Transient Auxin Transport Inhibition and Localised Flavonoid Induction Occur During the Earliest Stages of Nodulation in White Clover

Abstract

We used microspectrofluoromctry, transgcnic white clover plants containing reporter gene constructs, ballistic microtargeting and spot inoculation to determine the role that auxin and endogenous flavonoids play in root nodule initiation and development (Hirsch, 1992). The first reporter construct (GH3:GUS) reliably measures the activity of auxin mediated responses (Larkin et al., 1996), whereas the second construct (CHS3:GUS) reports the activity of the chalcone synthase, CHS3, promoter. GH3.GUS expression was monitored after the addition to roots of Rhizobium leguminosarum bv. trifolii, auxin, NPA (synthetic auxin transport inhibitor), lipo chitin oligosaccharides (LCOs; Spaink, 1996), O-acetylated chitin oligosaccharides, chitin oligosaccharides, flavonoid aglycones and flavonoid glucosides. Flavonoid accumulation was monitored by microspectrofluorometry, by observing changes in CHS3:GUS expression and by the use of flavonoid specific dyes. Microspectrofluorometry allows excitation and emission spectra of intracellular fluorescent compounds to be determined with little interference from cell wall autofluorescence.