Nod Bj-V (C18:1, MeFuc) production by Bradyrhizobium japonicum (USDA110, 532C) at suboptimal growth temperatures

Nod factors (Lipo-chitooligosaccharides, or LCOs) act as bacteria-to-plant signal molecules that modulate early events of the Bradyrhizobium-soybean symbiosis. It is known that low root zone temperature inhibits the early stages of this symbiosis; however, the effect of low soil temperature on bacteria-to-plant signaling is largely uninvestigated. We evaluated the effect of low growth temperatures on the production kinetics of Nod factor (LCO) by B. japonicum. Two strains of B. japonicum, 532C and USDA110, were tested for ability to synthesize Nod Bj-V (C(18:1), MeFuc) at three growth temperatures (15, 17 and 28 degrees C). The greatest amounts of the major Nod factor, Nod Bj-V (C(18:1), MeFuc), were produced at 28 degrees C for both strains. At 17 and 15 degrees C, the Nod factor production efficiency, per cell, of B. japonicum 532C and USDA110 was markedly decreased with the lowest Nod factor concentration per cell occurring at 15 degrees C. Strain 532C was more efficient at Nod factor production per cell than strain USDA 110 at all growth temperatures. The biological activity of the extracted Nod factor was unaffected by culture temperature. This study constitutes the first demonstration of reduced Nod factor production efficiency (per cell production) under reduced temperatures, suggesting another way that lower temperatures inhibit establishment of the soybean N(2) fixing symbiosis.     

A host-specific bacteria-to-plant signal molecule (Nod factor) enhances germination and early growth of diverse crop plants

Lipo-chitooligosaccharides (LCOs), or Nod factors, are host-specific bacteria-to-plant signal molecules essential for the establishment of a successful N(2)-fixing legume-rhizobia symbiosis. At submicromolar concentrations Nod factors induce physiological changes in host and non-host plants. Here we show that the Nod factor Nod Bj V(C18:1,MeFuc) of Bradyrhizobium japonicum 532C enhances germination of a variety of economically important plants belonging to diverse botanical families: Zea mays, Oryza sativa (Poaceae), Beta vulgaris (Chenopodaceae), Glycine max, Phaseolus vulgaris (Fabaceae), and Gossypium hirsutum (Malvaceae), under laboratory, greenhouse and field conditions. Similar increases in germination were observed for filtrates of genistein-induced cultures of B. japonicum 532C, while non-induced B. japonicum, induced Bj 168 (a nodC mutant of B. japonicum deficient in Nod factor synthesis) or the pentamer of chitin did not invoke such responses, demonstrating the role of Nod factor in the observed effects. In addition, three out of four synthetic LCOs evaluated also promoted germination of corn, soybean and Arabidopsis thaliana seeds. LCO also enhanced the early growth of corn seedlings under greenhouse conditions. These findings suggest the possible use of LCOs for improved crop production.     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

In vitro induction of lipo-chitooligosaccharide production in Bradyrhizobium japonicum cultures by root extracts from non-leguminous plants

Bradyrhizobium japonicum can form a N2-fixing symbiosis with compatible leguminous plants. It can also act as a plant-growth promoting rhizobacterium (PGPR) for non-legume plants, possibly through production of lipo-chitooligosaccharides (LCOs), which should have the ability to induce disease resistance responses in plants. The objective of this work was to determine whether non-leguminous crop plants can induce LCO formation by B. japonicum cultures. Cultures treated with root extracts of soybean, corn, cotton or winter wheat were assayed for presence and level of LCO. Root extracts of soybean, corn and winter wheat all induced LCO production, with extracts of corn inducing the greatest amounts. Root washings of corn also induced LCO production, but less than the root extract. These results indicated that the stimulation of non-legume plant growth by B. japonicum could be through the production of LCOs, induced by materials excreted by the roots of non-legume plants.     

