Autoregulation of soybean–Bradyrhizobium nodule symbiosis is controlled by shoot or/and root factors

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.     

Root Isoflavonoid Response to Grafting between Wild-Type and Nodulation-Mutant Soybean Plants

It was previously reported that the hypernodulating soybean (Glycine max [L.] Merr.) mutants, derived from the cultivar Williams, had higher root concentration of isoflavonoid compounds (daidzein, genistein, and coumestrol) than did Williams at 9 to 12 days after inoculation with Bradyrhizobium japonicum. These compounds are known inducers of nod genes in B. japonicum and may be involved in subsequent nodule development. The current study involving reciprocal grafts between NOD1-3 (hypernodulating mutant) and Williams showed that root isoflavonoid concentration and content was more than twofold greater when the shoot genotype was NOD1-3. When grafted, NOD1-3 shoots also induced hypernodulation on roots of both Williams and NOD1-3, while Williams shoots induced normal nodulation on both root genotypes. This shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by the shoot. In contrast, the nonnodulating characteristic of the NN5 mutant was strictly root controlled, based on reciprocal grafts. Delayed inoculation (7 days after planting) resulted in greater nodule numbers on both NOD1-3 and Williams, compared with a seed inoculation treatment. The nodulation pattern of grafted plants was independent of whether the shoot portion was derived from inoculated seed or uninoculated seed, when grafted at day 7 onto seedling roots derived from inoculated seed. This observation, coupled with the fact that no difference existed in nodule number of NOD1-3 and Williams until after 9 days from seed inoculation, indicated that if isoflavonoids play a role in differential nodulation of the hypernodulating mutant and the wild type, the effect is on advanced stages of nodule ontogeny, possibly related to autoregulation, rather than on initial infection stages.     

Concentration-dependent influence of quercetin on nodulation process and the main characteristics of soybean inoculated with Bradyrhizobium japonicum

Soybean plants cv. Corsoy were grown in greenhouse conditions on sterilized quartz sand. They were inoculated with Bradyrhizobium japonicum, strain 542. The plants were treated with different concentrations of quercetin (ranging from 10 nM to 1M) at regular intervals during the experiment. The experiment was terminated at flower development. The following parameters, important for symbiosis efficiency were determined: shoot, root and nodule weights, nodule number, total leghemoglobin in the nodules,total nitrogen and soluble protein concentrations in shoots and roots, as well as chlorophyll concentration in the leaves.The results obtained partly confirmed the earlier findings that quercetin inhibits nodulation since increasing quercetin concentration decreased the number of nodules. However, at very low concentrations, quercetin stimulated the number of nodules. Quercetin also exerted a stimulating influence on other characteristics of the plant and nodules which did not correlate with nodule number and quantity of N fixed. These are: nodule weight, leghemoglobin concentration, total soluble protein content in shoots and roots as well as shoot and root weight.     

Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors

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.     

Bradyrhizobium strains and the nodulation,nodule efficiency and growth of soybean (Glycine max L.) in Egyptian soils

Six strains and a commercial inoculant ofBradyrhizobium japonicum were evaluated in association withGlycine max (L.) cultivar Clark. Inoculated and uninoculated plants were grown in pot and field experiments. Nodules were counted and weighed and roots and shoots were separated and analysed for total nitrogen. In pot experiments, two of six bacterial strains were superior to the other four, and to the commercial inoculant (Nitragin) in promoting greater root and top growth and plant nitrogen accumulation. In the field experiment, there were indications that environmental conditions may have affected nodulation by the bacteria. The strains could be divided into three groups according to nodule efficiencies, accumulation of plant dry matter, and total nitrogen content. The greater variations in nodule efficiencies of the tested strains could be attributed to the quantities of bacteroid, cytosol protein and leghaemoglobin in the nodules.     

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Effect of abscisic acid application on root isoflavonoid concentration and nodulation of wild-type and nodulation-mutant soybean plants

Isoflavonoids (daidzein, genistein, and coumestrol) are involved in induction of nod genes in Bradyrhizobium japonicum and may be involved in nodule development as well. Abscisic acid (ABA) may also impact nodulation since ABA is reportedly involved in isoflavonoid synthesis. The current study was conducted to evaluate whether ABA plays a role in differential nodulation of a hypernodulated soybean (Glycine max L. Merr.) mutant and the Williams parent. Exogenous ABA application resulted in a decrease in nodule number and weight in both lines. Isoflavonoid concentrations were also markedly decreased in response to ABA application in both inoculated and noninoculated soybean roots. The inoculation treatment itself resulted in a marked increase in isoflavonoid concentrations of NOD1-3, regardless of ABA levels, while only slight increases occurred in Williams. The nodule numbers of both soybean lines across several ABA concentration treatments were highly correlated with the concentration of all three isoflavonoids. However, differences in internal levels of ABA between lines were not detected when grown in the absence of external ABA additions. It is concluded that differential nodule expression between the wild type and the hypernodulated mutant is not likely due to differential ABA synthesis.     

