Effect of exogenous flavonoids on nodulation of pea (Pisum sativum L.)

Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv. viciae were tested for their effect on symbiosis formation with garden pea as the host. A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation. The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30. A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 micro g ml(-1) nutrient solution. At 10 micro g ml(-1), the highest concentration tested, naringenin was already inhibitory. By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 micro g ml(-1), as well as by another tested nod gene inducer, hesperetin. The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth. Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules. The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms. Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin-conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity. By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity. Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 microg ml(-1), however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation-inhibiting high flavonoid concentrations. The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations. This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes. The losses were 99, 96.4, and 90% of the initial concentration of 10 micro g ml(-1) for naringenin, hesperetin, and quercetin, respectively.     

Jasmonic acid stimulates the expression of nod genes in Rhizobium

Jasmonates and salicylic acid are considered to be signal molecules that induce a variety of plant genes involved in wound or defence response, as well as affecting nos promoter activity. In this paper we examined whether these chemicals could also affect nod genes from isogenic rhizobia strains. Isogenic strains contain the Rhizobium leguminosarum nodA promoter fused to the lacZ gene of Escherichia coli and differ only in the source of the regulatory nodD gene. Naringenin, jasmonic acid and methyl jasmonate induced expression of nod genes in strain RBL1284 and salicylic acid showed no activity alone or when used in combination with other compounds; addition of naringenin + jasmonic acid produced a synergistic effect. Results obtained with strain RBL5284 were similar to those for RBL1284 albeit the combination of naringenin with the other compounds markedly inhibited nod gene expression. Whereas RBL5283 responded to naringenin with a strong induction, jasmonic acid, methyl jasmonate or salicylic acid showed no significant responses. The inhibitory effect of salicylic acid on nod gene expression indicates that the induction mechanism of jasmonic acid, methyl jasmonate, N-propyldihydrojasmonate and naringenin is probably different from that of salicylic acid.     

The relationship between nodulin gene expression and the Rhizobium nod genes in Vicia sativa root nodule development

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.     

Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells.

Reporter strains of soil-borne bacteria were used to test the hypothesis that chemicals released by root border cells can influence the expression of bacterial genes required for the establishment of plant-microbe associations. Promoters from genes known to be activated by plant factors included virE, required for Agrobacterium tumefaciens pathogenesis, and common nod genes from Rhizobium leguminosarum bv viciae and Rhizobium meliloti, required for nodulation of pea (Pisum sativum) and alfalfa (Medicago sativum), respectively. Also included was phzB, an autoinducible gene encoding the biosynthesis of antibiotics by Pseudomonas aureofaciens. The virE and nod genes were activated to different degrees, depending on the source of border cells, whereas phzB activity remained unaffected. The homologous interaction between R. leguminosarum bv viciae and its host, pea, was examined in detail. Nod gene induction by border cells was dosage dependent and responsive to environmental signals. The highest levels of gene induction by pea (but not alfalfa) border cells occurred at low temperatures, when little or no bacterial growth was detected. Detached border cells cultured in distilled water exhibited increased nod gene induction (ini) in response to signals from R. leguminosarum bv viciae.     

在结瘤基因nodA启动子内发现了两个不同功能的结构区域

在豌豆根瘤菌（ＲｈｉｚｏｂｉｕｍＬｅｇｕｍｉｎｏｓａｒｕｍ）结瘤基因ｎｏｄＡ的启动子内发现了具有两个不同功能的结构区域,其一我们称为Ｒｉｐ,在ｎｏｄＡ诱导表达中起着关键作用,可能识别经诱导剂作用而发生构象变化的调控蛋白ＮｏｄＤ,另一为ＲＩＰ缺失后留下的,我们称为ＲＰ区,只要ＲＰ存在,不需要诱导剂,ＮｏｄＤ蛋白即能导致结瘤基因ｎｏｄＡ的表达。因此该区可能识别原始构象的调控蛋白ＮｏｄＤ。     

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.     

Rhizobia catabolize nod gene-inducing flavonoids via C-ring fission mechanisms.

