Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase

Abstract

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry. Similar reduction of lignin levels by down-regulation of i-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.There is currently intense interest in modifying the content and / or composition of the cell wall structural polymer lignin as a means of improving the efficiency of the paper pulping process for forest trees or of increasing digestibility of forages for ruminant animals (Whetten and Sederoff, 1991; Boudet and Grima-Pettenati, 1996; Campbell and Sederoff, 1996).Recent studies have concentrated on attempts to downregulate the levels of enzymes involved in the reactions specific for lignin monomer synthesis by expression of homologous or heterologous antisense genes in transgenic plants (Dwivedi et al., 1994;Halpin et al., 1994;Ni et al., 1994; Atanassova et al., 1995;Van Doorsselaere et al., 1995;Sewalt et al., 1997). Although the biosynthetic pathway to lignin monomers is relatively well understood, involving consecutive hydroxylation and O-methylation reactions leading from p-coumaric acid via ferulic acid (the monomethoxylated precursor of the G residues of lignin) to sinapic acid (the dimethoxylated precursor of the S residues of lignin), it has recently been suggested that parallel pathways of monomer hydroxylation and methylation could occur at the level of the COA thioesters (Ye et al., 1994) or even at the level of the aldehydes formed after the first reduction of the COA thioesters (Matsui et al., 1994; Fig. 1).The existence of a metabolic grid for the O-methylation of monolignols would complicate the interpretation of experiments in which a single enzyme of the pathway was down-regulated. Indeed, severa1 reports of the effects of antisense inhibition of enzymes involved in the late reactions of monolignol biosynthesis have presented unpredicted and sometimes contradictory results. Ni et al. (1994) reported that modest down-regulation of COMT activity in transgenic tobacco (Nicofiana fabacum) leads to a small reduction in lignin content with no significant change in lignin composition. However, other groups have shown that strong down-regulation of COMT in tobacco or poplar (Populu...

A Small Decrease of Plastid Transketolase Activity in Antisense Tobacco Transformants Has Dramatic Effects on Photosynthesis and Phenylpropanoid Metabolism

“…The latter might be related to the reduced lignin content or to decreased cross-linking of polysaccharide components by hydroxycinnamic acids (Chapple and Carpita, 1998). Similar changes in cell wall digestibility occur in transformants with decreased activity of phenylalanine ammonia-lyase, caffeic acid:3-O-methyltransferase (Sewalt et al, 1997), and cinnamyl alcohol dehydrogenase (Bernard-Vailhe et al, 1998; Baucher et al, 1999).…”

“…Our results indicate that TK has a remarkably high flux control coefficient (Ͼ0.7) for phenylpropanoid metabolism. This is similar to or larger than the effects of the decreased expression of phenylalanine ammonia-lyase (Bate et al, 1994;Howles et al, 1996;Sewalt et al, 1997), 4-coumarate:CoA lyase (Lee et al, 1997), and cinnamate-4-hydroxylase (Sewalt et al, 1997), three enzymes that build a regulatory unit that responds to a range of developmental and environmental factors (Chapple and Carpita, 1998;Koopman et al, 1999).…”

“…The latter might be related to the reduced lignin content or to decreased cross-linking of polysaccharide components by hydroxycinnamic acids (Chapple and Carpita, 1998). Similar changes in cell wall digestibility occur in transformants with decreased activity of phenylalanine ammonia-lyase, caffeic acid:3-O-methyltransferase (Sewalt et al, 1997), and cinnamyl alcohol dehydrogenase (Bernard-Vailhe et al, 1998; Baucher et al, 1999).The simplest explanation for these changes is that flux into the shikimate pathway and phenylpropanoid metabolism is restricted by the supply of Ery4P and aromatic amino acids, respectively. Independent support for the latter is provided by comparison with a study by Yao et al (1995) in which a similar decrease of Trp and Phe due to ectopic overexpression of tryptophan decarboxylase led to a marked decrease of CGA and lignin.…”

Cultura de células em suspensão como ferramenta para estudos em parede celular secundária em gramí­neas C4

“…A redução da expressão de genes que codificam enzimas da via de biossíntese em lignina de modo a limitar o fluxo de carbono para a produção deste polímero é a estratégia mais largamente utilizada para a produção de matérias-primas otimizadas (Sewalt et al, 1997;Hu et al, 1999;Goujon et al, 2003;Chen & Dixon, 2007;Novaes et al, 2010;Xu et al, 2011;Min et al, 2012;Van Acker et al, 2014). Não apenas o conteúdo, mas a composição da lignina também pode afetar o processo de sacarificação da biomassa lignocelulósica (Marriott et al, 2016).…”

“…A lignina imobiliza celulases e enzimas associadas, impedindo o acesso aos seus substratos e afetando negativamente a liberação de monossacarídeos durante a hidrólise enzimática dos polissacarídeos de parede, processo conhecido como sacarificação (Vanholme et al, 2012). Por isso, estratégias de engenharia genética têm focado no desenvolvimento de plantas que acumulam menos lignina ou que sintetizam um polímero mais ameno à degradação.A redução da expressão de genes que codificam enzimas da via de biossíntese em lignina de modo a limitar o fluxo de carbono para a produção deste polímero é a estratégia mais largamente utilizada para a produção de matérias-primas otimizadas (Sewalt et al, 1997;Hu et al, 1999;Goujon et al, 2003;Chen & Dixon, 2007; Novaes et al, 2010;Xu et al, 2011; Min et al, 2012;Van Acker et al, 2014 respectivamente, e é chamado de bioetanol de primeira geração (Fig. 3).…”