MICROSPECTROSCOPIC IMAGING OF NODULATION FACTOR-BINDING SITES ON LIVING VICIA-SATIVA ROOTS USING A NOVEL BIOACTIVE FLUORESCENT NODULATION FACTOR

A novel bioactive fluorescent nodulation (Nod) factor, NodRlv-IV(BODIP Y FL-C-16), has been synthesized by attaching a BODIPY FL-C-16 acyl ch ain to the primary amino group of chitotetraose deacetylated at the no nreducing terminus by recombinant NodB. The binding of the fluorescent Nod factor to root systems of Vicia sativa was investigated with fluo rescence spectral imaging microscopy (FSPIM) and fluorescence ratio im aging microscopy (FRIM). Spatially resolved fluorescence spectra of li ving and labeled Vicia sativa root systems were measured by FSPIM. Str ong autofluorescence, inherent to many plant systems when excited at 4 88 nm, was corrected for by utilizing the difference in fluorescence e mission spectra of the autofluorescence and NodRlv-IV(BODIPY FL-C-16). A methodology is presented to break down the in situ fluorescence emi ssion spectra into spatially resolved autofluorescence and BODIPY FL f luorescence spectra. Furthermore, an FRIM method was developed for cor recting autofluorescence in fluorescence micrographs for this system. After autofluorescence correction it was shown that NODRlv-IV(BODIPY F L-C-16) was co root hairs, but was also bound to other parts of the ro ot surface.