Syringyl Lignin Is Unaltered by Severe Sinapyl Alcohol Dehydrogenase Suppression in Tobacco

Abstract

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

“…Although they both have similar catalytic efficiencies toward most hydroxycinnamaldehydes , there has been some dispute over whether or not angiosperms have evolved CADs that specifically utilize sinapaldehyde and whether this neofunctionalization explains the occurrence of S lignin biosynthesis in flowering plants (Li et al, 2001;Peter and Neale, 2004;Bomati and Noel, 2005;Chiang, 2006;Barakate et al, 2011). Although Li et al (2001) identified a CAD homolog, which they termed sinapyl alcohol dehydrogenase (SAD), from Populus tremuloides that has a high catalytic efficiency toward sinapaldehyde and found that this enzyme is missing in gymnosperms, which are deficient in S lignin, Barakate et al (2011) thoroughly demonstrated that S lignin is dependent on CAD activity and not SAD. Even though our data clearly show a differential contribution of the two CAD isoforms to S lignin biosynthesis in Arabidopsis, neither CADC nor CADD can be considered an ortholog of the SAD protein discussed by Li et al (2001).…”

Lignification and Lignin Manipulations in Conifers

“…Suppression studies in M. sativa indicated that biosynthesis of S-type lignin might involve methyltransferases other than CCoAOMT Nakashima et al, 2008). In addition, angiosperm species contain CAD isozymes that efficiently convert sinapaldehyde to sinapyl alcohol (Barakate et al, 2011;Sibout et al, 2003), and this seems not to apply to conifers (O'Malley et al, 1992;Wagner et al, unpublished results). In addition, peroxidases specific for the oneelectron oxidation of sinapyl alcohol have been identified in angiosperms (Gómez Ros et al, 2007;Ros Barceló et al, 2007), but are unlikely to exist in conifers.…”

Characterization of three cinnamyl alcohol dehydrogenases from Carthamus tinctorius

“…However, CtCAD1 and CtCAD3 maintained the activity towards sinapaldehyde at an equal level to that of coniferaldehyde, although the homology modeling analysis showed that the GLY302/LEU122 residue combination in PtreSAD were substituted by ASN300/MET120 and ALA302/LEU122 in CtCAD1 and CtCAD3, respectively (Table 3). In addition, NtSAD2 with ALA301/MET122 did not show strong preference towards either coniferaldehyde or sinapaldehyde (Barakate et al 2011). The significant sinapaldehyde reducing activity of the CtCADs and NtSAD2 can be rationalized by the multiple effects of amino acid substitution on their kinetic behavior.…”

“…Although AtELI3-2 (AtCAD8 or At4g37990) showed a high similarity to PtreSAD, the recombinant protein expressed from the gene showed similar specific activities towards both coniferaldehyde and sinapaldehyde (Kim et al 2004). This is also the case for Nicotiana tabacum SAD2 as demonstrated by Barakate et al (2011). Nevertheless, research on sinapaldehyde-specific CADs are important and can lead to strategies for increasing syringyl lignin content, which can significantly improve effectiveness in kraft pulping (Bugos et al 1991).…”