The Last Step of Syringyl Monolignol Biosynthesis in Angiosperms Is Regulated by a Novel Gene Encoding Sinapyl Alcohol Dehydrogenase

Abstract

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.

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

“…Williamson et al (1995) and Somssich et al (1996) demonstrated that ELI-3 was neither a CAD nor a malate dehydrogenase but rather a benzyl alcohol dehydrogenase, which accepts various benzaldehyde substrates. In our phylogenetic analysis, these proteins (AtCAD-A, AtCAD-B1, and -B2) fall within the same cluster as PtSAD, which has been recently identified and characterized in poplar (Li et al, 2001). PtSAD was shown to be highly specific for sinapaldehyde and was proposed to be responsible for S-unit deposition in lignins of poplar fibers.…”

“…AtCAD-1, -E, and -F make up a subfamily with MsaCAD-1 (Brill et al, 1999). AtCAD-B1, AtCAD-B2, and AtCAD-A belong to the same subfamily as PtSAD (Li et al, 2001). AtCAD-C and AtCAD-D belong to the wellcharacterized CAD group.…”

“…Initially, CAD was believed to be multispecific, catalyzing the reduction of the different cinnamyl-aldehydes. The discovery of isozymes in Eucalyptus gunii (GrimaPettenati et al, 1993), alfalfa (Medicago sativa; Brill et al, 1999), and aspen (Populus tremuloides; Li et al, 2001) with different affinities for various substrates has lead to the hypothesis that multiple substrate specificities were related to various physiological roles. In parallel, expression of CAD cDNAs in Escherichia coli or yeast (Saccharomyces cerevisiae) was carried out to determine substrate specificity of some cloned CAD cDNA, which shared high sequence similarity to known CAD proteins.…”

Differential Substrate Inhibition Couples Kinetically Distinct 4-Coumarate:Coenzyme A Ligases with Spatially Distinct Metabolic Roles in Quaking Aspen

“…During aspen stem development, G lignin is deposited within the primary growth internodes, whereas S-G lignin is deposited in maturing xylem and phloem fibers of secondary growth internodes (Osakabe et al, 1996;Li et al, 2001). Based on our kinetic, in situ, and promoter analyses, aspen 4CL1 is strictly coupled with lignification during secondary vascular development.…”

“…New findings have revealed a pathway for S lignin biosynthesis in angiosperms that branches away from the pathway for G lignin biosynthesis at coniferaldehyde, and that operates parallel to and independently of the G lignin pathway ( Fig. 6; Osakabe et al, 1999;Li et al, 2000Li et al, , 2001. It has also been concluded from in vitro experiments that intermediates of the S lignin pathway can modulate the upstream methylation and hydroxylation of CA and FA (Fig.…”