The roles of flavonoids in the nodulation of alfalfa by Rhizobium meliloti: The effect of R. meliloti on chalcone synthase and chalcone isomerase gene expression patterns

In the Rhizobium-legume symbiosis, flavonoids act as inducers of rhizobial nodulation (nod) genes. In this work, the roles of flavonoids in the Rhizobium meliloti-alfalfa symbiosis were investigated, not only as nod gene inducers, but also as auxin transport inhibitors and as stress-related compounds. Expression patterns of genes encoding two key enzymes in flavonoid biosynthesis, chalcone synthase (CHS) and chalcone isomerase (CHI), were examined in roots of alfalfa. Clones corresponding to four distinct CHS gene family members, as well as a clone for CHI, were isolated from alfalfa and used to examine the spatial and temporal patterns of gene expression using in situ hybridization, northern, and RNase protection analysis. In uninoculated plants, both CHS and CHI are expressed at high levels in young root tissue.; The effect of R. meliloti on transcript levels of CHS and CHI was examined at three stages of the symbiosis. Inoculation with wild-type R. meliloti or either Nod{dollar}sp-{dollar} or Fix{dollar}sp-{dollar} R. meliloti led to a transient increase in CHS levels approximately 6 h after inoculation. This induction appears to be a non-specific defense response, related to ethylene production by the plant. At 2 to 6 d post-inoculation, a second peak in transcript levels was seen. At this time, transcripts were detected in the root epidermis and root hairs, sites consistent with a role in nod gene induction. A two-fold increase in nod gene-inducing (Ini) activity of root exudates from inoculated plants as compared to uninoculated plants was correlated with the increase in transcript levels. Transcripts corresponding to CHS were also found associated with emerging nodule and lateral root primordia, reflecting a possible wounding response. No evidence for a role of flavonoids in nodule development was observed.; Levels of flavonoids as well as CHS and CHI transcripts were higher in nodules induced by a number of defined Fix{dollar}sp-{dollar} mutants of R. meliloti, including those defective in an exopolysaccharide synthesis (exoB) and nitrogenase regulation (nifA), than in nodules induced by wild-type R. meliloti. The induction of flavonoid synthesis is correlated with the stage to which the symbiosis advances and therefore may be triggered by cellular deterioration associated with ineffective symbioses. This suggests that this induction may also be a wounding response.