FOURIER-TRANSFORM INFRARED AND RAMAN-SPECTROSCOPIC EVIDENCE FOR THE INCORPORATION OF CINNAMALDEHYDES INTO THE LIGNIN OF TRANSGENIC TOBACCO (NICOTIANA-TABACUM-L) PLANTS WITH REDUCED EXPRESSION OF CINNAMYL ALCOHOL-DEHYDROGENASE

Xylem from stems of genetically manipulated tobacco plants which had h ad cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity down-re gulated to a greater or lesser degree (clones 37 and 49, respectively) by the insertion of antisense CAD cDNA had similar, or slightly highe r, lignin contents than xylem from wild-type plants. Fourier-transform infrared (FT-IR) microspectroscopy indicated that down-regulation of CAD had resulted in the incorporation of moieties with conjugated carb onyl groups into lignin and that the overall extent of cross-linking, particularly of guaiacyl (4-hydroxy-3-methoxyphenyl) rings, in the lig nin had altered. The FT-Raman spectra of manipulated xylem exhibited m axima consistent with the presence of elevated levels of aldehydic gro ups conjugated to a carbon-carbon double bond and a guaiacyl ring. The se maxima were particularly intense in the spectra of xylem from clone 37, the xylem of which exhibits a uniform red coloration, and their a bsolute frequencies matched those of coniferaldehyde. Furthermore, xyl em from clone 37 was found to have a higher content of carbonyl groups than that of clone 49 or the wild-type (clone 37: clone 49: wild-type ; 2.4:1.6:1.0) as measured by a degradative chemical method. This is t he first report of the combined use of FT-IR and FT-Raman spectroscopi es to study lignin structure in situ. These analyses provide strong ev idence for the incorporation of cinnamaldehyde groups into the lignin of transgenic plants with down-regulated CAD expression. In addition, these non-destructive analyses also suggest that the plants transforme d with antisense CAD, in particular clone 37, may contain lignin that is less condensed (cross-linked) than that of the wild-type.