Metabolite Profiling Reveals a Role for Atypical Cinnamyl Alcohol Dehydrogenase CAD1 in the Synthesis of Coniferyl Alcohol in Tobacco Xylem

Abstract

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes.

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism

“…Extraction, GC-MS profiling, and the subsequent processing of GC-MS data were conducted as described previously (Kaplan et al, 2004;Damiani et al, 2005;Desbrosses et al, 2005;Dauwe et al, 2007) using an HP6890 series mass spectrometry system coupled to a 5973 mass selective detector (Agilent Technologies). The identification of primary metabolites was based on the quadrupole mass spectral and retention time index library (i.e.…”

Downregulation of Cinnamoyl-Coenzyme A Reductase in Poplar: Multiple-Level Phenotyping Reveals Effects on Cell Wall Polymer Metabolism and Structure

“…Four to eight ramets were analyzed for each poplar line. In vitro propagation, growth conditions, sampling, extraction, HPLC analysis of the soluble phenolics from poplar xylem tissue, and LC-MS/MS analysis of the cinnamic acids and cinnamaldehydes were as previously described (Morreel et al, 2004a(Morreel et al, , 2004bDamiani et al, 2005).…”

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis

“…Another type of CAD, named abnormal or atypical CAD, has been reported in Eucalyptus gunnii and tobacco (Nicotiana tabacum; Goffner et al, 1998;Damiani et al, 2005), and a homolog At5g19440 exists in Arabidopsis. Because of its low sequence similarity with bona fide CAD, At5g19440 was excluded from the systematic characterization of CADs in Arabidopsis (Costa et al, 2003;Kim et al, 2004).…”