichon et al. 1994). ...
The spatio-temporal expression pattern of the Medicago truncatula ENOD20 gene during early stages of nodulation has been analyzed with transgenic alfalfa (M. varia) expressing a pMtENOD20-GUS chimeric fusion, Our results show that transcriptional activation of this gene occurs initially in dividing inner cortical cells corresponding to sites of nodule primordium formation and subsequently in root hairs containing infection threads. Use of Sinorhizobium meliloti nod gene mutants that uncouple nodule organogenesis from infection has confirmed that early MtENOD20 transcription is tightly linked to cortical cell activation (CCA). Furthermore, these experiments have revealed that an S. meliloti nodH mutant, defective in Nod factor sulfation and epidermal cell activation, is nevertheless able to elicit low-level CCA. Purified S. meliloti Nod factors trigger MtENOD20 transcription very rapidly (within 12 to 24 h) in the root cortex, and studies with transgenic alfalfa show that Nod factors are able to elicit gene expression coupled to CCA at concentrations as low as 10(-11) M. Finally, we have made use of a range of synthetic lipo-chitooligosaccharides to show that fatty acid chain length is an important structural parameter in the capacity of Nod factors to elicit CCA, Taken together, our results suggest that pMtENOD20-GUS transgenic lines should prove valuable tools in future studies of Rhizobium and Nod factor-elicited CCA.
... initiation of cortical cell division centres (CDs), which represent potential nodule meristems The number of nodules forn:ied on legume roots in (Caetano-Anolles & Gresshoff, 19916). By contrast, response to infection by rhizobia is apparently in soybean the response controls the maturation of controlled by an intrinsic plant regulatory mech-CDs to advanced stages of nodule ontogeny (Calvert anism by which early initiated nodules suppress the et al , Francisco & Akao, 1993. ...
summaryA split-root growth system was used to study photosynthate partitioning to developing nodules and roots of soybean (Glycine max L., Merr). Opposite sides of the root systems were inoculated with Bradyrhizobium japonicum at 8 and 12 d after planting (early/delayed inoculation treatment) or, alternatively, only one side was inoculated 8 d after planting (early/uninoculated treatment). Plants were incubated with 14CO2 at 24-h intervals from early inoculation until the onset of N2 fixation (acetylene reduction). After staining with Eriochrome black, root and nodule meristematic structures were excised under a dissecting microscope and their radioactivity determined by scintillation counting. The specific radioactivity of nodule structures increased with nodule development, and was as much as 4 times higher in early nodules than in roots and nodules on half-roots receiving delayed inoculation By the time that N2 fixation could be measured in the first mature nodules, the early inoculated half-root contained over 70% of the radioactivity recovered from the entire root systems of both early/delayed and early/uninotulated treatments. These results suggest that developing nodules create a strong sink for photosynthate, and that nodules and roots compete for current photosynthate. Early initiated nodules might develop at the expense of late initiated nodules, as well as at the expense of the roots themselves.
... This process involves suppression of nodule emergence from ontogenetically younger root tissues by previously formed nodules on older parts of the root system [25,30]. It has been proposed that once a critical number of subepidermal cell divisions in the root cortex are initiated, a precursor molecule from the root is transported to the shoot, where it is converted into the shoot-derived inhibitor, which, in turn, is transported back to the root and suppresses the later-formed subepidermal cell divisions from developing into emergent nodules [7]. Supernodulating and hypernodulating mutants of soybean that appear to have altered this autoregulation have been derived from Bragg and Williams parents [9,13]. ...
Two strains of Bradyrhizobium japonicum wereevaluated with five commercial cultivars of soybean(Clark, Crauford, Davis, Centaur, and Nessen) and onehypernodulating mutant NOD1-3. The hypernodulatingNOD1-3 produced 30–50 times more nodules thancommercial cultivars either inoculated with B.japonicum strain USDA 123 or RCR 3409. The currentexperiments were extended to determine if therestricted nodulation of commercial cultivars could be overcome by grafting them to a hypernodulated shoot (NOD1-3). Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. The shoots of Clark, Davis, Centaur and Nessen significantlyinhibited nodule formation on the root of NOD1-3,while Crauford shoots did not alter nodule formationon the roots of NOD1-3 as compared with self-grafts ofNOD1-3. It appears that the shoot of NOD1-3 has theability to alter autoregulatory control of nodulationof Clark and Davis cultivars, but did not withCrauford, Centaur and Nessen. The results suggestedthat the regulation of nodulation in soybean cultivarsClark and Davis is controlled by the shoot factors,while the Crauford was root controlled.Reciprocal-grafts between NOD1-3 and Centaur or Nessenindicate that both shoot and root factors involved inregulation of nodulation and the regulation ofnodulation did not depend on bradyrhizobial strains. Isoflavonoid analyses from extracts of grafted plantsshowed that NOD1-3 shoots had markedly higher rootisoflavonoid concentrations in roots of both Clark andNOD1-3. The shoot control of hypernodulation may becausally related to differential root isoflavonoidlevels, which are also controlled by the shoot. Thecurrent work was extended to investigate the effect ofapplication of an isoflavonoid (daidzein) on nodulationand nitrogen fixation of soybean cultivars Clark andCentaur as well as in vitro growth of Bradyrhizobium japonicum. Application of theisoflavonoid (daidzein) significantly enhanced thenodulation and nitrogenase activity of Clark but notof Centaur indicating that this character is notrelated to isoflavonoids. Therefore, autoregulationin Clark and Centaur plants may be separate events inlegume-rhizobia symbiosis and regulated by differentkinds of signals. Addition of daidzein to yeastmannitol broth medium promoted the growth of B.japonicum strain USDA 123 and RCR 3409. It seemsthat this compound is able to help the nodulation ofsoybean cv Clark by a Bradyrhizobium strain. Understanding the signaling pathways between rhizobiaand their host plants may allow modifications of thisinteraction to improve symbiotic performance.
... While on plants like Vicia the trimer-forming chitinase is sufficient for inactivation of the cognate Nod signals, on Medicago only the dimer-forming enzyme can inactivate NodRm-IV(S), and this might explain the higher activity of this enzyme in Medicago roots. In alfalfa, nodulation is under feedback control (Anolles and Bauer, 1988) and this is exerted during the initiation of cortical cell divisions ( Anolles and Gresshoff, 1991) and the infection process (Vasse et a/., 1993). Interestingly, in the latter case a hypersensitive response, including the accumulation of chitinases, has been correlated with the abortion of infection threads, and Nod signals were proposed as possible targets for the chitinases. ...
Acylated chitooligosaccharide signals (Nod factors) trigger the development of root nodules on leguminous plants and play an important role in determining host specificity in the Rhizobium-plant symbiosis. Here, the ability of plant chitinases to hydrolyze different Nod factors and the potential significance of the structural modifications of Nod factors in stabilizing them against enzymatic inactivation were investigated. Incubation of the sulfated Nod factors of Rhizobium meliloti, NodRm-IV(S) and NodRm-V(S), as well as their desulfated derivatives NodRm-IV and NodRm-V, with purified chitinases from the roots of the host plant Medicago and the nonhost plant Vicia resulted in the release of the acylated lipotrisaccharide NodRm-III from NodRm-V, NodRm-IV and NodRm-V(S), whereas NodRm-IV(S) was completely resistant to digestion by both chitinases. Kinetic analysis showed that the structural parameters determining host specificity, the length of the oligosaccharide chain, the acylation at the nonreducing end and the sulfatation at the reducing end of the lipooligosaccharide, influence the stability of the molecule against degradation by chitinases. When the Nod factors were incubated in the presence of intact roots of Medicago, as well as of Vicia, the acylated lipotrisaccharide was similarly released in vivo from all Nod factors except NodRm-IV(S). In addition, a dimer-forming activity was observed in intact roots which also cleaved NodRm-IV(S). This activity was much greater in Medicago than in Vicia and increased upon incubation. The initial overall degradation rate of the Nod factors on Medicago was inversely correlated with their biological activities on Medicago roots. These results open the possibility that the activity of Nod factors on Medicago may partly be determined by the action of chitinases.
... Supernodulating mutants with increased nodule number were isolated in several legume species, including Mt (review by Kinkema et al., 2006;OkaKira & Kawaguchi, 2006). These mutants are impaired in the 'autoregulation of nodule number' (AON) which is the systemic feedback repression of nodulation by the pre-existing nodules (Kosslak & Bohlool, 1984;Caetano-Anollés & Gresshoff, 1991). Different complementation groups may display this phenotype, but until now most of the mutations identified at the molecular level belong to one group. ...
Adaptation of Medicago truncatula to local nitrogen (N) limitation was investigated to provide new insights into local and systemic N signaling. The split-root technique allowed a characterization of the local and systemic responses of NO(3)(-) or N(2)-fed plants to localized N limitation. (15)N and (13)C labeling were used to monitor plant nutrition. Plants expressing pMtENOD11-GUS and the sunn-2 hypernodulating mutant were used to unravel mechanisms involved in these responses. Unlike NO(3)(-)-fed plants, N(2)-fixing plants lacked the ability to compensate rapidly for a localized N limitation by up-regulating the N(2)-fixation activity of roots supplied elsewhere with N. However they displayed a long-term response via a growth stimulation of pre-existing nodules, and the generation of new nodules, likely through a decreased abortion rate of early nodulation events. Both these responses involve systemic signaling. The latter response is abolished in the sunn mutant, but the mutation does not prevent the first response. Local but also systemic regulatory mechanisms related to plant N status regulate de novo nodule development in Mt, and SUNN is required for this systemic regulation. By contrast, the stimulation of nodule growth triggered by systemic N signaling does not involve SUNN, indicating SUNN-independent signaling.
... This regulatory circuit is called 'autoregulation of nodulation' (AON). Initial studies with split-root systems demonstrated that the suppression of nodulation is a systemic feedback response (Kosslak and Bohlool, 1984), which is active in a variety of legumes such as soybean (Delves et al., 1986; Olsson et al., 1989 ), subterranean clover (Sargent et al., 1987), alfalfa (Anollés and Bauer, 1988; Caetano-Anollés and Gresshoff, 1991b), white bean (Park and Buttery, 1988) and Lotus (Suzuki et al., 2008). A key gene (nodule autoregulation receptor kinase – NARK, a leucine-rich-repeat (LRR) receptor kinase) controlling AON signalling has been isolated from soybean (GmNARK), pea (PsSym29), Medicago (MtSUNN) and Lotus (LjHar1) (Krusell et al., 2002; Nishimura et al., 2002; Searle et al., 2003; Schnabel et al., 2005). ...
To define the signalling events required for the activation of AON, we utilised approach grafts between wild-type pea plants and their mutants defective at successive stages of nodule formation. AON signalling strength was monitored by prior inoculation of mutant root portions (as so-called 'sensor') and quantifying nodule formation on connected roots of delayed inoculated wild type (the 'reporter'). Detectable AON sensing and associated signal exchange between root and shoot started after root hair curling but before the initiation of visible cortical and pericycle cell divisions. The strength of AON signalling was correlated with the stage of nodule development and size of nodule, with mature nitrogen-fixing nodules possessing the strongest AON-inducing signal. We demonstrated that the pea supernodulating mutant nod3 may function pre-NARK in the root. A model for the activation of AON signalling and its potential relationship with cell division, nitrogen fixation and/or cytokinin signal transduction are presented.
Legumes form a symbiosis with atmospheric nitrogen (N 2 )‐fixing soil rhizobia, resulting in new root organs called nodules that enable N 2 ‐fixation. Nodulation is a costly process that is tightly regulated by the host through autoregulation of nodulation (AON) and nitrate‐dependent regulation of nodulation. Both pathways require legume‐specific CLAVATA/ESR‐related (CLE) peptides.
Nitrogen‐induced nodulation‐suppressing CLE peptides have not previously been investigated in Medicago truncatula , for which only rhizobia‐induced MtCLE12 and MtCLE13 have been characterised. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control.
The nodulation‐suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum . Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE‐domain. MtCLE35 was found to inhibit nodulation in a SUNN‐ and RDN1‐dependent manner via overexpression analysis.
Together, our findings indicate that MtCLE12 and MtCLE13 have a specific role in AON, while MtCLE35 regulates nodule numbers in response to both rhizobia and nitrate. MtCLE34 likely had a similar role to MtCLE35, but its function was lost due to a premature nonsense mutation.
In diesem Kapitel beschränken wir uns auf die exemplarische Darstellung von 3 Symbiosetypen, die für die Primärproduktion in Ökosystemen eine entscheidende Rolle spielen. Die Flechten als Primärproduzenten, die zur Besiedelung der unterschiedlichsten Substrate sogar unter extremen Bedingungen fähig sind. Die biotische Stickstofffixierung, die ökologisch wichtige eukaryotische Primärproduzenten zur Aufnahme atmosphärischen Stickstoffs befähigt und für den Antrieb des N‐Kreislaufs terrestrischer Ökosysteme essentiell ist. Die Mykorrhizen als ökologisch obligate Symbiosen für die Ernährung und Wasserversorgung der Höheren Pflanzen. Sie prägen den Nährstoffkreislauf und die Struktur der Vegetation. Es werden nach einigen prinzipiellen Erörterungen zum Wesen der Symbiose das Verständnis der Symbiose als System hervorgehoben, die Rolle von Symbiosen in Ökosystemen und die Auswirkungen von Symbiosen auf die Bildung von Ökosystemen beschrieben.
Because of their enormously large range of plant hosts and role in plant nutrition, arbuscular mycorrhizal (AM) fungi represent an extraordinarily fascinating field of study. Plant growth promotion effects by AM fungi were described as early as 1900 (Sthal 1900) and several data obtained in the second half of the last century support the idea that these microrganisms can act as biocontrol agents (BCA).
The extent of root colonization is variable in different plants and under different environmental conditions (Giovannetti and Hepper 1985). Some effects of AM colonization on plants have been reported to be dependent on the degree of root colonization, while others have not. Root exudation and pH are modified by the presence of AM fungi (Bansal and Mukerji 1994; Bago et al. 1996), therefore AM fungi can affect the growth of rhizobacteria. Similarly, both root colonization by AM fungi and their effects on the plant can be affected by the presence of rhizobacteria, which can be plant growth-promoting, mycorrhiza helper or biocontrol agents.
Among microorganisms which interact with plants, some are pathogenic and elicit either disease on a susceptible (compatible) host or disease resistance on a resistant (incompatible) plant. In the latter case, plant defence mechanisms are induced and a hypersensitive reaction, H.R., often occurs. The H.R. is a localized plant reaction resulting in the necrosis of the two partners and the arrest of the progression of the invader. In contrast to pathogenic associations, symbiotic interactions result in the ability of the two partners to establish a beneficial relationship. In the Rhizobium-legume symbiosis a series of events, recognition between symbionts, plant invasion by the bacterium and induction of plant cortical cell division, lead to the organogenesis of a new organ, the root nodule, in which molecular nitrogen is reduced to ammonia by the bacteria.
A complex interplay, involving multiple signal exchange between the legume host and its rhizobial partner, is required for the induction and subsequent development of the N2-fixing symbiotic root nodule. In particular, it has been shown that a sulphated lipo-oligosaccharide (NodRm), purified from the supernatant of Rhizobium meliloti (Lerouge et al., 1990), can act as a specific symbiotic signal to elicit root hair deformations and nodule organogenesis on alfalfa (Medicago sativa) plants (Truchet et al.,1991). It is now established that other Rhizobium species produce different specific symbiotic signal molecules, the so-called Nod factors, with a core structure similar to NodRm (Spaink et al., 1991). Rhizobial nodulation (nod) genes are responsible for the synthesis of Nod factors (Denarié, Roche, 1992). A detailed analysis of the host response to these Nod factors requires the identification of plant genes which can serve as molecular markers for the earliest stages of the recognition, infection and nodule organogenetic triggering processes. Recently, Scheres et al. (1990) have reported that transcripts of a pea gene (PsENOD12), which encodes a proline-rich protein, are present in a variety of cell types involved in the early stages of infection.
Respected and known worldwide in the field for his research in plant nutrition, Dr. Horst Marschner authored two editions of Mineral Nutrition of Higher Plants. His research greatly advanced the understanding of rhizosphere processes and trace element uptake by plants and he published extensively in a variety of plant nutrition areas. While doing agricultural research in West Africa in 1996, Dr. Marschner contracted malaria and passed away, and until now this legacy title went unrevised. Despite the passage of time, it remains the definitive reference on plant mineral nutrition. Great progress has been made in the understanding of various aspects of plant nutrition and in recent years the view on the mode of action of mineral nutrients in plant metabolism and yield formation has shifted. Nutrients are not only viewed as constituents of plant compounds (constructing material), enzymes and electron transport chains but also as signals regulating plant metabolism via complex signal transduction networks. In these networks, phytohormones also play an important role. Principles of the mode of action of phytohormones and examples of the interaction of hormones and mineral nutrients on source and sink strength and yield formation are discussed in this edition. Phytohormones have a role as chemical messengers (internal signals) to coordinate development and responses to environmental stimuli at the whole plant level. These and many other molecular developments are covered in the long-awaited new edition. Esteemed plant nutrition expert and Horst Marschner's daughter, Dr. Petra Marschner, together with a team of key co-authors who worked with Horst Marschner on his research, now present a thoroughly updated and revised third edition of Marschner's Mineral Nutrition of Higher Plants, maintaining its value for plant nutritionists worldwide. A long-awaited revision of the standard reference on plant mineral nutrition Features full coverage and new discussions of the latest molecular advances Contains additional focus on agro-ecosystems as well as nutrition and quality.
Primary expression of the Rhizobium meliloti-induced peroxidase gene rip1 occurs prior to nodule morphogenesis, specifically at the site of impending rhizobial infection (D. Cook, D. Dreyer, D. Bonnet, M. Howell, E. Nony, K. VandenBosch [1995] Plant Cell 7:43–55). We examined the distribution and structure of rip1 transcript throughout nodule development. We determined that expression of rip1 in root tips is correlated with the competence of this zone for symbiotic association, whereas after rhizobial infection rip1 transcript is specifically associated with the zone of nodule development, including nascent nodule primordia. rip1 transcripts are characterized by multiple polyadenylation sites distributed within 200 to 400 bp of the translation stop site, and a single major transcription initiation site in close proximity to the rip1 open reading frame. Thus, rip1 expression is likely to be mediated through effects on a single transcription unit. Immediately 5[prime] of the rip1 transcription unit DNA sequence analysis identified a 377-bp DNA element containing extensive repeat structure that is widely distributed in the Medicago truncatula genome.
Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule‐inducing rhizobia. The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation. Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system. In this work, split‐ root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation systemically influences AMF root colonization and vice versa. Nodules on one half of the split‐root system suppressed subsequent AMF colonization on the other half. Conversely, root systems pre‐colonized on one side by AMF exhibited reduced nodule formation on the other side. An inhibition effect was also observed with Nod factors (lipo‐chito‐oligosaccharides). NodSm‐IV(C16:2, S) purified from S. meliloti systemically suppressed both nodule formation and AMF colonization. The application of Nod factors, however, did not influence the allocation of ¹⁴C within the split‐root system, excluding competition for carbohydrates as the regulatory mechanism. These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.
Nodulation in alfalfa (Medicago sativa L.) is controlled by a systemic feedback regulatory mechanism that suppresses nodule initiation in younger portions of the root system. Excision of primary root nodules induced by wild-type Rhizobium meliloti stimulates the formation of new nodules on lateral roots. In similar experiments we found that excision of bacteria-free nodules from primary roots induced by mutants of R. meliloti deficient in exopolysaccharide synthesis allowed nodules to reappear in lateral roots especially around the root tip at the time of nodule removal. Our results suggest that organized nodular structures trigger autoregulatory responses in legumes, even in the absence of bacterial infection.
