Rhizobium inoculation induces condition-dependent changes in the flavonoid composition of root exudates from Trifolium subterraneum

Rapid induction of chalcone synthase (predominantly CHS5) gene expression occurs within 6 h following the inoculation of Rhizobium leguminosarum bv. trifolii strain ANU843 on Trifolium subterraneum or wounding of plants (C. G. R. Lawson, M. A. Djordjevic, J. J. Weinman and B. G. Rolfe. 1994. Molecular Plant-Microbe Interactions 7, 498-507). Experiments were conducted under the same conditions to examine the time of onset of synthesis and excretion of flavonoids that might result from this early CHS expression. Flavonoids in root tissues and root exudates were examined by HPLC analysis and the ability of fractionated and unfractionated material to induce nodulation gene expression in Rhizobium measured. There were no detectable changes in nod-gene-inducing activity of individual HPLC fractions of root exudates of 1 day dark-grown roots after Rhizobium inoculation. In contrast, after 3 days exposure to Rhizobium, analysis of specific HPLC fractions showed the presence of an additional nod-gene-inducing compound which the data indicate was 4′,7-dihydroxyflavone. A different and additional nod gene inducer was found in inoculated 5 day samples of root exudate of light-grown plants indicating that light exposure changes the HPLC profiles as well as the nod-gene-inducing compound(s). Exudates collected from wounded plants were considerably different from those from Rhizobium-inoculated and uninoculated plants and contained no detectable nod gene inducers. The late detection (at day 3) of Rhizobium-induced flavonoid excretion may occur too late to be directly correlated with the observed expression of CHS 6 h after inoculation. In addition, the data suggest that although the CHS5 promotor responds to both wounding and Rhizobium inoculation, the biochemical consequences of CHS5 induction resulting from these treatments are different.