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

Abstract

Xylem from stems of genetically manipulated tobacco plants which had had cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity down-regulated to a greater or lesser degree (clones 37 and 49, respectively) by the insertion of antisense CAD cDNA had similar, or slightly higher, 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 carbonyl groups into lignin and that the overall extent of cross-linking, particularly of guaiacyl (4-hydroxy-3-methoxyphenyl) rings, in the lignin had altered. The FT-Raman spectra of manipulated xylem exhibited maxima consistent with the presence of elevated levels of aldehydic groups conjugated to a carbon-carbon double bond and a guaiacyl ring. These maxima were particularly intense in the spectra of xylem from clone 37, the xylem of which exhibits a uniform red coloration, and their absolute frequencies matched those of coniferaldehyde. Furthermore, xylem 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 the first report of the combined use of FT-IR and FT-Raman spectroscopies to study lignin structure in situ. These analyses provide strong evidence 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 transformed with antisense CAD, in particular clone 37, may contain lignin that is less condensed (cross-linked) than that of the wild-type.

The maize Brown midrib1 locus affects cell wall composition and plant development in a dose-dependent manner

“…The value of A 1510 of the bm1 mutant is con-siderably lower compared with the wild type and bm1 heterozygote and there is a statistically significant difference between genotypes (P = 0.0007). A high ratio of A 1665 : A 1510 and A 1595 : A 1510 is indicative of the incorporation of coniferaldehyde residues in the cell wall (Mackay et al, 1997;Stewart et al, 1997). Both ratios are higher in the bm1 mutant, confirming the results from Wiesner reactions (Figure 2).…”

“…The peak at 1665 cm −1 represents C=O stretching in the carbonyl moiety of conjugated aldehydes or ketones (Mackay et al, 1997;Stewart et al, 1997) and based on an ANOVA the difference in absorbance that is observed for the three genotypes is statistically significant (P = 0.0004). The peaks at 1595 and 1510 cm −1 are indicative of lignin and hydroxycinnamic acids (Stewart et al, 1997). The value of A 1510 of the bm1 mutant is con-siderably lower compared with the wild type and bm1 heterozygote and there is a statistically significant difference between genotypes (P = 0.0007).…”

“…The difference between the genotypes at this frequency is significant (P = 0.02) and the bm1 heterozygote appears to have an absorbance that is distinct from the two homozygotes. The large peak with a maximum at 1048 cm −1 is due to the presence of polysaccharides (Séné et al, 1994;Stewart et al, 1997). Each of the three genotypes appears to have a distinct absorbance, and the overall difference between the three genotypes is statistically significant (P = 0.003).…”

“…Various functional groups have characteristic absorbances and their presence can be used for diagnostic Heredity purposes. FT-IR spectra of ligno-cellulosic materials are typically complex, because of overlap in signal between various functional groups (Faix, 1992;Séné et al, 1994;Stewart et al, 1997).…”

Lignin and Lignan Biosynthesis: Distinctions and Reconciliations

“…Instead, the molecular approach has primarily been devoted to attempts at determining the effects of differentially expressing genes encoding putative regulatory enzymes in the lignin biosynthetic pathway (Figure 3) (56)(57)(58)(59)(60)(61)(62). As can be seen, there are essentially only four types of transformations beyond cinnamic acid, namely aromatic hydroxylations (63)(64)(65), O-methylations (66-68), CoA ligations and (consecutive) NADPH dependent reductions (69).…”