Changes in the concentration of free and conjugated ABA, zeatin riboside (ZR), and IAA in response to Bradyrhizobium inoculation and subsequent nodulation were monitored in xylem sap, phloem sap, and leaves of soybean [Glycine max (L.) Merr. cv. Williams 82] and its hypernodulating mutant, NOD1-3. In this study, pre-inoculation concentrations of phloem and xylem sap ABA and ZR were lower in NOD1-3 than in Williams 82, a difference that was accentuated in phloem after inoculation. The concentration of xylem ABA increased within 6 h of inoculation, while the concentration of phloem and leaf ABA did not change until 48-96 h after inoculation. Leaf uptake of [3H]ABA and distribution to phloem sap was greater in Williams 82 than in NOD1-3 during 48-72 h after inoculation. Inoculation resulted in similar increases in phloem and leaf IAA concentrations in both cultivars. While inoculation increased xylem sap ZR in both lines, the concentration of ZR increased much earlier in NOD1-3. Of particular interest is that ratios between hormones were altered during nodulation. Leaf and phloem ABA/IAA ratios were higher in Williams 82 than in the hypernod mutant, while the phloem IAA/ZR was greater from inoculation until nodulation in the NOD1-3 hypernod mutant. The xylem ABA/ZR ratio, as well as phloem ABA/ZR ratio, decreased in Williams 82 following inoculation, and leaf ABA concentration was elevated. The most noteworthy results of this study, therefore, came from an examination of the ratios between hormones in xylem and phloem sap, and the demonstration that hormone transport may play an important role in autoregulation of root nodulation.
The symbiotic association with N-fixing bacteria facilitates the growth of leguminous plants under nitrogen-limiting conditions. The establishment of the symbiosis requires signal exchange between the host and the bacterium, which leads to the formation of root nodules, inside which bacteria are hosted. The formation of nodules is controlled through local and systemic mechanisms which involves root-shoot communication. Our study was aimed at investigating the proteomic changes occurring in shoots and concomitantly in roots of Medicago truncatula at an early stage of Sinorhizobium meliloti infection. The principal systemic effects consisted in alteration of chloroplast proteins, induction of proteins responsive to biotic stress and changes in proteins involved in hormonal signaling and metabolism. The most relevant local effect was the induction of proteins involved in the utilization of photosynthates and C-consuming processes (such as sucrose synthase and fructose-bisphosphate aldolase). In addition, some redox enzymes such as peroxiredoxin and ascorbate peroxidase showed an altered abundance. The analysis of local and systemic proteome changes suggests the occurrence of a stress response in the shoots and the precocious alteration of energy metabolism in roots and shoots. Furthermore, our data indicate the possibility that ABA and ethylene participate in the communicative network between root and shoot in the control of rhizobial infection.
Rhizobium nodulation genes can produce active extracellular signals for legume nodulation. The R. meliloti host-range nodH gene has been postulated to mediate the transfer of a sulfate to a modified lipo-oligosaccharide, which in its sulfated form is a specific nodulation factor for alfalfa (Medicago sativa L.). We found that alfalfa was capable of effective nodulation with signal-defective and non-nodulating nodH mutants (Nnr) defining a novel gene-for-gene interaction that conditions nodulation. Bacteria-free nodules that formed spontaneously at about a 3-5% rate in unselected seed populations of alfalfa cv 'Vernal' in the total absence of Rhizobium (Nar) exhibited all the histological, regulatory and ontogenetic characteristics of alfalfa nodules. Inoculation of such populations with nodH mutants, but not with nodA or nodC mutants, produced a four- to five-fold increase in the percentage of nodulated plants. Some 10-25% of these nodulated plants formed normal pink nitrogen-fixing nodules instead of white empty nodules. About 70% of the S1 progeny of such Nnr(+) plants retained the parental phenotype; these plants were also able to form nodules in the absence of Rhizobium. If selected Nar(+) plants were self-pollinated almost the entire progeny exhibited the parental Nar(+) phenotype. Segregation analysis of S1 and S2 progeny from selected Nar(+) plants suggests that the Nar character is monogenic dominant and that the nodulation phenotype is controlled by a gene dose effect. The inoculation of different S1 Nar(+) progeny with nodH mutant bacteria gave only empty non-fixing nodules. Our results indicate that certain alfalfa genotypes can be selected for suppression of the non-nodulation phenotype of nodH mutants. The fact that the Nnr plant phenotype behaved as a dominant genetic trait and that it directly correlated with the ability of the selected plants to form nodules in the absence of Rhizobium suggests that the interaction of plant and bacterial alleles occurs early during signal transduction through the alteration of a signal reception component of the plant so that it responds to putative signal precursors.
In Lupinus luteus L. the infection occurred via curled root hair. Bradyrhizobia penetrated its cell wall probably due to the localised digestion and then multiplicated into the interior of the root hair. Some rhizobia escaped the penetration site before the host cell built the new wall around it. Escaped bacteria passed the distance to the root hair cell base probably in the space between cell wall and plasma membrane. At the root hair cell base the cell wall penetration and matrix escape occurred again. After escaping matrix rhizobia were endocytotically directed to the interior of dedifferentiated cortex cell, and during subsequent mitosis were segregated to the proximal derivative. Thus the cell became the bacteroid tissue initials. The bacteria, which remained within the penetration site, were immobilised here due to the production of cell wall around them. Internalised bacteria were initially associated with large numbers of vesicles bearing cell wall like matrix. In abortive primordia the bacteria penetrated the cell wall of competent cells and the associations of bacteria and cell wall-like matrix surrounded with a membrane were produced. At this stage the symbiosis was arrested and symbiosomes were degraded. The necrosis or lysis of some primordium cells was observed.
Root ethylene biosynthesis has been studied in soybean (Glycine max [L.] Merr.) cv. Bragg plants and its supernodulating (nts382 and nts1007) and non-nodulating (nod49 and nod139) mutants. Regardless of NO3 treatment, inoculation with Bradyrhizobium japonicum significantly increased root ethylene evolution rate, reaching a plateau between 24 and 48 h after inoculation, with the rates being significantly higher in 8 mmol L−1 fed roots (high) than in those given 1 mmol L-1 (low) during the time of experiment. This Inoculation Stimulated Ethylene Release (ISER) response appears to be related to the infection process and nodule development, as treatment with Ag+ (an inhibitor of ethylene action) at the moment of inoculation markedly increased nodule number of Bragg plants under both high and low NO3 concentrations. Compared with the parental Bragg, the near-isogenic nodulating mutants used in this study showed normal ethylene biosynthesis ability (ethylene evolution and ACC oxidase activity), although significant quantitative differences were detected among them. Whether these differences are causally related to the nodulation phenotype is not known. Our previous observations on the involvement of endogenous ethylene in the control of nodule number in alfalfa are therefore also applicable to soybean, a determinate nodule type legume. The results further suggest that effects other than an alteration of ethylene biosynthesis might have also been caused by the nts mutations.
Combined nitrogen [nitrate (NO3‐), ammonium (NH4+), and urea] will inhibit all components of symbiotic nitrogen (N2) fixation if present in sufficient concentrations. It is generally accepted that nitrate is particularly inhibitory to nodule growth and nitrogenase activity, and somewhat less inhibitory to the infection process. This project examined whether providing low (0.1 ‐ 0.5 mM) static concentrations of NO3‐ to pea (Pisum sativum L. cv. Express), seedlings could avoid the period of N hunger experienced prior to the establishment of N2 fixation, without delaying or reducing the symbiotic N2 fixation. All concentrations of NO3 tested significantly inhibited all measured components of N2 fixation. The nodulation process as measured by nodule number was inhibited to a similar degree as the other parameters. A concentration dependent response was evident, with 0.1 mM NO3 causing less inhibition than the 0.2 or 0.5 mM concentrations. Our results indicate the within the concentrations of 0.1 mM and 0.5 mM NO3 , it is not possible to stimulate the growth of pea plants without inhibiting nodulation and N2 fixation.
This chapter discusses the Cell and molecular biology of Rhizobium-plant interactions. Soil bacteria, referred to as rhizobia belonging to the genera Rhizobium, Bradyrhizobium, and Azorhizobium, have the unique ability to induce nitrogen-fixing nodules on the roots or stems of leguminous plants. Nodule development consists of several stages determined by different sets of genes both in the host and symbiont. At least at the very early steps of symbiosis, the bacterial and plant genes are activated consecutively by signal exchanges between the symbiotic partners. First, flavonoid signal molecules exuded by the host plant root induce the expression of nodulation (nod, nol) genes in Rhizobium in conjunction with the bacterial activator NodD protein. Then, in the second step, lipooligosaccharide Nod factors with various host-specific structural modifications are produced by the bacterial Nod proteins. The Nod factors induce various plant reactions, such as root hair deformation, initiation of nodule meristems, and induction of early nodulin genes, leading to nodule formation. Other classes of bacterial genes are required for successful infection and for nitrogen fixation. This chapter includes only the early events of communication between rhizobia and their host plants, that is, the perception of flavonoid signals by the bacteria, the production of Nod signals by rhizobia, and the early plant responses to the bacteria.
Azorhizobium caulinodans ORS571 and Sinorhizobium teranga ORS51 and ORS52 are symbionts of the same host plant Sesbania rostrata. In nature, A. caulinodans nodulates more competitively the stem-located infection sites of Sesbania rostrata. Sinorhizobium strains, although frequently present in root nodules, are seldom found in stem nodules. One probable explanation for this phenomenon is the more abundant presence of Azorhizobium on the leaf and stem surfaces of the host plant. Work presented here hints at other plausible factors that determine the greater "stem specificity" of Azorhizobium. We found that under experimental conditions in which roots are not inoculated, all strains nodulated stems very well. However, ORS51 and ORS52 were much more sensitive than ORS571 to suppression of stem nodulation by previous root inoculation. The introduction of the regulatory nodD gene from A. caulinodans diminished the sensitivity to this suppression, probably by enhanced nod gene expression and subsequent Nod factor production. Our hypothesis is that the greater infectivity of ORS571 is due to a more efficient production of mitogenic Nod factors at stem-located infection sites, thereby more readily overcoming systemic suppression caused by previous root inoculations.Key words: autoregulation, nitrogen fixation, rhizobial ecology, systemic suppression of nodulation.