Gas chromatographic and mass spectrometric analyses of derivatized culture medium extracts were used to identify the products of flavonoid metabolism by rhizobia. A number of Rhizobium species and biovars degraded their nod gene-inducing flavonoids by mechanisms which originated in a cleavage of the C-ring of the molecule and which yielded conserved A- and B-ring products among the metabolites. In contrast, Pseudomonas putida degraded quercetin via an initial fission in its A-ring, and Agrobacterium tumefaciens displayed a nonspecific mode of flavonoid degradation which yielded no conserved A- or B-ring products. When incubated with rhizobia, flavonoids with OH substitutions at the 5 and 7 positions yielded phloroglucinol as the conserved A-ring product, and those with a single OH substitution at the 7 position yielded resorcinol. A wider range of structures was found among the B-ring derivatives, including p-coumaric, p-hydroxybenzoic, protocatechuic, phenylacetic, and caffeic acids. The isoflavonoids genistein and daidzein were also degraded via C-ring fission by Rhizobium fredii and Rhizobium sp. strain NGR234, respectively. Partially characterized aromatic metabolites with potential nod gene-inducing activity were detected among the products of naringenin degradation by Rhizobium leguminosarum bv. viciae. The initial structural modification of nod gene-inducing flavonoids by rhizobia can generate chalcones, whose open C-ring system may have implications for the binding of inducers to the nodD gene product.     

Inoculation of pea (Pisum sativum L.) by Rhizobium leguminosarum bv. viceae preincubated with naringenin and hesperetin or application of naringenin and hesperetin directly into soil increased pea nodulation under short season conditions

In short season areas, low soil temperature is the major limiting factor for symbiotic nitrogen fixation of legume. One greenhouse and four field experiments were conducted in 1999 to determine whether the pre-incubation of Rhizobium leguminosarum bv. viceae with hesperetin and naringenin or application of these compounds onto the seed surface or into the seed furrow at the time of planting can increase pea nodulation and final grain yield. The results from these experiments clearly indicated that application of naringenin and hesperetin by either pre-incubating R. leguminosarum bv. viceae prior to inoculation of plant or directly applying onto the seed surface or into seed furrow at the time of planting can increase pea nodulation, and plant pod numbers. Interactions existed between symbiotic signal compounds and pea cultivars or R. leguminosarum bv. viceae strains. However, there was no impact on the final grain yield by the treatments from the field experiments. The effects of these treatments on the final grain yield have to be farther tested.     

A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin

Rhizobium bacteria produce different surface polysaccharides which are either secreted in the growth medium or contribute to a capsule surrounding the cell. Here, we describe isolation and partial characterization of a novel high molecular weight surface polysaccharide from a strain of Rhizobium leguminosarum that nodulates Pisum sativum (pea) and Vicia sativa (vetch) roots. Carbohydrate analysis showed that the polysaccharide consists for 95% of mannose and glucose, with minor amounts of galactose and rhamnose. Lectin precipitation analysis revealed high binding affinity of pea and vetch lectin for this polysaccharide, in contrast to the other known capsular and extracellular polysaccharides of this strain. Expression of the polysaccharide was independent of the presence of a Sym plasmid or the nod gene inducer naringenin. Incubation of R. leguminosarum with labelled pea lectin showed that this polysaccharide is exclusively localized on one of the poles of the bacterial cell. Vetch roots incubated with rhizobia and labelled pea lectin revealed that this bacterial pole is involved in attachment to the root surface. A mutant strain deficient in the production of this polysaccharide was impaired in attachment and root hair infection under slightly acidic conditions, in contrast to the situation at slightly alkaline conditions. Our data are consistent with the hypothesis that rhizobia can use (at least) two mechanisms for docking at the root surface, with use of a lectin-glycan mechanism under slightly acidic conditions.     

Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.     

Rhizobium leguminosarum bv. viciae genotypes interact with pea plants in developmental responses of nodules, roots and shoots

The variability of the developmental responses of two contrasting cultivars of pea (Pisum sativum) was studied in relation to the genetic diversity of their nitrogen-fixing symbiont Rhizobium leguminosarum bv. viciae. A sample of 42 strains of pea rhizobia was chosen to represent 17 genotypes predominating in indigenous rhizobial populations, the genotypes being defined by the combination of haplotypes characterized with rDNA intergenic spacer and nodD gene regions as markers. We found contrasting effects of the bacterial genotype, especially the nod gene type, on the development of nodules, roots and shoots. A bacterial nod gene type was identified that induced very large, branched nodules, smaller nodule numbers, high nodule biomass, but reduced root and aerial part development. The plants associated with this genotype accumulated less N in shoots, but N concentration in leaves was not affected. The results suggest that the plant could not control nodule development sustaining the energy demand for nodule functioning and its optimal growth. The molecular and physiological mechanisms that may be involved are discussed.     

Flavonoids induce Rhizobium leguminosarum to produce nodDABC gene-related factors that cause thick, short roots and root hair responses on common vetch.

Rhizobium leguminosarum produced a factor(s) that caused thick, short roots (Tsr phenotype) as well as root hair induction (Hai phenotype) and deformation (Had phenotype) in Vicia sativa plants upon incubation with root exudate or with one of the nod gene inducers naringenin or apigenin; this was a nodDABC gene-dependent process. Detection of the Hai and Had phenotypes was much more sensitive than that of the Tsr phenotype.