Expression Pattern of Two Paralogs Encoding Cinnamyl Alcohol Dehydrogenases in Arabidopsis. Isolation and Characterization of the Corresponding Mutants

Abstract

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at C␤. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units.Lignin is a complex phenolic polymer whose structure is vital to functions such as imparting rigidity to plant organs and as a physical barrier to invading pests. Its presence in cell wall confers to vessels hydrophobic properties that facilitate conduction of water, photo-assimilates, and minerals to different parts of the plant. Lignin structure and composition differ widely at the interspecies level as well as cell types and at the subcellular cell wall level (Donaldson, 2001). Striking differences are mostly observable between gymnosperms and angiosperms. These taxa contain different qualitative and quantitative proportions of monolignols or cinnamyl alcohols representing the main lignin monomers. The formation of cinnamyl alcohols from the corresponding cinnamoylCoA esters requires two enzymatic modifications of the carbonate chain of the phenolic precursors. The first step is catalyzed by cinnamoyl CoA reductase, and the second step is catalyzed by cinnamyl alcohol dehydrogenase (CAD). CAD leads to the conversion of hydroxy-cinnamaldehydes to the corresponding alcohols. The relative proportions of these cinnamyl alcohols is an important factor for lignin structural traits and mechanical properties (Baucher et al., 1998;Mellerowicz et al., 2001).CAD was one of the first enzymes studied in the lignin syn...

A coumaroyl-ester-3-hydroxylase Insertion Mutant Reveals the Existence of Nonredundant meta-Hydroxylation Pathways and Essential Roles for Phenolic Precursors in Cell Expansion and Plant Growth

“…The analyses of wild-type Ws roots confirmed that, compared to the corresponding stem lignin, roots contain relatively less S units and correlatively more G and H units (Sibout et al, 2003), with H units contributing 5% to 7% to the total lignin thioacidolysis monomers, depending on the developmental stage of wild-type plants (Table I). Similar to stems, roots of the cyp98A3 insertion mutants provided a lower total thioacidolysis yield and a much higher proportion of H monomers (43%) compared to the wild-type roots.…”

FLEXIBLE CULM 1 encoding a cinnamyl-alcohol dehydrogenase controls culm mechanical strength in rice

“…The supernatant was decanted and about 20 lg of total proteins were used for the CAD and SAD activity assays. Assays of crude enzyme were carried out in the reverse direction using coniferyl alcohol and sinapyl alcohol as the substrates, and the formation of hydroxy-cinnamaldehydes was monitored spectrophotometrically (DU640; Backman, Fullerton, USA) at 400 nm using the following molar extinction coefficients: coniferaldehyde 2.10 9 10 -4 M -1 cm -1 and sinapaldehyde 1.68 9 10 -4 M -1 cm -1 , as described by Sibout et al (2003). Assays were carried out at 30°C for 10 min in 100 ll of 100 mM Tris-HCl (pH 8.8), 100 lM NADP, and 250 lM coniferyl alcohol or sinapyl alcohol.…”

“…1.195) is an enzyme in the lignin biosynthetic pathway that catalyzes the final reduction of the hydroxyl-cinnamaldehydes to the corresponding alcohols (Mansell et al 1974). The relative proportions of these cinnamyl alcohols are important factors that determine the structural and mechanical properties of lignin (Mellerowicz et al 2001;Sibout et al 2003). CAD genes in different species display distinct features depending on their phylogenetic origin.…”

“…In Arabidopsis, Sibout et al (2003) reported that there were nine CAD-like proteins distributed among four classes based on their amino acid similarity. Studies on AtCADC and AtCADD revealed that both acted as the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis by supplying both coniferyl and sinapyl alcohols (Sibout et al 2005).…”

Monolignol Biosynthesis and its Genetic Manipulation: The Good, the Bad, and the Ugly

“…The Atcad-c and Atcad-d mutants of Arabidopsis have reduced CAD activities, but only Atcad-d shows a (slight) reduction in lignin content, reduction of S units and incorporation of sinapaldehyde in lignin (Sibout et al, 2003). According to Kim et al, (2004), CAD mutants of Arabidopsis attain wild-type lignin levels and composition by maturity.…”