A new perfusion system for the measurement and characterization of potential rates of soil nitrification

The effect of two isoflavonoids, coumestrol and daidzein which are present in aseptically grown roots and root exudates of soybean, was tested on some rhizospheric microorganisms. It was found that coumestrol promotes the growth ofR. japonicum USDA 138 (about 30%) andR. leguminosarum (about 15%) whereas it inhibits the growth ofAgrobacterium tumefaciens (about 50%) andPseudomonas sp. (about 20%). The following microorganisms were unaffected by this molecule:R. japonicum W505,Agrobacterium radiobacter, Micrococcus luteus andCryptococcus laurentii. It was found that daidzein promotesR. japonicum USDA 138 growth (about 20%) and inhibitsPseudomonas sp. growth (about 20%); other microorganisms were unaffected. In addition, coumestrol favoured the formation of ‘coccoids’ cells byRhizobium japonicum USDA 138 which could be the infective state of this strain. It seems that this compound is able to help nodulation of soybean by aRhizobium strain. This result supports the work of Peterset al. (1986) and Redmondet al. (1986) who show that flavones present in plant exudates induces expression of nodulation genes in Rhizobium.     

Shoot versus Root Signal Involvement in Nodulation and Vegetative Growth in Wild-Type and Hypernodulating Soybean Genotypes

Grafting studies involving Williams 82 (normally nodulating) and NOD1-3 (hypernodulating) soybean (Glycine max [L.] Merr.) lines and Lablab purpureus were used to evaluate the effect of shoot and root on nodulation control and plant growth. A single- or double-wedge graft technique, with superimposed partial defoliation, was used to separate signal control from a photosynthate supply effect. Grafting of hypernodulated soybean shoots to roots of Williams 82 or L. purpureus resulted in increased nodule numbers. Grafting of two shoots to one root enhanced root growth in both soybean genotypes, whereas the nodule number was a function of shoot genotype but not of the photosynthetic area. In double-shoot, single-root-grafted plants, removing trifoliolate leaves from either Williams 82 or NOD1-3 shoots decreased root and shoot dry matter, attributable to decreased photosynthetic source. Concurrently, Williams 82 shoot defoliation increased the nodule number, whereas NOD1-3 shoot defoliation decreased the nodule number on both soybean and L. purpureus roots. It was concluded that (a) soybean leaves are the dominant site of autoregulatory signal production, which controls the nodule number; (b) soybean and L. purpureus have a common, translocatable, autoregulatory control signal; (c) seedling vegetative growth and nodule number are independently controlled; and (d) two signals, inhibitor and promoter, may be involved in controlling legume nodule numbers.     

Water use and chemical composition of ryegrass (Lolium) cultivars

Summary Significant differences in total dry matter yields of shoots and roots were found between 11 ryegrass (Lolium) cultivars grown in a glasshouse. Although shoot yield varied significantly between individual cultivars there was no overall difference between the annual and perennial cultivars; whereas for roots, the yields of the perennial plants were much smaller than those of the annual types. Water use (g H₂O g total DM^–1) also varied significantly between cultivars. However, there was no relationship between efficient water use and dry matter production.No significant differences were found in shoot composition between the cultviars for nitrogen, phosphorus, and potassium; however, concentrations of sulphur, magnesium, calcium, and sodium varied significantly. Sodium concentrations were generally higher in the annual compared to the perennial cultivars. For roots only nitrogen, phosphorus, and sulphur differed significantly between cultivars. Of the elements only calcium in the shoots was shown to be related to water use. Thus cultivars which were low users of water also had significantly lower calcium concentrations in their shoots. Water use appeared to affect the absorption of calcium by the root to a far greater extent than the transport from roots to shoot. An apparent relationship between magnesium concentration in the shoots and water use was shown to be due to the close association of magnesium with calcium in the plant.     

The autoregulation of nodulation mechanism is related to leaf development

To understand the autoregulation of nodulation (AON) system, in which leguminous plants control the nodule number, we examined the details of the characteristics of hypernodulation soybean mutants NOD1-3 and NOD3-7. A microscopic study showed that NOD1-3 and NOD3-7 produced small-size leaves due to the smaller number of leaf cells, compared with the Williams parent. These phenotypes were not affected by inoculation with bradyrhizobia or nitrate supply. The AON signaling might be related to the control system of leaf cell proliferation. This hypothesis was strongly supported by the finding that activation of AON in wild types by inoculation leads to an increase in the cell number of leaves.     