Nod factor enhances calcium uptake by soybean

Inoculation with rhizobia or application of Nod factors (lipo-chitooligosaccharides, LCOs) causes transient increases in cytosolic calcium concentration in root hairs of legume plants. We conducted experiments to evaluate whether application of LCO and inoculation with rhizobia improved (45)CaCl(2) uptake into soybean (Glycine max [L.] Merr.) leaves. Roots of soybean seedlings with one developing trifoliolate were immersed in Murashige and Skoog (MS) basal liquid medium containing treatment solutions and (45)CaCl(2), and the plants were incubated under continuous light. After 24 h, leaf samples were taken, and their radioactivity levels were determined. Addition of NodBj-V (C18:1 MeFuc) at a concentration of 10(-7) M increased (45)Ca(2+) uptake. Inoculation with genistein-induced Bradyrhizobium japonicum strain 532C and USDA3 also increased (45)Ca(2+) uptake; whereas, inoculation with strain Bj-168, a nodC-mutant incapable of producing LCO, did not. Rhizobia that do not normally nodulate soybean, i.e. Rhizobium leguminosarum, and Sinorhizobium meliloti did not affect calcium uptake, nor did the tetramer or pentamer of chitosan, or lumichrome. Surprisingly, Rhizobium sp. NGR234, which can nodulate some types of soybean, although without effective N(2)-fixation, also did not affect calcium uptake. This work suggests that the rhizobial symbiosis, in addition to its known role in provision of nitrogen fixation, also improves early calcium uptake into soybean plants.     

Isolation and characterization of the major nod factor of Bradyrhizobium japonicum strain 532C

Bradyrhizobium japonicum 532C nodulates soybean effectively under cool Canadian spring conditions and is used in Canadian commercial inoculants. The major lipo-chitooligosaccharide (LCO), bacteria-to-plant signal was characterized by HPLC, FAB-mass spectroscopy MALDI-TOF mass spectroscopy and revealed to be LCO Nod Bj-V (C18:1, MeFuc). This LCO is produced by type I strains of B. japonicum and is therefore unlikely to account for this strains superior ability to nodulate soybean under Canadian conditions. We also found that use of yeast extract mannitol medium gave similar results to that of Bergerson minimal medium.     

Differential response of soybean (Glycine max (L.) Merr.) genotypes to lipo-chito-oligosaccharide Nod Bj V (C(18:1) MeFuc)

Lipo-chito-oligosaccharides (LCOs) are bacteria-to-plant signal molecules essential for the establishment of rhizobia-legume symbioses. LCOs invoke a number of physiological changes in the host plants, such as root hair deformation, cortical cell division and ontogeny of complete nodule structures. The responses of five soybean cultivars to Nod BJ: V (C(18:1) MeFuc) isolated from Bradyrhizobium japonicum strain 532C were studied with a new technique. Two distinct types of root hair deformation were evident (i) bulging, in which root hairs were swollen at the tip or at the base depending on the cultivars and (ii) curling. The nodulating capacity of B. japonicum 532C varied among cultivars. Cultivars that produced a bulging reaction when treated with LCO had fewer nodules and the roots had low phenol contents. Cultivars that produced curling had higher numbers of nodules and the roots had higher amounts of phenol. Further, the roots of cultivars that showed root hair bulging were able to degrade LCO much faster than cultivars that manifested curling. The results of the present study establish relationships among the type of LCO-induced root hair deformation, root system LCO-degrading ability and nodulation capacity of soybean cultivars.     

Evidence for the production of chemical compounds analogous to nod factor by the silicate bacterium Bacillus circulans GY92

Silicate bacteria are generally placed in the species Bacillus circulans and are widely used in biological fertilisers and biological leaching. The bacteria can form conspicuous amounts of extracellular polysaccharides in nitrogen-free media or in the presence of substrates with large C/N ratios. Using high performance liquid chromatography, we have shown that B. circulans produced a new peak/compound when induced with the plant-to-bacteria signal molecule genistein. This material co-eluted with the lipo-chitooligosaccharide (Nod Bj-V (C18:1, MeFuc)) of Bradyrhizobium japonicum. This compound exhibited root hair deformation activity on soybean, which is characteristic of lipo-chitooligosaccharides (LCOs). We propose that this might be an LCO or closely related compound with similar biological activity.     

Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti

Medicago truncatula is a model legume plant that interacts symbiotically with Sinorhizobium meliloti, the alfalfa symbiont. This process involves a molecular dialogue between the bacterium and the plant. Legume roots exude flavonoids that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range. In turn, nod genes control the synthesis of lipo-chito-oligosaccharides (LCOs), Nod factors, which are bacteria-to-plant signal molecules mediating recognition and nodule organogenesis. M. truncatula roots or seeds have been treated with Nod factors and hydroponically growing seedlings have been inoculated with a limiting population of S. meliloti. It has been shown that submicromolar concentrations of Nod factors increase the number of nodules per plant on M. truncatula. Compared with roots, this increase is more noticeable when seeds are treated. M. truncatula seeds are receptive to submicromolar concentrations of Nod factors, suggesting the possibility of a high affinity LCO perception system in seeds or embryos as well.     

Response of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots to lipo-chitooligosaccharide from <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and other chitin-like compounds

Lipo-chitooligosaccharides (LCOs) are bacteria-to-plant signals required for the establishment of rhizobia–legume nitrogen fixing symbioses. The ability of LCO [Nod Bj V (C_18:1, MeFuc)] isolated from B. japonicum (strain 532C), and of oligomers of chitosan (tetramer, pentamer) and chitin (pentamer) to affect the developmental morphology of roots in Arabidopsis thaliana (L.) Heynh ecotype Columbia (Col-0) was assessed using an interactive scanner-based image analysis system. LCOs have been shown to play a role in plant organogenesis at nanomolar concentrations. LCO and the chitin pentamer promoted root growth and development in Arabidopsis at concentrations of 10 nM and 100 μM, respectively. The LCO treated Arabidopsis plants had about 35% longer roots than untreated control plants. Similarly, treatment with 100 μM chitin pentamer (CHIT5) resulted in 26% longer roots than the untreated plants; however, chitosan oligomer (CH4 or CH5) treated plants did not differ from the control plants at either concentration (100 or 1 μM). Both LCOs and the chitin pentamer at higher concentrations increased root surface area, mean root diameter and number of root tips. However, leaf area increase was observed only in plants treated with LCO at 10 nM.     

Nod factors stimulate seed germination and promote growth and nodulation of pea and vetch under competitive conditions

Nod factors are lipochitooligosaccharide (LCO) produced by soil bacteria commonly known as rhizobia acting as signals for the legume plants to initiate symbiosis. Nod factors trigger early symbiotic responses in plant roots and initiate the development of specialized plant organs called nodules, where biological nitrogen fixation takes place. Here, the effect of specific LCO originating from flavonoid induced Rhizobium leguminosarum bv. viciae GR09 culture was studied on germination, plant growth and nodulation of pea and vetch. A crude preparation of GR09 LCO significantly enhanced symbiotic performance of pea and vetch grown under laboratory conditions and in the soil. Moreover, the effect of GR09 LCOs seed treatments on the genetic diversity of rhizobia recovered from vetch and pea nodules was presented.     

NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals

The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2- O -methylfucose residue at the reducing-terminal N -acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro ( Macroptilium atropurpureum ). Here we show that transfer of nodZ to Rhizobium leguminosarum biovar (bv) viciae , which produces LCOs that are not modified at the reducing-terminal N -acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N -acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureum Glycine soja Vigna unguiculata and Leucaena leucocephala . Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.     

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host-specificity gene

Rhizobia secrete specific lipo-chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N -acetyl- d -glucosamine of LCOs is substituted at C₆ with 2- O -methyl- l -fucose which can be acetylated or sulphated. We identified a flavonoid-inducible locus on the symbiotic plasmid pNGR234 a that contains a new nodulation gene, noeE which is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non-sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum . Furthermore, mutation of noeE (NGRΔ noeE  ) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus . The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRΔ noeE leads to the production of a mixture of LCOs that are sulphated on C₆ of the reducing terminus and sulphated on the 2- O -methylfucose residue. Together, these findings show that noeE is a host-specificity gene which probably encodes a fucose-specific sulphotransferase.     