The initiation and development of nitrogen (N2) fixing nodules in the roots of leguminous plants occurs by the induction of cell division and redifferentiation in the root cortex, followed by the formation of a meristem and progressive differentiation of specialized cells and tissues. During this process, competent rhizobia invade the root and become specialized N2-fixing endosymbionts. The onset of the symbiosis is largely mediated by an exchange of diffusible signals, bacterial lipo-oligosaccharides being the main determinants of specificity and the initial inducers of plant responses. It is however the host which controls most facets of the nodulation process, including nodule morphology, efficiency, specificity and function. The dissection of plant mechanisms underlying signal-transduction during nodulation may be crucial to understand and then manipulate the symbiosis. Positional cloning or gene targeting offer strategies that promise the identification of crucial plant genes determining nodulation. The search for the nts — 1 gene that controls nodulation in soybean illustrates the challenges and limitations of positional cloning. It also shows how biotechnology can offer tools to help in the breeding of plant traits important to agriculture. Molecular dissection of the symbiosis will ultimately be used to improve N2 fixation by molecular breeding and genetic engineering in legumes.
A laboratory study was conducted to determine the effects of defoliation and denodulation on compensatory growth of Medicago sativa (L.). Plants grown hydroponically in clear plastic growth pouches were subjected to 0 and 50% nodule pruning, and 0, 25, 50, and 75% defoliation by clipping trifoliate leaves. An additional experiment was conducted to determine if clipping leaves simulated herbivory by Hypera postica (Gyllenhal) larvae. Previously, we determined that nodule pruning accurately simulated herbivory by Sitona hispidulus (L.) larvae (Quinn & Hall, 1992). Results indicated that denodulation stimulated nodule growth and caused exact compensation in standing and total number of nodules per plant within 15 days and in standing nodule biomass within 22 days of treatment. Denodulation caused a significant reduction (13%) in final shoot biomass, but did not affect significantly final root biomass. Percentage of change in number of trifoliate leaves per plant increased with the level of defoliation. Within 22 days of treatment, total number of trifoliate leaves per plant was similar to controls. However, final standing shoot biomasses were significantly less that controls, indicating undercompensatory growth. Shoot biomasses of the 25-, 50-, and 75%-defoliated plants were 18, 20, and 36% lower than controls, respectively. Nodule biomass per plant was reduced by 24 and 32% in 50- and 75%-defoliated plants, respectively, but was not affected significantly by 25% defoliation. Root biomass was affected by all levels of defoliation. Clipping trifoliate leaves accurately simulated defoliation by H. postica larvae. Our results indicated that partial defoliation affected shoot, root, and nodule biomass of M. sativa, but that partial denodulation only affected shoot biomass.
We have studied regulation of nodulation in Alnus incana (L.) Moench using double inoculations in plastic pouches and a slide technique to observe root hair deformation. Initially, the distribution of nodules between main and lateral roots appeared quite constant, independent of the concentration of inoculum (1 to 250 μg of crushed nodules plant−1). Susceptibility to infection after the second inoculation was restricted to lateral roots after the initial infections developed. When pre-existing nodules were excised before the second inoculation, subsequent nodules appeared to arise where infections had arrested at stages earlier than actual nodule emergence. We observed that root hairs formed postinoculation were very crowded and short with a pronounced deformation. No nodules were found later on this region of the root, suggesting a loss of susceptibility in this region. Split-root experiments with delays between inoculation of the first and second side of the root system showed irreversible, systemic inhibition of nodulation on the second side starting between 3 and 6 days after the inoculation of the first side. Only when compatible, infective strains were used in the first inoculation, was nodule formation inhibited after the second inoculation. We conclude that autoregulation of nodulation operates in Alnus incana and on a time scale similar to what is found in some legumes.
The supernodulating mutants of legumes lack the internal regulation of the number of symbiotic root nodules that harbour N2-fixing nodule bacteria. On one hand, these mutants represent an efficient tool for dramatic increase in the degree of rhizobial symbiosis development. The trait of released nodulation is often associated with the desirable resistance of nodule initiation and functioning to the inhibition by ambient nitrate. On the other hand, the more intense and stable atmospheric nitrogen fixation of supernodulated plants is devalued by plant growth depression that results from the disproportion between the photosynthetic capacity of the shoot and the catabolic demands of symbiotic nodules. The deleterious effects of excessive nodulation can be neutralised or alleviated by a breeding strategy aimed at creating an ideotype of N2-fixing legume. The growth depression can be diminished by the reduction in the nodule number typical for supernodulators, that is, 6–10-fold of the wild type, to the level found permissive for the particular crop. This shift should be accompanied with breeding aimed at the increased photosynthetic capacity of the shoot. Forage varieties of legumes represent a reserve of high photosynthetic and shoot growth capacity, thanks to a long-term breeding history for green biomass accumulation. Moreover, the deleterious effects of supernodulation are less perceived after introgression into the background of forage varieties in view of different criteria in their evaluation, such as nitrogen accumulation and biomass production per crop area unit. The growth of supernodulators can be further corrected by breeding for auxiliary traits such as long-vine shoot architecture, a longer vegetation period and late flowering. The same strategy is applicable to the compensation for inherent pleiotropic changes in plant development, which are often associated with primarily symbiotic mutations. Supporting evidence for the efficiency of the described approach has already been reported.
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.
Significance of Nodulation and Nitrogen Fixation Nodulation Autoregulation Mutants Agronomic Evaluation of Supernodulation Soybeans Conclusions, Models, and Future Perspectives Literature Cited
The development of spontaneous nodules, formed in the absence ofRhizobium and combined nitrogen, on alfalfa (Medicago sativa L. cv. Vernal) was investigated at the light and electron microscopic level and compared to that ofRhizobium-induced normal nodules. Spontaneous nodules were initiated from cortical cell divisions in the inner cortex next to the endodermis, i.e., the site of normal nodule development. These nodules, on uninoculated roots, were white multilobed structures, histologically composed of nodule meristems, cortex, endodermis, central zone and vascular strands. Nodules were devoid of intercellular or intracellular bacteria confirming microbiological tests. Early development of spontaneous nodules was initiated by series of anticlinal followed by periclinal divisions of dedifferentiated cells in the inner cortex of the root. These cells formed the nodular meristem from which the nodule developed. The cells in the nodule meristems divided unequally and differentiated into two distinct cell types, one larger type being filled with numerous membrane-bound starch grains, and the other smaller type with very few starch grains. There were no infection threads or bacteria in the spontaneous nodules at any stage of development. This size differentiation is suggestive of the different cell sizes seen inRhizobium-induced nodules, where the larger cell type harbours the invading bacteria and the smaller type is essential in supportive metabolic roles. The ontogenic studies further support the claim that these structures are nodules rather than aberrant lateral roots, and that plant possess all the genetic information needed to develop a nodule with distinct cell types. Our results suggest that bacteria and therefore theirnod genes are not necessarily involved in the ontogeny and morphogenesis of spontaneous and normal nodules in alfalfa.
Two strains of Bradyrhizobium japonicum were evaluated with five commercial cultivars of soybean (Clark, Crauford, Davis, Centaur, and Nessen) and one hypernodulating mutant NOD1-3. The hypernodulating NOD1-3 produced 30–50 times the number of nodules of commercial cultivars either inoculated with B. japonicum strain USDA 123 or RCR 3409. Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. In contrast, the shoots of Clark, Davis, Centaur and Nessen significantly inhibited nodule formation on the root of NOD1-3. However, Crauford shoots did not alter nodule formation on the roots of NOD1-3 as compared with self-grafts of NOD1-3. It appears that the shoot of NOD1-3 has the ability to alter autoregulatory control of nodulation of Clark and Davis cultivars, but not of Crauford, Centaur and Nessen. The results suggest that the regulation of nodulation in soybean cultivars Clark and Davis is controlled by the shoot factors, while the Crauford was root controlled. Reciprocal grafts between NOD1-3 and Centaur or Nessen indicate that both shoot and root factors are involved in regulation of nodulation. The results suggested that the regulation of nodulation did not depend on bradyrhizobial strains. The shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by shoot. Application of daidzein significantly enhanced the nodulation and nitrogenase activity of soybean cv. Clark. Root control of restricted nodulation of soybean cv. Centaur did not respond to the addition of daidzein in nutrient solution indicating that this character is not related to isoflavonoids. Therefore, autoregulation in Clark and Centaur plants may be separate events in legume–rhizobia symbiosis and regulated by different kinds of signals.
Two-dimensional gel electrophoresis of pea root and root hair proteins revealed the existence of at least 10 proteins present at elevated levels in root hairs. One of these, named RH2, was isolated and a partial amino acid sequence was determined from two tryptic peptides. Using this sequence information oligonucleotides were designed to isolate by PCR an RH2 cDNA clone. In situ hybridization studies with this cDNA clone showed that rh2 is not only expressed in root hairs, but also in root epidermal cells lacking these tubular outgrowths. During post-embryonic development the gene is switched on after the transition of protoderm into epidermis and since rh2 is already expressed in a globular pea embryo in the protoderm at the side attached to the suspensor, we conclude that the expression of rh2 is developmentally regulated. At the amino acid level RH2 is 95% homologous to the pea PR protein I49a. These gene encoding I49a is induced in pea pods upon inoculation with the pathogen Fusarium solani [12]. We postulate that rh2 contributes to a constitutive defence barrier in the root epidermis. A similar role has been proposed for chalcone synthase (CHS) and chitinase, pathogenesis-related protein that are also constitutively present in certain epidermal tissues.
