Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa: Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin

Abstract

Transgenic alfalfa plants were generated harboring caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) cDNA sequences under control of the bean phenylalanine ammonia-lyase PAL2 promoter. Strong downregulation of COMT resulted in decreased lignin content, a reduction in total guaiacyl (G) lignin units, a near total loss of syringyl (S) units in monomeric and dimeric lignin degradation products, and appearance of low levels of 5-hydroxy guaiacyl units and a novel dimer. No soluble monolignol precursors accumulated. In contrast, strong downregulation of CCOMT led to reduced lignin levels, a reduction in G units without reduction in S units, and increases in beta-5 linked dimers of G units. Accumulation of soluble caffeic acid beta-d-glucoside occurred only in CCOMT downregulated plants. The results suggest that CCOMT does not significantly contribute to the 3-O-methylation step in S lignin biosynthesis in alfalfa and that there is redundancy with respect to the 3-O-methylation reaction of G lignin biosynthesis. COMT is unlikely to catalyze the in vivo methylation of caffeic acid during lignin biosynthesis.

Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa ( Medicago sativa L.)

“…Down-regulation of COMT results in a strikingly reduced S͞G ratio, whereas downregulation of CCoAOMT reduces G lignin, but not S lignin, in alfalfa (32). Acid detergent fiber and NDF were slightly reduced in all transgenic as compared with control lines (Table 3).…”

“…Forage quality analysis was performed on stem material from highly down-regulated lines (Table 3), and on previously generated lines, in the same genetic background, down-regulated in COMT, CCoAOMT, or both enzymes (32). Down-regulation of COMT results in a strikingly reduced S͞G ratio, whereas downregulation of CCoAOMT reduces G lignin, but not S lignin, in alfalfa (32).…”

“…Alfalfa stem sections were made by using a vibratome (series 1000, Ted Pella, Inc., Redding, CA) and subjected to Maule staining as described in ref. 32. Photographs were taken by using an Olympus SZX stereomicroscope system with a SPOT RT color camera.…”

A Novel Mg2+-dependent O-Methyltransferase in the Phenylpropanoid Metabolism of Mesembryanthemum crystallinum

“…In angiosperms, this task may also be performed by class II OMTs that are specific for caffeic acid, caffeyl aldehyde, or caffeyl alcohol (COMT) (9 -11). This apparent redundancy results in a cell-and tissue-specific metabolic grid that is essential for plants to regulate lignin composition and structure (12)(13)(14)(15). In gymnosperms, the situation could be more complex, with a bifunctional caffeic acid/caffeoyl-CoA methylating activity (AEOMT) and an additional specific caffeoyl-CoA activity found in loblolly pine (Pinus taeda; Refs.…”

Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

“…Subsequent coupling of the phenoxy radicals to each other, or to the growing polymer during lignification forms the lignin polymer. The cross-coupling of these phenoxy radicals in planta generates different inter-unit bonds, including the most frequent ␤-O-4 (␤-aryl ether) linkages, and less frequent 5-O-4 ether linkages (4,7). Apparently, the generation of radical intermediates and formation of ether linkages between the phenylpropane units requires unsubstituted 4-hydroxyl (supplemental Fig.…”