Flavonoid-responsive nodY-lacZ expression in three phylogenetically different Bradyrhizobium groups

Previously, restriction fragment length polymorphism analysis using the nodD1YABC gene probe showed the genetic diversity of common nodD1ABC gene regions of Bradyrhizobium japonicum , Bradyrhizobium elkanii , and the Thai soybean Bradyrhizobium. The nodD1 sequences of representative strains of the 3 groups differed phylogenetically, suggesting that responses of NodD1 proteins of the 3 Bradyrhizobium groups to diverse flavonoids may differ. To confirm this hypothesis, 6 representative strains were chosen from the 3 Bradyrhizobium groups. Six reporter strains were constructed, all carrying the pZB32 plasmid, which contains a nod box and the nodY-lacZ fusion of B. japonicum USDA 110. Differences in nodY-lacZ expression among the strains in response to 37 flavonoid compounds at various concentrations were evaluated. Of those compounds, prunetin (4′,5-dihydroxy-7-methoxyisoflavone) and esculetin (6,7-dihydroxycoumarin) were identified as Bradyrhizobium group-specific nod gene inducers. Esculetin showed nod gene induction activities unique to Thai Bradyrhizobium strains. The levels of nodY-lacZ induction among B. japonicum and Thai Bradyrhizobium strains increased with increasing concentration of prunetin, whereas, those in B. elkanii strains did not.

Aldonic Acids: A Novel Family of nod Gene Inducers of Mesorhizobium loti, Rhizobium lupini, and Sinorhizobium meliloti

Molecular signals, such as flavonoids (or nonflavonoid type), nod gene-inducers, and bacterial lipochitin oligosac-charides (LCOs) act as modulators of species specificity during early stages of infection in Rhizobium spp.-legume interactions. The fact that signaling in Lupinus albus remains to be determined prompted us to investigate the flavonoid signal responsible for nod gene induction in Rhizobium lupini. A screening method was used based on the measurement of β-galactosidase activity of R. lupini strains harboring nodC::lacZ fusions in the presence of (i) authentic lupin isoflavones, (ii) carbohydrate-like inducers, and (iii) high-pressure liquid chromatography (HPLC)-fractionated lupin seed effusates and root exudates, as putative nod gene inducers. The results indicate that both erythronic and tetronic acids (4-C sugar acids) led to low but significant increases in β-galactosidase activities, compared with the controls. In addition, lupi-wighteone, a monoprenylated isoflavone, exerts a synergistic effect with the carbohydrate-like inducers, compared with other isoflavone treatments. The natural occurrence of aldonic acids in L. albus root exudates and seed effusates has been demonstrated by HPLC analysis. When tested with nodC::lacZ fusions, tetronic acid resulted in nod gene induction in Sinorhizobium meliloti. In addition, a combination of luteolin and tetronic acid promotes further increases in S. meliloti nod gene expression, as shown by β-galactosidase assays. Incorporation studies with [14C]LCO precursors confirmed the inductive role of both erythronic and tetronic acids in promoting LCO biosynthesis in R. lupini cultures, and of tetronic acid in Mesorhizobium loti and S. meliloti. Hydrolysis of the LCOs with various enzymes substantiated their putative identities. These results are discussed in relation to the impact of these unusual signal molecules on our knowledge of flavonoid signaling in Rhizobium-legume symbiosis.

The adaptation of Medicago polymorpha to a range of edaphic and environmental conditions: effect of temperature on growth, and acidity stress on nodulation and nod gene induction

Three experiments were conducted with Medicago polymorpha collected from a diverse range of sites in Sardinia, to investigate the relationship between edaphic and environmental conditions at collection sites, and the growth and nodulation of the genotype. Growth response to 2 temperature regimes (20/15�C, 11/9�C) was greater for genotypes collected from high altitude than for those from 1350 m. Shoot yield was more depressed by low temperature and more stimulated by high temperature in high altitude than low altitude genotypes. All genotypes nodulated when exposed to considerable acidity stress (pH 5.4) and 1 mmol Ca/L in solution culture. However, there were differences between genotypes in nodulation reduction caused by exposure to acidity. These differences were not well related to the soil pH at the site of collection, or to the effects of pH on the ability of root exudates from the genotypes to stimulate nod gene expression in Rhizobium meliloti.

Acid-Tolerant Species of Medicago Produce Root Exudates at Low pH Which Induce the Expression of Nodulation Genes in Rhizobium meliloti

An acid rhizosphere may interfere with the transcription of nodulation genes in Rhizobium spp. by modifying the production of legume root exudates, or the rhizobial response to them. Certain annual species of Medicago (M. murex, M. polymorpha) can nodulate better in acid soils and solutions than other species (M. truncatula, M. littoralis). The mechanisms of this acid tolerance in nodulation are poorly understood. Root exudates collected at pH 5.8 and pH 6.0 from acid-tolerant species of annual medics induced the expression of nodulation genes in Rhizobium meliloti as indicated from a lacZ gene fusion, whereas exudates from acid-sensitive species grown at these pH values displayed decreased induction activity. For the acid-sensitive host, M. truncatula, increasing the Ca concentration from 0.5 to 5.0 mM at pH 5.8 increased the nod-gene induction activity of its exudates, but there was no effect at higher pH. There was no effect of Ca or pH on the nod-gene induction activity of exudates collected from M. murex. These results indicate a likely mechanism underlying differences in ability to nodulate under acid stress among annual species of Medicago.