Submitted to: Phytochemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/30/2004
Publication Date: 2/1/2004
Citation: Lapiette, C., Pilate, G., Pollet, B., Mila, I., Leple, J., Jouanin, L., Kim, H., Ralph, J. 2004. Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins. Phytochemistry. 65:313-321. Interpretive Summary: There is an enzyme used by all plants in the last step for production of monolignols, the building blocks from which the polymer lignin is produced. (It is lignin that holds fibers together in woody and forage plants; lignin is essential for the plant, but is a limit to plant cell wall digestibility by ruminant animals and an impediment in industrial pulping to make paper. Consequently there is a lot of effort directed toward selection and genetic methods for altering lignins). Some plants are naturally deficient in this enzyme, CAD, and genetic engineers have also recently been able to manipulate the level of the enzyme by regulation of the gene. However, whereas the enzyme levels and gene expression can be measured, the degree of effect on the plant's biochemistry has been difficult to ascertain, except by very detailed and laborious methods. We recently disclosed the discovery by our French collaborators at INRA of a 'molecular marker compound' for CAD, and our work elucidating the structure of the marker. We also deduced that it was derived from diagnostic structures in the lignin arising from incorporation of the aldehyde monolignol precursors. The availability of the marker allows researchers to rapidly assess the effect of CAD levels on plant lignin composition, and therefore provides a more streamlined tool. Here we demonstrate that the marker level tracks very well with the degree of CAD-deficiency in a series of variably CAD-downregulated poplar transgenics, providing proof of concept. Also, the structures of the lignins were examined in more detail. And we showed that the lignins were much more soluble in alkali than that from normal plants, suggesting that the lignin deposition and molecular weight were altered in these transgenics. These studies are part of a broad program aimed at improving plant cell wall utilization.
Technical Abstract: A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8'O'4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and b'O'4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains.