Abstract

The symbiotic interaction between Rhizobium lupini and Lupinus albus results in the formation of root nodules where nitrogen fixation takes place. In these associations, molecular signals such as flavonoids (or non-flavonoid type), nod gene-inducers, and bacterial lipochitooligosaccharides (LCOs) are known to act as modulators of species-specificity in the early stages of infection. Of the several known Rhizobium -legume symbioses, signalling in Lupinus remains to be determined. Using a screening method based on the in vitro ability of the flavonoid inducer to adsorb onto the membranes of its symbiont, indicated that derrone, wighteone and lupiwighteone were adsorbed most onto R. lupini membranes. A second screening method, based on the ability of the inducer molecule to stimulate the growth rate of the symbiont, showed that 0.5 oM each of derrone, lupalbigenin, genistein monoprenyls, licoisoflavone A and lupinalbin A, resulted in a significant growth stimulation of R. lupini when cultivated in a minimal medium. A third strategy involved monitoring Ý-galactosidase activity of R. lupini strains harboring nodC :: lacZ fusions, in the presence of (a) authentic lupin isoflavones, (b) carbohydrate-like inducers, and (c) HPLC-fractionated lupin seed effusates and root exudates as putative nod gene inducers. The results indicated that both erythronic and tetronic acids (C4 sugar acids) led to low, but significant increases in Ý-galactosidase activities, as compared with the controls. In addition, lupiwighteone, a monoprenylated isoflavone, seems to exert a synergistic effect with the carbohydrate-like inducers, as compared with other isoflavone treatments. Incorporation studies of [ 14 C]LCO-precursors into induced R. lupini cultures, confirmed the inductive role of erythronic and tetronic acids, as well as their synergistic effect when combined with lupiwighteone or derrone. Enzymatic hydrolysis of the R. lupini LCOs with various enzymes substantiated their putative identities. Similar incorporation experiments were carried out on other Rhizobium spp . in order to assess the role of aldonic acids in promoting LCO biosynthesis. These results are discussed in relation to the impact of these unusual signal molecules on our knowledge of signalling in Rhizobium -legume symbiosis by flavonoids and LCOs.