Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase

Abstract

Isolated lignins from alfalfa deficient in caffeic acid 3-O-methyltransferase contained benzodioxanes resulting from the incorporation of the novel monomer, 5-hydroxyconiferyl alcohol. Due to the high level incorporated into the soluble lignin fraction and the use of sensitive NMR instrumentation, unique structural features were revealed. A new type of end-unit, the 5-hydroxyguaiacyl glycerol unit, was identified. It was possible to establish that coniferyl alcohol, sinapyl alcohol, and the novel 5-hydroxyconiferyl alcohol can cross-couple with the 5-hydroxyguaiacyl units that are formed in the lignin, the latter giving rise to extended chains of benzodioxane units. There is also evidence that 5-hydroxyconiferyl alcohol couples with normal (guaiacyl or syringyl) lignin units. Lignin in the alfalfa deficient in caffeoyl CoA 3-O-methyltransferase was structurally similar to the control lignin but the transgenic exhibited a dramatic decrease in lignin content (approximately 20%) and modest increase in cellulose (approximately 10%) reflecting a 30% increase in cellulose:lignin ratio. The compositional changes in both transgenics potentially allow enhanced utilization of alfalfa as a major forage crop by increasing the digestibility of its stem fraction.

Functional and Structural Characterization of a Cation-dependent O-Methyltransferase from the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…An enzymatic activity such as that described here for SynOMT could therefore be detrimental to lignin formation. Tremendous efforts have been undertaken to manipulate the composition of lignin in various plant species with the focus on methylating enzymes (7,39,40) or those involved in earlier biosynthetic steps, e.g. cinnamyl alcohol dehydrogenase and cinnamoyl-CoA reductase (41).…”

“…They are therefore referred to as catechol OMTs (COMT) and have been investigated as potential targets to cure degenerate brain diseases (5). In plants, caffeoyl-coenzyme A O-methyltransferases (CCoAOMTs), named after their preferred in vitro substrate, in conjunction with a second group of cation-independent caffeic acid OMTs, are crucial for determining the structural integrity of lignin in plant vascular tissues (6,7). Specific subtypes of CCoAOMT-like proteins also methylate, besides caffeoyl-CoA, other phenylpropanoids, preferentially flavonoids, with vicinal dihydroxy groups (2).…”

Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula

“…S1A designates COMT as primarily responsible for the 5-O-methylation of 5-hydroxyconiferaldehyde (8), consistent with the reduction in S lignin, but not G lignin, in plants with down-regulated COMT expression (5,(9)(10)(11). A route via caffeyl aldehyde has been suggested (12,13), but its physiological relevance is currently unclear, as is whether a specific form of cinnamoyl CoA reductase (CCR) is required for formation of caffeyl aldehyde.…”

Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl CoA reductase deficiency)

“…Evidence is now indisputable, from diagnostic NMR data and from thioacidolysis markers, that hydroxycinnamaldehydes do indeed incorporate into lignins, by endwise radical coupling mechanisms, particularly in CADdeficient angiosperms (Kim et al, 2002(Kim et al, , 2003Lapierre et al, 2004;Ralph et al, 1998Ralph et al, , 2001. Similarly, as again revealed by thioacidolysis and NMR, in COMT-deficient angiosperms, the novel monomer 5-hydroxyconiferyl alcohol (partially) substitutes for the sinapyl alcohol whose production is thwarted, producing novel benzodioxane structures in the lignins (Jouanin et al, 2000;Marita et al, 2001Marita et al, , 2003Morreel et al, 2004;Ralph et al, 2001). Contrary to claims that incorporation of non-monolignol monomers has been 'unequivocally disproven' (Anterola and Lewis, 2002;Lewis, 1999;Patten et al, 2005), monomer substitution is well authenticated in lignification, and evidence continues to mount that natural and mutant/transgenic lignins may derive from quite an array of phenolic precursors Boudet, 1998;Lu et al, 2004;Ralph, 2006;Ralph et al, 2004bRalph et al, , 2007Ralph et al, , 2008Sederoff et al, 1999).…”