Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii.

Rhizobium trifolii mutants containing Escherichia coli lac gene fusions to specific nodulation (nod) genes were used to characterise phenolic compounds secreted from the roots of white clover (Trifolium repens) plants. These compounds either had stimulatory or inhibitory effects upon the induction of the nod genes. The stimulatory compounds were hydroxylated flavones and the most active compound was 7,4'-dihydroxyflavone. The inhibitory compounds present in white clover root exudates were umbelliferone (a coumarin) and formononetin (an isoflavone). Transcriptional activation of nod gene promoters in response to short exposures (3 h) of 7,4'-dihydroxyflavone was growth phase dependent; cells in early log phase were highly responsive to flavone additions in vitro and nod gene induction could be detected within 20 min of exposure at 5 x 10 M. Cells in other growth phases were generally unresponsive. A 10-fold molar excess of umbelliferone to 7,4'-dihydroxyflavone resulted in complete inhibition of nod gene induction. Some commercially-obtained flavones were found to have weak stimulatory activity but could also inhibit nod gene induction by more effective stimulatory compounds. Strong stimulatory and inhibitory compounds all possessed a 7-hydroxy moiety and showed other structural similarities. This suggested that there was one binding site for these compounds. Because the response to these compounds was rapid, we propose that these phenolics act at the bacterial membrane or that an active uptake system is involved.