The region of the root of soybean (Glycine max [L.] Merr. cv. Bragg) susceptible to nodule initiation by Bradyrhizobium japonicum Jordan USDA110 was examined by serial section and light microscopy to study the control of nodule development. Three successive susceptible regions separated by 24-h intervals were examined. Infection foci were catalogued within defined stages of nodule development (as defined by Calvert et al., Can. J. Bot., 62 (1984) 2375–2384) and their number and location determined after short (72 h) and long (13 days) post-inoculation periods. The overall distributions of cell division stages were similar for both short and long periods, indicating that regulatory mechanisms were active throughout root development. Early cell division foci with no visible meristems (stages I–II) appeared with similar frequency in each susceptible region. In contrast, the frequency of more mature cell division events showing developing meristems (stagesIII–IV) was decreased in the third susceptible region for both periods. The constant frequency of cell division stages I–II within the feedback regulated reduction coupled with the reduction of later stages implies that nodule initiation is slowed and that the transition from cell division stage II to stage III is also regulated.
Pea (Pisum sativum L.) cv. Frisson and its two supernodulating mutants P64 and P88 were inoculated with two different Rhizobium strains and nodule development was followed using a cytological clearing method. Supernodulating plant mutants were characterized by a faster appearance of nodules and a more extensive nodule distribution over the root system. General characteristics of nodule development were similar to the wild-type Frisson. The formation of nodule meristem was identified as a limiting stage of nodule development arrest. The microsymbiont was found to have an effect on the meristem activity of nodules, increasing nodule dry matter accumulation and branching.
Common bean (Phaseolus vulgaris L.) is a traditional crop in much of Latin America, where it is often planted into soils containing numerous, sometimes ineffective, indigenous rhizobia. The presence of these indigenous organisms can limit response to inoculation. Because of this, we have sought bean cultivars that will nodulate preferentially with the inoculant strain, and have previously reported on the preference between the bean cultivar RAB39 and strains of Rhizobium tropici. We have detailed this interaction using the inoculant-quality strain UMR1899. In the present study the root tip marking (RTM) technique was used to demonstrate that this preference in nodulation was evident, even when inoculation with UMR1899 was delayed up to 8 relative to that with Rhizobium etli UMR1632. In contrast to studies with other legumes, roots of RAB39 were not predisposed to nodulate with UMR1632, even though preexposed to this strain for considerable periods of time. The presence of UMR1899 actually reduced nodulation by UMR1632 substantially, even when inoculation with UMR1899 was significantly delayed. When UMR1899 and UMR1632 were applied to separate halves of a split-root system, the number of nodules on the side receiving UMR1632 was less than for the half root inoculated with UMR1899, but the differences were not significant. This suggests that the preference response is not systemic but requires proximity between the strains involved. UMR1899 produced more than 50% of the nodules even when the ratio of UMR1632:UMR1899 in the inoculant was 10:1. The results are further evidence of a stable and marked preference of RAB39 for UMR1899, which warrants a more detailed study at the field level.Key words: Phaseolus vulgaris L., common bean, delayed inoculation, strain preference, cell proportions.
A supernodulating and Nts (nitrate-tolerant symbiosis) symbiotic mutation of pea (Pisum sativum L.) line RisfixC was found to retain its expression in the distant genetic background of pea lines Afghanistan L1268, Zhodino E900, and cv. Arvika. This finding allowed for reliable scoring for the trait in mapping crosses. The RisfixC mutation was localized 8.2cM apart from SYM2 and cosegregated with molecular markers for SYM2-NOD3 region Psc923 and OA-1. Grafting experiments showed that supernodulation is root-determined, consistently with mutants in the NOD3 locus. Therefore, the mutation of RisfixC can be localized in gene NOD3. Like in other published nod3 alleles, the RisfixC mutation determines supernodulation when it is expressed in the root but not in the shoot. Supernodulated adventitious roots that are spontaneously formed in the wild-type scions on mutant rootstocks indicate that the descending systemic signal, which is inhibitory to nodule formation, is absent in this type of chimeric plants. Since the descending signal production in the wild-type shoot reflects the presence of the ascending root signal, the nod3-associated lesion must be located in the beginning of the systemic circuit regulating nodule number.
Root-nodule bacteria (rhizobia) are of great importance for nitrogen acquisition through symbiotic nitrogen fixation in a wide variety of leguminous plants. These bacteria differ from most other soil microorganisms by taking dual forms, i.e. a free-living form in soils and a symbiotic form inside of host legumes. Therefore, they should have a versatile strategy for survival, whether inhabiting soils or root nodules formed through rhizobia-legume interactions. Rhizobia generally contain large amounts of the biogenic amine homospermidine, an analog of spermidine which is an essential cellular component in most living systems. The external pH, salinity and a rapid change in osmolarity are thought to be significant environmental factors affecting the persistence of rhizobia. The present review describes the regulation of homospermidine biosynthesis in response to environmental stress and its possible functional role in rhizobia. Legume root nodules, an alternative habitat of rhizobia, usually contain a variety of biogenic amines besides homospermidine and the occurrence of some of these amines is closely associated with rhizobial infections. In the second half of this review, novel biogenic amines found in certain legume root nodules and the mechanism of their synthesis involving cooperation between the rhizobia and host legume cells are also described.
CLE peptides are potentially involved in nodule organ development and in the autoregulation of nodulation (AON), a systemic
process that restricts nodule number. A genome-wide survey of CLE peptide genes in the soybean glycine max genome resulted in the identification of 39 GmCLE genes, the majority of which have not yet been annotated. qRT-PCR analysis indicated two different nodulation-related CLE
expression patterns, one linked with nodule primordium development and a new one linked with nodule maturation. Moreover,
two GmCLE gene pairs, encoding group-III CLE peptides that were previously shown to be involved in AON, had a transient expression
pattern during nodule development, were induced by the essential nodulation hormone cytokinin, and one pair was also slightly
induced by the addition of nitrate. Hence, our data support the hypothesis that group-III CLE peptides produced in the nodules
are involved in primordium homeostasis and intertwined in activating AON, but not in sustaining it.
Nitrogen (N) is the mineral nutrient required in the greatest amount and its availability is a major factor limiting growth
and development of plants. As sessile organisms, plants have evolved different strategies to adapt to changes in the availability
and distribution of N in soils. These strategies include mechanisms that act at different levels of biological organization
from the molecular to the ecosystem level. At the molecular level, plants can adjust their capacity to acquire different forms
of N in a range of concentrations by modulating the expression and function of genes in different N uptake systems. Modulation
of plant growth and development, most notably changes in the root system architecture, can also greatly impact plant N acquisition
in the soil. At the organism and ecosystem levels, plants establish associations with diverse microorganisms to ensure adequate
nutrition and N supply. These different adaptive mechanisms have been traditionally discussed separately in the literature.
To understand plant N nutrition in the environment, an integrated view of all pathways contributing to plant N acquisition
is required. Towards this goal, in this review the different mechanisms that plants utilize to maintain an adequate N supply
are summarized and integrated.
Systemic autoregulation of nodulation in legumes involves a root-derived signal (Q) that is perceived by a CLAVATA1-like leucine-rich repeat receptor kinase (e.g. GmNARK). Perception of Q triggers the production of a shoot-derived inhibitor that prevents further nodule development. We have identified three candidate CLE peptide-encoding genes (GmRIC1, GmRIC2, and GmNIC1) in soybean (Glycine max) that respond to Bradyrhizobium japonicum inoculation or nitrate treatment. Ectopic overexpression of all three CLE peptide genes in transgenic roots inhibited nodulation in a GmNARK-dependent manner. The peptides share a high degree of amino acid similarity in a 12-amino-acid C-terminal domain, deemed to represent the functional ligand of GmNARK. GmRIC1 was expressed early (12 h) in response to Bradyrhizobium-sp.-produced nodulation factor while GmRIC2 was induced later (48 to 72 h) but was more persistent during later nodule development. Neither GmRIC1 nor GmRIC2 were induced by nitrate. In contrast, GmNIC1 was strongly induced by nitrate (2 mM) treatment but not by Bradyrhizobium sp. inoculation and, unlike the other two GmCLE peptides, functioned locally to inhibit nodulation. Grafting demonstrated a requirement for root GmNARK activity for nitrate regulation of nodulation whereas Bradyrhizobium sp.-induced regulation was contingent on GmNARK function in the shoot.
Root hairs, seen as an extension and surface enlargement of root epidermal cells, so-called trichoblasts, serve for the uptake of nutrients and provide anchorage into the soil. In addition, the young emerging root hairs are the first cells of legumes with which symbiotic soil bacteria of several genera, collectively called rhizobia, interact (Bhuvaneswari et al. 1980; Broughton and Perret 1999; Turgeon and Bauer 1985). This interaction leads to root nodules, new organs that are formed within the cortex of the root. Here, the bacteria are hosted in order to fix atmospheric nitrogen into ammonia for the benefit of the plant. In return, the host plant pays back by supplying the bacteria with organic nutrients. In several areas soil nitrogen is a limiting factor, and there symbiosis makes leguminous species significantly more competitive than other species.
In Rhizobium-legume symbiosis, the plant host controls and optimizes the nodulation process by autoregulation. Tn5 mutants of Rhizobium leguminosarum bv. phaseoli TAL 182 which are impaired at various stages of symbiotic development, were used to examine autoregulation in the common bean (Phaseolus vulgaris L.). Class I mutants were nonnodulating, class II mutants induced small, distinct swellings on the roots, and a class III mutant formed pink, bacterium-containing, but ineffective nodules. A purine mutant (Ade-) was nonnodulating, while a pyrimidine mutant (Ura-) formed small swellings on the roots. Amino acid mutants (Leu-, Phe-, and Cys-) formed mostly empty white nodules. Each of the mutants was used as a primary inoculant on one side of a split-root system to assess its ability to suppress secondary nodulation by the wild type on the other side. All mutants with defects in nodulation ability, regardless of the particular stage of blockage, failed to induce a suppression response from the host. Only the nodulation-competent, bacterium-containing, but ineffective class III mutant induced a suppression response similar to that induced by the wild type. Suppression was correlated with the ability of the microsymbiont to proliferate inside the nodules but not with the ability to initiate nodule formation or the ability to fix nitrogen. Thus, the presence of bacteria inside the nodules may be required for the induction of nodulation suppression in the common bean.
The time course of early infection events in Glycine max following inoculation with Rhizobium japonicum is described. Bacteria became attached to epidermal cells and root hairs within minutes of inoculation. Marked root hair curling occurred within 12 h. Infection thread formation was visible at the light microscope level of resolution about 24 h after inoculation. Infections were observed in short, tightly curled root hairs. These root hairs had not yet emerged at the time of inoculation. Infection threads appeared to originate in pockets formed by contact of the cell wall of the curled root hair with itself. Infection threads in the hairs were multiple and (or) branched. By 48 h, the infection thread(s) had progressed to the base of the root hair but had not yet penetrated into the cortex. Increases in cortical cell cytoplasm and in mitotic division occurred in advance of the penetrating infection thread. A nodule meristem developed in the outer cortex next to the infected root hair by 4 days and was accompanied by cell division across the cortex.
The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.
Alfalfa roots infected with four nodulation defective (Nod-) mutants of Rhizobium meliloti which were generated by transposon Tn5 mutagenesis were examined by light and electron microscopy. In one class of Nod- mutants, which we can nonreactive, the bacteria did not induce root hair curling or penetrate host cells. In a second class of Nod- mutants, which we call reactive, the bacteria induced some root hair curling and entered root epidermal cells, although no infection threads were formed. In addition, reactive Nod- mutants induced extensive root hair proliferation and hypertrophied roots. This study presents the details of the phenotype of the association between each mutant strain and alfalfa roots.
Subterranean clover plants possessing two equally infectible and robust lateral root systems ("split roots") were used in conjunction with several specific mutant strains (derived from Rhizobium trifolii ANU843) to investigate a systemic plant response induced by infective Rhizobium strains. This plant response controls and inhibits subsequent nodulation on the plant. When strain ANU843 was inoculated onto both root systems simultaneously or 24, 48, 72, or 96 h apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum but only when the delay period between inocula was greater than 24 h. Equal numbers of nodules were generated on both roots when ANU843 was inoculated simultaneously or 24 h apart. The ability to infect subterranean clover plants was required to initiate the plant inhibitory response since preexposure of one root system to non-nodulating strains did not retard the ability of the wild-type strain to nodulate the opposing root system (even when the delay period was 96 h). Moreover, the use of specific Tn5-induced mutants subtly impaired in their ability to nodulate demonstrated that the plant could effectively and rapidly discriminate between infections initiated by either the parent or the mutant strains. When inoculated alone onto clover plants, these mutant strains were able to infect the most susceptible plant cells at the time of inoculation and induce nitrogen-fixing nodules. However, the separate but simultaneous inoculation on opposing root systems of the parent and the mutant strains resulted in the almost complete inhibition of the nodulation ability of the mutant strains. We concluded that the mutants were affected in their competitive ability, and this finding was reflected by poor nodule occupancy when the mutants were coinoculated with the parent strain onto a single root system. Thus the split-root system may form the basis of a simple screening method for the ranking of competitiveness of various rhizobia on small seeded legumes.
Rhizobium nod genes are essential for root hair deformation and cortical cell division, early stages in the development of nitrogen-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 mimic the activity of compound(s) made upon the induction of the Rhizobium nod genes.
Soybean seeds [Glycine max (L.) Merr. ev. Bragg] were mutagenized with ethyl methanesulfonate. The M(2) progeny (i.e., the first generation after mutagenesis) of these seeds were screened for increased nodulation under high nitrate culture conditions. Fifteen independent nitrate-tolerant symbiotic (nts) mutants were obtained from 2500 M(2) families. In culture on sand with KNO(3), nodule mass and nodule number in mutant lines were several-fold those of the wild type cultured under the same conditions. Inheritance of the nts character through to subsequent generations was observed in the 10 mutants tested. Mutant nts382 also nodulated more than the wild type in the absence of nitrate. Furthermore, nitrate stimulated growth in both the wild type and nts382, and these lines had similar nitrate reductase activity. These results indicate that nts382 is affected in a nodule-development regulatory gene and not in a gene related to nitrate assimilation.
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 1 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 nodulation characteristics of soybean (Glycine max) mutant nts382 are described. The mutant nodulated significantly more than the parent cultivar Bragg in the presence and absence of several combined nitrogen sources (KNO(3), urea, NH(4)Cl, and NH(4)NO(3)). The number of nodules on the tap root and on lateral roots was increased in the mutant line. In the presence of KNO(3) and urea, nitrogenase activity was considerably higher in nts382 than in Bragg. Mutant plants were generally smaller than wild-type plants. Although nts382 is a supernodulator, inoculation with Rhizobium japonicum was necessary to induce nodule formation and both trial strains CB1809 (= USDA136) and USDA110 elicited the mutant phenotype. Segregation of M(3) progeny derived from a M(2) wild-type plant indicated that the mutant character is inherited as a Mendelian recessive. The mutant is discussed in the context of regulation of nodulation and of hypotheses that have been proposed to explain nitrate inhibition of nodulation.
The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts1116) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype.
Wild-type soybean (Glycine max [L] Merr. cv Bragg) and a nitrate-tolerant supernodulating mutant (nts382) were grown in split root systems to investigate the involvement of the autoregulation response and the effect of timing of inoculation on nodule suppression. In Bragg, nodulation of the root portion receiving the delayed inoculation was suppressed nearly 100% by a 7-day prior inoculation of the other root portion with Bradyrhizobium japonicum strain USDA 110. Significant suppression was also observed after a 24-hour delay in inoculation. Mutant nts382 in the presence of a low nitrate level (0.5 millimolar) showed little, if any, systemic suppression. Root fresh weights of individual root portions were similar for both wild type and nts382 mutant. When nts382 was grown in the absence of nitrate, a 7-day delay in inoculation resulted in only 30% suppression of nodulation and a significant difference in root fresh weight between the two sides, with the delayed inoculated side always being smaller. Nodulation tests on split roots of nts382, nts1116, and wild-type cultivars Bragg, Williams 82, and Clark demonstrated a difference in their systemic suppression ability. These observations indicate that (a) autoregulation deficiencies in mutant nts382 result in a reduction of systemic suppression of nodulation, (b) some suppression is detectable after 24 hours with a delayed inoculation, (c) the presence of low nitrate affects the degree of suppression and the root growth, and (d) soybean genotypes differ in their ability to express this systemic suppression.
The excision of nodules and root-tips from red clover inoculated with an effective strain of nodule bacteria leads to an increase in the number of nodules subsequently formed. This is thought to be due to the removal by excision of the source of an inhibitor. In the nodule the inhibitory activity appears to be centred in the growing-point and not in the bacterial tissue, since the excision of the nodular meristem only will stimulate further nodulation. The response to excision depends upon the number of excisions made and is inversely related to the inherent susceptibility of the individual plant.
Excision of nodules from plants inoculated with ineffective strains of bacteria has no influence on further nodulation, presumably because the growing-points of the nodules are abortive.
A field isolate of R. japonicum has recently been isolated that may help to answer the question of whether or not rhizobia need to attach to the host root surface in some particular way in order to infect and nodulate. This field isolate, designated 1007, was obtained from a large nodule in the crown region of a soybean plant from a local field (S.J. Vesper, T.V. Bhuvaneswari, W.D. Bauer, in preparation).
Clover and lucerne roots from plants grown in tube culture were examined for infection thread formation and nodule number. The number of infection threads was about equal to the number of nodules in Trifolium pratense L.; this relation was shown to hold for abundantly and sparsely nodulating plants and for bacterial inocula.nts producing large and small numbers of nodules.
We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1-10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4'-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21-24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a "hybrid" structure intermediate between a nodule and a lateral root.
When high dosages of wild-type Rhizobium meliloti RCR2011 were inoculated at two different times, 24 h apart, onto either the primary roots of alfalfa (Medicago sativa L.) seedlings or onto lateral roots on opposite sides of a split-root system, the number of nodules generated by the second inoculum was much smaller than the number generated by the first inoculum. These results provide evidence that alfalfa has an active, systemic mechanism for feedback control of nodulation. Non-nodulating mutants and delayed, weakly nodulating mutants did not elicit a discernable suppression of nodulation by subsequently inoculated wild-type cells. An appreciable number of Rhizobium infections thus seem required to elicit the suppressive response. Mutants in nodulation regions IIb and IIa nodulated extensively in the initially susceptible region of the root, but nodule initiation by these mutants was 100-1000 times less efficient, respectively, than the parent. Nodules formed by these mutants emerged 1 d later than normal. The IIb mutants elicited a relatively strong suppression of nodulation in younger parts of the root, but region-IIa mutants elicited only a weak response. These results indicate that elicitation of the regulatory response need not be proportional to nodule formation and imply that genes in region IIa play an important role in elicitation. At high dosages, the region-II mutants induced the development of thick, short roots in a considerably higher percentage of plants than the wild-type bacteria. Nodules generated by wild-type isolates and region-II mutants did not emerge in strict acropetal sequence, probably because some infections developed more slowly than others. Prior exposure of the root to non-nodulating mutants resulted in nodulation by the parent in regions of the root otherwise too mature to be susceptible, indicating that exposure to these mutants may affect the sequence of root development.
Root nodule tissues from an effective association and two ineffective associations between Medicago sativa (alfalfa) and Rhizobium meliloti were compared histologically. In nodule tissue formed by ineffective R. meliloti strain 1021726, rhizobia released into host cells sometimes became enveloped in large masses of an apparent polysaccaride-like material and did not develop into bacteroids. Bacteroids produced by this strain were smaller and showed fewer pleomorphic shapes than bacteroids in effective nodules. Nodules induced by this strain senesced much more rapidly than effective nodules.MnPL-480 is an alfalfa genotype that produces ineffective nodules with most effective strains of R. meliloti. These nodules were strikingly different than either effective or ineffective nodules induced by wild type R. meliloti strains. MnPl-480 nodules were tumour-like and most cells were filled with starch. In contrast to either effective or bacterial induced ineffective associations on other lines of alfalfa, MnPL-480 nodules had few infected cells and little proliferation of infection threads.Plant (MnPL-480) induced ineffectiveness appears to be expressed differently than bacterial (strain 1021726) induced ineffectiveness. In either association ineffectiveness may be expressed at more than one level within the nodule.
Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·103 bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (103 cells/plant) were co-inoculated with 106 cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular “signals” that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.
The infection of the root hairs of young seedlings of twelve species of Trifolium and of Vicia hirsuta was examined. The amount of infection (numbers of hairs containing infection threads) at 2 weeks varied much between species of host and was less affected by bacterial strain; host and strain differences were independent. In most hosts a high proportion of infections did not result in nodule formation. The relative rate of increase in numbers of infected hairs was constant before nodulation began. The duration of this pre-nodulation phase of exponential increase in infection, but not its rate, differed between species. Nodulation (and lateral root formation) caused an abrupt lowering of the initial rate of infection. Post-nodulation infection also increased exponentially. Low concentrations of nitrate nitrogen delayed nodulation and increased the number of hairs infected. Infected hairs were not randomly distributed along the root, infection beginning at a few well-separated points. Later infections occurred near these primary foci to give zones of infection which then spread up and down the root. The positions of nodules or lateral roots were not related to the primary foci of hair infection.
Summary Two methods have been developed in order to discriminate between lateral roots, nodules and root-derived structures which exhibit both root and nodule histological features and which can develop on legumes inoculated with certainRhizobium mutants. The first method, known as the “clearing method”, allows the observation by light microscopy of cleared undissected root-structures. The second, known as the “slicing method”, is a complementary technique which provides a greater degree of structural information concerning such structures. The two methods have proved invaluable in defining unequivocally the nature of the interaction between a rhizobial strain and a legume host.
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.
Roots of young soybean (Glycine max (L.) Merr.) seedlings inoculated with Rhizobium japonicum Kirchner USDA 110 ARS were examined in serial sections by light microscopy to ascertain the extent of infection. The location of each infection site was established in relation to the zones of root and root hair development at the time of inoculation. Each infection locus was classified as to its relative state of differentiation using a developmental scale encompassing the first 10 days of nodule development. Both the initiation and maturation of Rhizobium infections were found to be governed by the acropetal development of host root hairs. Regions of the root where mature root hairs were present at the time of inoculation were not susceptible to Rhizobium infection. Infections developed most frequently in root hairs which emerged shortly after inoculation. Many infections formed on the root but relatively few developed into nodules. Most infection loci which formed infection threads stopped developing at stages prior to meristem formation. A high proportion of the infection loci were pseudoinfections, i.e., localized areas of cortical cell division without infection thread formation. The maturation of infections in younger regions of the root was suppressed by prior exposure of older regions of the root to rhizobia. Development was suppressed at stages after meristem formation but before nodule emergence.
The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj
1) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj
1) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.
Root nodule initiation in Pisum sativum begins with cell divisions in the inner cortex at some distance from the advancing infection thread. After penetrating almost the entire cortex, the branches of the thread infiltrate the meristematic area previously initiated in the inner cortical cells. These cells are soon invaded by bacteria released from the infection thread and subsequently differentiate into non-dividing, bacteriod-containing cells. As the initial meristematic centre in the inner cortex is thus lost to bacteroid formation, new meristematic activity is initiated in neighbouring cortical cells. As development proceeds, more cortical layers contribute to the nodule, with the peripheral layer and apical meristem of the nodule not invaded by bacteria.
Lateral root primordia are initiated in a region separate from that in which nodules are formed, with the lateral primordia being closer to the root apex. This is interpreted to indicate that the physiological basis for nodule initiation is distinct from that for initiation of lateral roots. The role of a single tetraploid cell in nodule initiation is refuted, as is the existence of incipient meristematic foci in the root. It is suggested that the tetraploid cells in nodule meristems arise from pre-existing endoreduplicated cells, or by the induction of endoreduplication in diploid cortical cells by Rhizobium.
Separately grown soybean (Glycine max (L.) Merr. cv. Bragg) plants of identical or different nodulation genotype were approach-grafted just below the cotyledons. Five days later, their roots were inoculated at different times to study the effects of prior inoculation of one root system on nodulation of the other. Time-separated inoculation of isografts showed that nodulation in parental Bragg was strongly suppressed, while no suppression was observed in the supernodulating mutant nts382. In Bragg isografts, inoculation of one root system resulted in 50–59% decrease in nodulation on the other if inucolation of the latter was delayed by 3 days. Upon grafting the non-nodulating soybean derivative nod139, which fails to induce cortical cell divisions, did not elicit feedback suppressive responses in Bragg. Instead, nodules emerged 4–6 days earlier in grafted Bragg and nodulation was clearly stimulated in Bragg and nts382. In contrast the non-nodulating mutant nod49, which induces some cortical cell divisions that do not develop beyond the very early stages of nodule ontogeny, systemically suppressed nodule formation in Bragg, but did not have stimulatory effects on nodulation of nts382. These results suggest that: (a) cortical cell division foci of early ontogeny elicit feedback suppressive responses that control nodule number systemically; (b) feedback regulation is present in wild-type soybean but is defective in the supernodulating mutant; (c) a counteracting stimulatory response is induced early during pre-infection before the formation of a nodule primordia. A model for regulation of nodule formation in soybean is proposed, in which cortical cell division foci induce negative regulatory responses in the shoot through systemic signaling that ultimately results in blockage of further nodule development in the roots.
Legume root-nodules are differentiated organs composed of peripheral tissue containing vascular bundles, and a central tissue in which are located the nitrogen-fixing bacteroids. The morphogenesis of these eukaryotic organs is induced by a prokaryotic organism, Rhizobium, which is amenable to genetic analysis. Inoculation of lucerne seedlings with leucine-requiring (Leu⁻) mutants of R. meliloti resulted in the formation of ineffective nodules. In these nodules, bacteria were not released from the infection threads into the host cytoplasm. When urea was provided as a nitrogen source to compensate for the defect in nitrogen fixation, the nodules became anatomically similar to those of effective nodules induced by the wild-type strain. The fact that these nodules were induced by bacteria which remained sequestered in infection threads indicates that nodule morphogenesis can be triggered from a distance. We hypothesize the existence of a bacterial nodule organogenesis-inducing principle (NOIP) which can cross the plant cell wall and plasmalemma.
In nitrogen-fixing nodules the central tissue exhibited a ploidy gradient, while in ineffective Leu⁻ nodules it was found to be monosomatic. The initiation of nodule formation is therefore independent of polyploidy. Supplying the defective plant-bacterial system with l-leucine or one of its precursors, α-ketoisovalerate or α-ketoisocaproate, caused the release of rhizobia into the plant cytoplasm and a restoration of nitrogen fixation. In the central tissue infected cells were polyploid and enlarged, and uninfected cells remained small and contained small nuclei. Therefore induction of differentiation of the central tissue requires the presence of bacteria in the cytoplasm. We hypothesize the role of a bacterial central tissue differentiation inducing principle (CTDIP) which cannot pass from cell to cell.
The inhibiting effect of nitrate on nodulation and acetylene reduction of N2-fixing nodules is a well known phenomenon in legumes. By mutagenic treatment of seeds of the pea (Pisum sativum var. Rondo) variability was induced and in the M2-generation a mutant, efficiently nodulating in the presence of 15 mM KNO3, was selected. The mutant appeared to be monogenic and recessive and the gene was designated as nod3. Nodulation of mutant nod3 and wild-type cv. Rondo was investigated in the absence and the presence of KNO3 in the medium; the mutant showed much better nodulation under both culture conditions. The acetylene reduction per plant of mutant nod3, nodulated on nitrogen-free and on nitrate-containing medium, appeared to be much higher than that in cv. Rondo. The results, as well as the potential use of mutant nod3 in applied agricultural research, are discussed.
Pea mutants for nodulation have been obtained by treating seeds with ethyl methane sulfonate (EMS) followed by 2 screening procedures. In one, mutants resistant to nodulation (nod−), or with ineffective nodules (nod+, fix−) were obtained, whilst in the other 4 hypernodulated mutants (nod++) with 5–10 times more nodules than cv. Frisson and expressing a character of nitrate tolerant symbiosis (nts) were discovered. All mutations are under the control of single recessive genes. (nod−), (nod+, fix−) and (nod++, nts) mutations result from mutation events at 6, 7 and 1 different loci respectively.Grafting experiments showed the (nod−) and (nod+, fix−) phenotypes are associated with the root genotypes and that (nod++, nts) phenotype is associated with the shoot genotype.
Thesis (Ph. D. in Biology)--University of Dayton. Bibliography: leaves 129-138.
The nodB gene of Rhizobium meliloti encodes a 23.8-kDa protein that is conserved in several Rhizobium species. Monospecific polyclonal antibodies against NodB were used to localize this protein in the cytosol of R. meliloti and Escherichia coli cells containing nodABC genes. In comparison to the NodA and NodC proteins, NodB is synthesized in a disproportionately low amount. The NodA and NodB proteins are involved in generating small, heat-stable compounds that stimulate the mitosis of various plant protoplasts. Our experiments suggest that NodC is not involved in the synthesis of the factors. On the basis of their properties, we speculate that the factors are cytokinin-like substances.
Root cells of four common legumes were found to remain susceptible to nodulation by rhizobia for only a short period of time. Delayed inoculation experiments conducted with these legume hosts indicated that the initially susceptible region of the root became progressively less susceptible if inoculations were delayed by a few hours. Profiles of the frequency of nodule formation relative to marks indicating the regions of root and root hair development at the time of inoculation indicated that nodulation of Vigna sinensis (L.) Endl. cv California Black Eye and Medicago sativa L. cvs Moapa and Vernal roots was inhibited just below the region that was most susceptible at the time of inoculation. This result suggests the existence of a fast-acting regulatory mechanism in these hosts that prevents overnodulation. Nodulation in white clover may occur in two distinct phases. In addition to the transient susceptibility of preemergent and developing root hair cells, there appeared to be an induced susceptibility of mature clover root hair cells. A cell-free bacterial exudate preparation from Rhizobium trifolii cells was found to render mature root hair cells of white clover more rapidly susceptible to nodulation.
