Flavonoids synthesized in cortical cells during nodule initiation are early developmental markers in white clover

We examined the site-specific induction of the flavonoid pathway before and during nodule initiation in white clover with transgenic plants, fluorescence microscopy, and microspectrofluorometry to test if flavonoids play a role in nodule organogenesis. A chalcone synthase regulated β-glucuronidase (GUS) transgene (CHS:gusA) was upregulated from 3 h post inoculation (p.i.) until cell division (around 40 h p.i.) in inner cortex cells underlying the inoculation site. Intracellular fluorescence occurred in vacuoles of those inner cortex cells from 13 h p.i. until the fluorescent cells divided. Fluorescence emission spectra of contents of individual fluorescing cortex cells were measured in situ and compared with emission spectra of compounds purified from root extracts. The fluorescing compound located in cells of the inner cortex after Rhizobium leguminosarum bv. trifolii infection was identified as a water-soluble derivative of 7,4'-dihydroxyflavone. Nodule primordium cells contained a different fluorescent compound, identified as the isoflavonoid formononetin. CHS3:gusA expression and flavonoid accumulation were only induced in inner cortex cells by a modulating Rhizobium strain and by clover-specific lipo-chitinoligosaccharides, but not by non-nodulating rhizobia. Fluorescence was also induced by compatible rhizobia in other legumes such as alfalfa, pea, and siratro in the cells that participate in nodule initiation. Our results show that fluorescent flavonoids are useful markers in nodule organogenesis in clover and may have direct roles in nodule formation.