Rice cultivar evaluation for phosphorus use efficiency

Phosphorus deficiency is one of the most growth-limiting factors in acid soils in various parts of the world. The objective of this study was to screen 25 rice cultivars (Oryza sativa L.) at low, medium, and high levels of soil P. Number of tillers, root length, plant height, root dry weight and shoot dry weight were related to tissue P concentrations, P uptake and P-use efficiency. Shoot weight was found to be the plant parameter most sensitive to P deficiency. Significant cultivar differences in P use efficiency were found. Phosphorus use efficiency was higher in roots than shoots and decreased with increasing levels of soil P. Positive correlations were found among growth parameters such as plant height, tillers, root and shoot weight, and P content of roots and shoots. These results indicate selection of rice cultivars for satisfactory performance under low P availability can be carried out using shoot and root dry weight as criteria.     

Shoot apex removal does not alter autoregulation of Modulation in soybean

The suppression of new nodule development in soybean (Glycine max (L.) Merr.) has been previously demonstrated to involve the shoot through reciprocal grafts between the wild-type cultivar Bragg and its supernodulating mutant nts382. Using the same grafting technique, but modified through the excision of the shoot apex region and emerging lateral shoots, we show here that autoregulation of nodule number still existed despite apex removal. This radical treatment lowered total nodule number per plant as well as root, shoot and nodule dry weight. Bragg shoots grafted onto nts382 roots gave wild-type nodulation (26 nodules, 15mg total nodule mass) as compared to nts382 shoots grafted onto Bragg roots (340 nodules, 277 mg total nodule mass). Specific nodule mass differed between supernodulating (about 0·5-1·0mg per nodule) and wild-type nodulating (2·3 mg per nodule) plants. In contrast to other growth characteristics, apex removal did not affect specific nodule size, except in plants with wild-type shoots and nts382 (supernodulation) roots. Apex removal only slightly affected the percentage of nodule weight per total root weight in nts382, but had a severe effect in wild type. Growth reductions varied between the normal and supernodulating plants. The fact that autoregulation of nodulation still functions in plants devoid of functional shoot apices suggests that the autoregulation signal may not be derived from the apex regions and that the leaf may be a likely source.     

Occurence and regulation of Calvin cycle enzymes in non-autotrophic Beggiatoa strains

A field isolate of Bradyrhizobium japonicum which failed to attach polarly or firmly to soybean roots was compared with the laboratory isolate I-110 for its relative rate of growth, piliation, attachment and nodulation. Both isolates grew at a comparable rate in yeast extract-gluconate medium as well as in soybean root exudate, produced comparable amounts of soybean lectin binding polysaccharide, infected through curled root hairs and developed effective nodules. Approximately 5% of cells in cultures of 110 possessed pili but none were detected in cultures of 1007 by electron microscopy. Light microscopic observations of root hairs from roots exposed to 1007 and 110 inoculum showed no polarly attached cells of 1007 and approximately 100 cells of 110 polarly attached per mm root hair length. Plate counting of firmly bound cells released by sonication indicated that the number of 1007 cells firmly adhering was at least 1000-fold lower than the number of 110 cells attached. The significance of polar, firm and weak attachment in the initiation of symbiotic interactions is discussed.Dedicated to the menory of Harry E. Calvert     

When Does the Self-Regulatory Response Elicited in Soybean Root after Inoculation Occur?

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.     

Exclusion of inefficient Bradyrhizobium japonicum serogroups by soybean genotypes

Soybean [Glycine max (L.) Merr.] forms a symbiosis with serogroups of Bradyrhizobium japonicum that differ in their dinitrogen fixing abilities. The objectives of this study were to identify soybean genotypes that would restrict nodulation by relatively inefficient serogroups indigenous to a large portion of the southeastern USA, and then characterize the nodulation responses of selected genotypes with specific bradyrhizobial strains under controlled conditions. From field screening trials followed by controlled single and competitive inoculations of serogroups USDA 31, 76 and 110, twelve soybean genotypes out of 382 tested were identified with varying levels of exclusion abilities. Soybean nodule occupancies and nodulation characteristics were influenced by plant genotype, environment (i.e. field or greenhouse), bradyrhizobial serogroup, and location of nodules (i.e. tap or lateral root). The cultivar Centennial sustains high seed yields even though it nodulates to a high degree with the inefficient serogroup USDA 31. In contrast, data from the released cultivars Braxton, Centennial and Coker 368 indicate that they may have been selected to exclude the inefficient serogroup USDA 76 from their tap root nodules, possibly contributing to high seed yield.     