The number of nodules which develop on the primary root of soybean seedlings (Glycine max L. Merr) after inoculation with Rhizobium japonicum is substantially diminished in the region of the root developmentally 10 to 15 hours younger than the region maximally susceptible to nodulation at the time of inoculation. This rapid inhibition of nodulation has been investigated by inoculating soybean seedlings with rhizobia at two different times, 15 hours apart. Living R. japonicum cells, but not heterologous rhizobia or UV-killed cells of the homologous bacterium, were capable of eliciting the rapid inhibitory response. Nodulation responses to varying inoculum concentrations showed that bacterial dosages could be superoptimal, resulting in reduced nodulation and reduced inhibition of nodulation. When suspensions of R. japonicum were dripped uniformly onto the root surfaces, the degree of inhibition of nodulation in developmentally younger regions of the root was correlated with the number of nodules formed in the older and initially most susceptible region of the root. Nodulation in the developmentally younger region of the root, however, was affected very little if the first inoculum was restricted to contact with root cells in the region initially most susceptible to nodulation. The rapid regulatory response may be an important factor contributing to the clustering of nodules in the crown region of soybean roots in field-grown plants and the sparse nodulation commonly observed in younger regions of the root.
In a split-root system of soybeans (Glycine max L. Merr), inoculation of one half-side suppressed subsequent development of nodules on the opposite side. At zero time, the first side of the split-root system of soybeans received Rhizobium japonicum strain USDA 138 as the primary inoculum. At selected time intervals, the second side was inoculated with the secondary inoculum, a mixture of R. japonicum strain USDA 138 and strain USDA 110. In a short-day season, nodulation by the secondary inoculum was inhibited 100% when inoculation was delayed 10 days. Nodulation on the second side was significantly suppressed when the secondary inoculum was delayed for only 96 hours. In a long-day season, nodule suppression on the second side was highly significant, but not always 100%. Nodule suppression on the second side was not related to the appearance of nodules or nitrogenase activity on the side of split-roots which were inoculated at zero time. When the experiments were done under different light intensities, nodule suppression was significantly more pronounced in the shaded treatments.
The inoculation of soybean (Glycine max L.) roots with Bradyrhizobium japonicum produces a regulatory response that inhibits nodulation in the younger regions of the roots. By exposing the soybean roots to live homologous bacteria for only a short period of time, the question of whether or not early interactions of rhizobia with root cells, prior to infection, elicit this regulatory response has been explored. B. japonicum cells mixed with infective bacteriophages were applied to the roots and then 6 or 24 hours later roots were again inoculated with phage-resistant rhizobia. Mixing of the rhizobia and bacteriophages caused bacterial lysis in 6 to 8 hours and allowed the bacteria to act as live symbionts on the root for only a few hours. However, the interaction of live homologous bacteria with the soybean roots for a few hours did not cause inhibition of nodulation in the younger regions of the roots. Results of these experiments indicate that the self-regulatory response in soybean is not rapidly produced by the early, pre-infection, interactions between rhizobia and the root cells.
Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells.
Since NO(3) (-) availability in the rooting medium seriously limits symbiotic N(2) fixation by soybean (Glycine max [L.] Merr.), studies were initiated to select nodulation mutants which were more tolerant to NO(3) (-) and were adapted to the Midwest area of the United States. Three independent mutants were selected in the M(2) generation from ethyl methanesulfonate or N-nitroso-N-methylurea mutagenized Williams seed. All three mutants (designated NOD1-3, NOD2-4, and NOD3-7) were more extensively nodulated (427 to 770 nodules plant(-1)) than the Williams parent (187 nodules plant(-1)) under zero-N growth conditions. This provided evidence that the mutational event(s) affected autoregulatory control of nodulation. Moreover, all three mutants were partially tolerant to NO(3) (-); each retained greater acetylene reduction activity when grown hydroponically with 15 millimolar NO(3) (-) than did Williams at 1.5 millimolar NO(3) (-). The NO(3) (-) tolerance did not appear to be related to an altered ability to take up or metabolize NO(3) (-), based on solution NO(3) (-) depletion and on in vivo nitrate reductase assays. Enhanced nodulation appeared to be controlled by the host plant, being consistent across four Bradyrhizobium japonicum strains tested. In general, the mutant lines produced less dry weight than the control, with root dry weights being more affected than shoot dry weights. The nodulation trait has been stable through the M(5) generation in all three mutants.
The development of root nodule symbioses. The infection process Biology of Nitrogen Fixation . North-Holland Pub Co Regulation of the soybean-Rhizobium synthesis by shoot and root factors
- Pj Dart
- A Mathews
- Da Day
- As Carter
- Gresshoff
Dart PJ (1974) The development of root nodule symbioses. The infection process. In A Quispel, ed, Biology of Nitrogen Fixation. North-Holland Pub Co, Amsterdam, pp 381-429 13. Delves AC, Mathews A, Day DA, Carter AS, Gresshoff PM (1986) Regulation of the soybean-Rhizobium synthesis by shoot and root factors. Plant Physiol 82: 588-590
Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti Distribution of Bradyrhizobium-induced cell divisions in soybean
- Me Dudley
- Tw Jacobs
- Long
- Sr
- G Caetano-Anolles
- Joshi
- Gresshoff
- G Roth
- Stacey
- Newton
Dudley ME, Jacobs TW, Long SR (1987) Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti. Planta 171: 289-301 17. Gerahty N, Caetano-Anolles G, Joshi PA, Gresshoff PM (1990) Distribution of Bradyrhizobium-induced cell divisions in soybean. In PM Gresshoff, LE Roth, G Stacey, WE Newton, eds, Nitrogen Fixation: Achievements and Objectives. Chapman and Hall, New York, p 737
Physiological studies on nodule formation. II. The influence of delayed inoculation on the rate of nodulation in red clover Nutman PS (1952) Studies on the physiology of nodule formation . III. Experiments on the excision of root-tip and nodules The relation between root hair infection by 372
- Nutman
Nutman PS (1949) Physiological studies on nodule formation. II. The influence of delayed inoculation on the rate of nodulation in red clover. Ann Bot N S 13: 261-283 28. Nutman PS (1952) Studies on the physiology of nodule formation. III. Experiments on the excision of root-tip and nodules. Ann Bot N S 16: 81-102 29. Nutman PS (1962) The relation between root hair infection by 372 Plant Physiol. Vol. 95, 1991 NODULATION CONTROL IN ALFALFA Rhizobium and nodulation in Trifolium and Vicia. Proc R Soc Lond 156: 122-137
Optimiza-tion ofsurface sterilization forlegumeseed
- G Caetano-Anolles
- G Favelukes
- Bauer
Caetano-Anolles G, Favelukes G, Bauer WD (1990) Optimiza-tion ofsurface sterilization forlegumeseed.CropSci 30: 708- 71
cv Vernal) seeds were pro-vided by R. Van Keuren, Agronomy Department Seeds were surface sterilized with ethanol and mercuric chloride and germinated in water-agar plates (4) This procedure was shown to reduce seed-borne contamination to a minimum
- Alfalfa
Alfalfa (Medicago sativa (L.) cv Vernal) seeds were pro-vided by R. Van Keuren, Agronomy Department, Ohio State University, Wooster, OH. Seeds were surface sterilized with ethanol and mercuric chloride and germinated in water-agar plates (4). This procedure was shown to reduce seed-borne contamination to a minimum (7).
The development of root nodule symbioses. The infection process Biology of Nitrogen Fixa-tion
- Dart
- Pj
Dart PJ (1974) The development of root nodule symbioses. The infection process. In A Quispel, ed, Biology of Nitrogen Fixa-tion. North-Holland Pub Co, Amsterdam, pp 381-429
Development of Bradyrhizobium infections in a supernodulating and non-no-dulating mutant of soybean
- Carroll Bj Gresshoff
- Pm
Mathews A, Carroll BJ, Gresshoff PM (1989) Development of Bradyrhizobium infections in a supernodulating and non-no-dulating mutant of soybean (Glycine max (L.) Merr.). Proto-plasma 150: 40-47
Distribution of Bradyrhizobium-induced cell divisions in soy-bean Nitrogen Fixation: Achievements and Objectives
- N G Gerahty
- Pa Joshi
- Pm Gresshoff
- Gresshoff
- Le Roth
- Stacey
- We
- Newton
Gerahty N, Caetano-Anolles G, Joshi PA, Gresshoff PM (1990) Distribution of Bradyrhizobium-induced cell divisions in soy-bean. In PM Gresshoff, LE Roth, G Stacey, WE Newton, eds, Nitrogen Fixation: Achievements and Objectives. Chapman and Hall, New York, p 737
Regulation of the soybean-Rhizobium synthesis by shoot and root factors
- Mathews A Day
- Carter As Da
- Gresshoff
- Pm
Delves AC, Mathews A, Day DA, Carter AS, Gresshoff PM (1986) Regulation of the soybean-Rhizobium synthesis by shoot and root factors. Plant Physiol 82: 588-590
ALFALFA Rhizobium and nodulation in Trifolium and Vicia
- Nodulation
- In
NODULATION CONTROL IN ALFALFA
Rhizobium and nodulation in Trifolium and Vicia. Proc R Soc
Lond 156: 122-137
The relation between root hair infection by 372
- Ps Nutman
Nutman PS (1962) The relation between root hair infection by
372
Distribution of Bradyrhizobium-induced cell divisions in soybean
- N Gerahty
- G Caetano-Anolles
- Pa Joshi
- Pm Gresshoff
- Le Gresshoff
- Roth
- Stacey
Gerahty N, Caetano-Anolles G, Joshi PA, Gresshoff PM (1990)
Distribution of Bradyrhizobium-induced cell divisions in soybean. In PM Gresshoff, LE Roth, G Stacey, WE Newton, eds,
Nitrogen Fixation: Achievements and Objectives. Chapman
and Hall, New York, p 737
Physiological studies on nodule formation. II. The influence of delayed inoculation on the rate of nodulation in red clover
- Ps Nutman
Nutman PS (1949) Physiological studies on nodule formation.
II. The influence of delayed inoculation on the rate of nodulation in red clover. Ann Bot N S 13: 261-283