Soybean (Glycine max) root lignification induced by ferulic acid. The possible mode of action.

Ferulic acid, in the form of feruloyl CoA, occupies a central position as an intermediate in the phenylpropanoid pathway. Due to the allelopathic function, its effects were tested on root growth, H(2)O(2) and lignin contents, and activities of cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) and peroxidase (POD, EC 1.11.1.7) from soybean (Glycine max (L.) Merr.) root seedlings. Three-day-old seedlings were cultivated in half-strength Hoagland's solution (pH 6.0), with or without 1.0 mM ferulic acid in a growth chamber (25 degrees C, 12/12 hr light/dark photoperiod, irradiance of 280 micromol m(-2) s(-1)) for 24 or 48 hr. Exogenously supplied ferulic acid induced premature cessation of root growth, with disintegration of the root cap, compression of cells in the quiescent center, increase of the vascular cylinder diameter, and earlier lignification of the metaxylem. Moreover, the allelochemical decreased CAD activity and H(2)O(2) level and increased the anionic isoform PODa5 activity and lignin content. The lignin monomer composition of ferulic acid-exposed roots revealed a significant increase of guaiacyl (G) units. When applied jointly with piperonylic acid (an inhibitor of the cinnamate 4-hydroxylase, C4H), ferulic acid increased lignin content. By contrast, the application of 3,4-(methylenedioxy) cinnamic acid (an inhibitor of the 4-coumarate:CoA ligase, 4CL) with ferulic acid did not. Taken together, these results suggest that ferulic acid may be channeled into the phenylpropanoid pathway (by the 4CL reaction) and, further, may increase the lignin monomer amount solidifying the cell wall and restricting the root growth.

High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in soybean roots.

This study proposes a simple, quick and reliable method for determining the cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity in soybean (Glycine max L. Merr.) roots using reversed-phase high performance liquid chromatography (RP-HPLC). The method includes a single extraction of the tissue and conduction of the enzymatic reaction at 30 degrees C with cinnamaldehydes (coniferyl or sinapyl), substrates of CAD. Disappearance of the substrates in the reaction mixture is monitored at 340 nm (for coniferaldehyde) or 345 nm (for sinapaldehyde) by isocratic elution with methanol/acetic acid through a GLC-ODS (M) column. This HPLC technique furnishes a rapid and reliable measure of cinnamaldehyde substrates, and may be used as an alternative tool to analyze CAD activity in enzyme preparation without previous purification.