The <Emphasis Type="Italic">Medicago truncatula SUNN</Emphasis> Gene Encodes a <Emphasis Type="Italic">CLV1</Emphasis>-like Leucine-rich Repeat Receptor Kinase that Regulates Nodule Number and Root Length

Four Medicago truncatula sunn mutants displayed shortened roots and hypernodulation under all conditions examined. The mutants, recovered in three independent genetic screens, all contained lesions in a leucine-rich repeat (LRR) receptor kinase. Although the molecular defects among alleles varied, root length and the extent of nodulation were not significantly different between the mutants. SUNN is expressed in shoots, flowers and roots. Although previously reported grafting experiments showed that the presence of the mutated SUNN gene in roots does not confer an obvious phenotype, expression levels of SUNN mRNA were reduced in sunn-1 roots. SUNN and the previously identified genes HAR1 (Lotus japonicus) and NARK (Glycine max) are orthologs based on gene sequence and synteny between flanking sequences. Comparison of related LRR receptor kinases determined that all nodulation autoregulation genes identified to date are the closest legume relatives of AtCLV1 by sequence, yet sunn, har and nark mutants do not display the fasciated clv phenotype. The M. truncatula region is syntenic with duplicated regions of Arabidopsis chromosomes 2 and 4, none of which harbor CLV1 or any other LRR receptor kinase genes. A novel truncated copy of the SUNN gene lacking a kinase domain, RLP1, is found immediately upstream of SUNN and like SUNN is expressed at a reduced level in sunn-1 roots.     

Effects of cadmium and nickel on in vivo carbon dioxide exchange rate of pigeon pea (Cajanus cajan L.)

Effects of acidic soil factors (Al, H-ion, Mo, and Mn) upon the soybean (Glycine max (L.) Merr. cv. Essex)/Bradyrhizobium japonicum symbiosis were examined in acidified soil. Plants were grown under full sunlight in pots containing N-deficient soil (pH 6.7) or similar soil amended with sufficient Al₂(SO₄)₃ or elemental S to give soil pH values of 4.8 and 4.6, respectively, and water-extractable Al levels of 30 and 14 M, respectively. Other treatments consisted of the addition of inorganic N or inoculation with commercial or locally-isolated B. japonicum. Acidification did not reduce shoot or root weights of plants receiving inorganic N but reduced (P0.05) shoot and root dry weights, nodule dry weights and numbers, shoot N concentrations, and chlorophyll levels of inoculated plants. Shoot dry weights and nodulation of inoculated plants were greater (P0.05) in Al₂(SO₄)₃-amended soil than in S-amended soil. Addition of Mo was not beneficial. It was concluded that reduced plant growth was caused by the effects of acidified soil on nodulation and that H-ion toxicity was probably the most limiting factor. Effects of Al, Mn, or Mo appeared less likely.     

Hybrid Weakness in Phaseolus vulgaris L. II. Disruption of Root-Shoot Integration

We have been examining the importance of the root system on shoot growth and development using a developmentally disabled hybrid of the common bean Phaseolus vulgaris L. Parental cultivars (P. Vulgaris cv. Redkloud of Mesoamerican origin, and P. vulgaris cv. Batt of Andean origin) grow normally, but crosses produce F1 hybrids exhibiting hybrid weakness associated with reduced root and shoot growth. In this study, applications of benzylaminopurine (BAP) to roots of F1 hybrids increased the number of root tips and leaves. Reciprocal grafting was used to study the effects of the root system on shoots. Grafting of roots of the Mesoamerican cultivar onto shoots of F1 hybrids increased the cytokinin concentrations in leaves of F1 hybrids and removed the characteristics associated with hybrid weakness. To determine whether factors in the xylem sap enhanced leaf growth, leaf discs were incubated on sap collected from Mesoamerican and Andean cultivars. Sap from Mesoamerican plants enhanced the growth of leaf discs excised from F1 hybrids more than sap collected from Andean cultivars. Estimates of the transport of zeatin riboside (ZR)–type cytokinins from roots of F1 hybrids indicated that transport out of hybrid roots was reduced compared with those transported out of Mesoamerican or Andean roots. Results suggest that ZR-type cytokinins are involved in hormonal integration between roots and shoots of P. vulgaris and that one of the barriers to hybridization between Andean and Mesoamerican landraces is related to hormone transport. Received October 15, 1998; accepted May 12, 1999