Suppression of O-Methyltransferase Gene by Homologous Sense Transgene in Quaking Aspen Causes Red-Brown Wood Phenotypes1

Abstract

Homologous sense suppression of a gene encoding lignin pathway caffeic acid O-methyltransferase (CAOMT) in the xylem of quaking aspen (Populus tremuloides Michx.) resulted in transgenic plants exhibiting novel phenotypes with either mottled or complete red-brown coloration in their woody stems. These phenotypes appeared in all independent transgenic lines regenerated with a sense CAOMT construct but were absent from all plants produced with antisense CAOMT. The CAOMT sense transgene expression was undetectable, and the endogenous CAOMT transcript levels and enzyme activity were reduced in the xylem of some transgenic lines. In contrast, the sense transgene conferred overexpression of CAOMT and significant CAOMT activity in all of the transgenic plants' leaves and sclerenchyma, where normally the expression of the endogenous CAOMT gene is negligible. Thus, our results support the notion that the occurrence of sense cosuppression depends on the degree of sequence homology and endogene expression. Furthermore, the suppression of CAOMT in the xylem resulted in the incorporation of a higher amount of coniferyl aldehyde residues into the lignin in the wood of the sense plants. Characterization of the lignins isolated from these transgenic plants revealed that a high amount of coniferyl aldehyde is the origin of the red-brown coloration-a phenotype correlated with CAOMT-deficient maize (Zea mays L.) brown-midrib mutants.Lignin, a complex aromatic polymer, is the major component of wood that must be degraded to extract cellulose fibers for paper making. In angiosperm dicots lignin is composed of G and S monomers that are derived from coniferyl ( Fig. 1, 14) and sinapyl ( Fig. 1, 16) alcohols (monolignols), respectively. It has been shown that wood-pulping efficiency is greatly influenced by lignin content, as well as its monomeric composition, since G lignin is more difficult to remove than is G-S lignin during chemical pulping (Chang and Sarkanen, 1973;Kondo et al., 1987;Chiang and Funaoka, 1990). Thus, genetic engineering of lignin biosynthesis in pulpwood species has the potential to generate new tree clones with altered lignin content and/or structure to facilitate wood pulping.The biosynthetic pathway of monolignols has been extensively studied revealing that at least 10 enzymatic steps are involved in converting Phe into coniferyl and sinapyl alcohols. S-Adenosyl-l-Met-dependent CAOMT (Fig. 1) is an enzyme in this pathway that catalyzes the methylation of phenylpropanoid precursors (Fig. 1, 3 and 5) for the synthesis of both G and S lignins in angiosperm dicots (Sarkanen, 1971;Higuchi, 1985). CAOMT has been suggested to play an important role in mediating the synthesis of S lignin (Higuchi, 1985;Li et al., 1997) and together with CAD ( Fig. 1) are the two enzymes found to be responsible for the low-lignin-content-associated maize (Zea mays L.) and sorghum (Sorghum bicolor L.) bm (brown-midrib) mutants that exhibit red coloration in their lignified tissues (for review, see Cherney et al., 1991). Therefore, both CAO...

Essential Role of Caffeoyl Coenzyme A O-Methyltransferase in Lignin Biosynthesis in Woody Poplar Plants

“…Because COMT was considered to be the sole enzyme involved in methylation, COMT had long been targeted for the reduction of lignin content in the hope of reducing pollution from pulping or improving digestibility of forages. However, transgenic plants with a reduction in COMT activity alone showed a dramatic reduction in syringyl lignin without an apparent decrease in total lignin content (Dwivedi et al, 1994;Atanassova et al, 1995;Doorsselaere et al, 1995;Tsai et al, 1998). This contradicted with the early presumption of COMT as the sole O-methyltransferase (OMT) involved in monolignol biosynthesis.…”

“…Xylem or wood in plants with repression of COMT or cinnamyl alcohol dehydrogenase often shows strong red coloration, which has been attributed to the accumulation of hydroxycinnamyl aldehydes (Halpin et al, 1994;Higuchi et al, 1994;Ralph et al, 1997;Stewart et al, 1997;Tsai et al, 1998). In plants with repression of 4-coumarate-coenzyme A ligase, xylem displayed a brownish color, probably due to accumulation of hydroxycinnamic acids (Kajita et al, 1997).…”

“…Coloration of xylem or wood has been reported in transgenic plants or mutants with repression of other lignin pathway genes such as COMT, 4-coumarate: coenzyme A ligase, and cinnamyl alcohol dehydrogenase (Halpin et al, 1994;Atanassova et al, 1995;Doorsselaere et al, 1995;Campbell and Sederoff, 1996;Kajita et al, 1997;Ralph et al, 1997;Tsai et al, 1998). Xylem or wood in plants with repression of COMT or cinnamyl alcohol dehydrogenase often shows strong red coloration, which has been attributed to the accumulation of hydroxycinnamyl aldehydes (Halpin et al, 1994;Higuchi et al, 1994;Ralph et al, 1997;Stewart et al, 1997;Tsai et al, 1998).…”

“…Therefore, the enzymatic steps involved in the methoxylation of hydroxycinnamic acids are critical in the synthesis of different monolignols, thus influencing lignin composition. The important roles of methoxylation in determining lignin composition have been clearly demonstrated in transgenic plants with alterations in the expression of genes involved in methoxylation (Dwivedi et al, 1994;Atanassova et al, 1995;Doorsselaere et al, 1995;Meyer et al, 1998;Tsai et al, 1998;Zhong et al, 1998).…”

Free Phenols in Maize Pith and Their Relationship with Resistance to <I>Sesamia nonagrioides</I> (Lepidoptera: Noctuidae) Attack

“…Previous studies have indicated impairing different steps in monolignol biosynthesis results in reddish brown to tan stem and stalk pigmentation, including the CAD and COMT mutants examined in this study (Mackay et al 1997, Tsai et al 1998, Sibout et al 2005, Zhang et al 2006). There are differences in pith and midrib (leaf vein) coloration between bmr6 and bmr12 (Porter et al 1978, Saballos et al 2009), probably because bmr6 and bmr12 block different steps of the monolignol biosynthetic pathway.…”