结瘤因子的研究进展

结瘤因子是由根瘤菌产生的一类信号分子,它们在结瘤的起始阶段发挥着十分重要的作用。新近的研究结果证明结瘤因子大分子骨架上的不同侧链基团是决定细菌与宿主植物间相互识别的关键因素,根瘤菌细胞中一系列结瘤基因编码能够合成Lipo-chio-oligosaccharides(LCOs)的各种酶类,进而确定结瘤信号分子的特定结构。目前,一系列令人兴奋的实验结果表明：LCOs不仅可促进豆科作物的生物固氮作用,对一些非豆科作物的细胞分裂作用等同样具有刺激作用。对根瘤菌结瘤因子的研究显然有助于进一步了解细菌与植物的相互作用机理,并进而为农业生产为直接利益。本文在综述这方面的研究进展同时,还就瘤菌,豆科作用和结瘤信号分子之间的相互作用机理,以及根际促生细菌,水杨酸和结瘤信号分子之间的可能关系进行了理论分析。     

Rhizobial Nod factors stimulate somatic embryo development in Picea abies

 Nod factors are lipochitooligosaccharides (LCOs) secreted by rhizobia. Nod factors trigger the nodulation programme in a compatible host. A bioassay was set up to test how crude (NGR234) and purified (NodS) Nod factors influence cell division and somatic embryogenesis in a conifer, Norway spruce (Picea abies). The Nod factors promoted cell division in the absence of auxin and cytokinin. More detailed studies showed that NodS stimulates development of proembryogenic masses from small cell aggregates and further embryo development. However, stimulation was only observed in low-density cell cultures. Our data suggest that rhizobial Nod factors substitute for conditioning factors in embryogenic cultures of Norway spruce. Received: 20 January 1999 / Revision received: 26 March 1999 / Accepted: 27 April 1999     

Aberrant nodulation response of Vigna umbellata to a Bradyrhizobium japonicum NodZ mutant and nodulation signals.

The (Brady)rhizobium nodulation gene products synthesize lipo-chitin oligosaccharide (LCO) signal molecules that induce nodule primordia on legume roots. In spot inoculation assays with roots of Vigna umbellata, Bradyrhizobium elkanii LCO and chemically synthesized LCO induced aberrant nodule structures, similar to the activity of these LCOs on Glycine soja (soybean). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast, the synthetic LCO-IV(C16:0), which has previously been shown to be active on G. soja, was inactive on V. umbellata. A B. japonicum NodZ mutant, which produces LCO without 2-O-methyl fucose at the reducing end, was able to induce nodule structures on both plants. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the NodZ mutant acted cooperatively to produce nodulelike structures on V. umbellata roots.     

昆明西山滇青冈林内滇青冈种子库动态的研究

通过对昆明西山滇青冈林内滇青冈种子库的跟踪取样调查和种子埋藏试验,对滇青冈种子库的动态进行了研究。昆虫在种子成熟前侵入种子,经种子雨进入种子库时已有71.8%的种子失去萌发能力。种子雨输入种子库的绝大部分种子停留在表面种子库,其中48.55%的种子被虫害,25.36%被某些非生物或生物搬运,17.39%的腐烂,8.7%的被动物当场取食,没有种子萌发,影响种子库动态的各种因子的作用大小在时间上是变化。被搬运的种子中,有4.9%的由表面种子库转移到埋藏种子库。土层是滇青冈种子的安全生境,土壤种子库的存在时间超过250天。埋入土壤的试验种子一直处于静止状态,到6月雨季后有80%种子萌发,20%的腐烂。萌发种子数是当年产种子的0.26%。滇青冈林内的滇青冈种子库是季节性的,种子库对种群个体的补充作用是有限的。     

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.     

Molecular mechanisms of Nod factor diversity

The rhizobia–legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo-chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non-allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co-evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on 'symbiosis islands', which are symbiotic regions associated with movable elements.