RESUMEN
To investigate the substrate preferences of O-methyltransferases in the monolignol biosynthetic pathways, caffeoyl and 5-hydroxy coniferyl aldehydes were synthesized by a new procedure involving a Wittig reaction with the corresponding hydroxybenzaldehydes. The same procedure can also be used to synthesize caffeoyl and 5-hydroxyconiferyl alcohols. Relative O-methyltransferase activities against these substrates were determined using crude extracts and recombinant caffeic acid O-methyltransferase from alfalfa (Medicago sativa), and crude extracts from the model legume Medicago truncatula, tobacco, wheat and tall fescue. Extracts from all these species catalyzed methylation of the various monolignol aldehydes and alcohols more effectively than the corresponding hydroxycinnamic acids.
Asunto(s)
Alcoholes/síntesis química , Aldehídos/síntesis química , Metiltransferasas/metabolismo , Plantas/enzimología , Cromatografía de Gases y Espectrometría de Masas , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Lignina/biosíntesis , Medicago sativa/enzimología , Metiltransferasas/genética , Plantas Modificadas Genéticamente/enzimología , Secuencia de Bases , Cartilla de ADN , Fabaceae/genética , Medicago sativa/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras GenéticasRESUMEN
Measurement of relative O-methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydroxyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O-methyltransferase (COMT) reduced activities against the non-esterifed substrates in the order: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest V(max)/K(m) values with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.
Asunto(s)
Catecol O-Metiltransferasa/metabolismo , Lignina/metabolismo , Medicago sativa/enzimología , Metiltransferasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Cinética , Metilación , Plantas Modificadas Genéticamente/enzimología , Especificidad por SustratoRESUMEN
Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Nicotiana/fisiología , Fenilanina Amoníaco-Liasa/metabolismo , Plantas Tóxicas , Northern Blotting , Southern Blotting , Sistema Enzimático del Citocromo P-450/genética , Retroalimentación , Regulación de la Expresión Génica de las Plantas , Oxigenasas de Función Mixta/genética , Fenilanina Amoníaco-Liasa/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Nicotiana/enzimología , Nicotiana/genética , Transcinamato 4-MonooxigenasaRESUMEN
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 L-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.
RESUMEN
Transcription of genes encoding L-phenylalanine ammonia-lyase (PAL), the first enzyme of the phenylpropanoid pathway, and caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT), enzymes involved in the synthesis of lignin and wall-esterified phenolic compounds, was strongly activated in elicitor-treated cell-suspension cultures of alfalfa (Medicago sativa L.). However, consequent changes in the extractable activities of COMT and CCOMT were small to nonexistent compared with a 15- to 16-fold increase in PAL activity. Only low levels of COMT and CCOMT transcripts were reflected in the total and polysomal RNA fractions compared with PAL transcripts. Elicited cell cultures did not accumulate lignin or the products of COMT and CCOMT in the soluble and wall-esterified phenolic fractions. In one alfalfa cell line in which elicitation resulted in very high PAL activity and increased deposition of methoxyl groups in the insoluble wall fraction, there was still no change in COMT and CCOMT activities. Overall, these results indicate that the initial gene transcription events in elicited cells may be less selective than the subsequent metabolic changes, highlighting the importance of posttranscriptional events in the control of phenylpropanoid biosynthesis.
RESUMEN
Both reduced glutathione (GSH) and oxidized glutathione elicited the phytoalexin response in cell-suspension cultures of bean (Phaseolus vulgaris L.) but had no effect in those of alfalfa (Medicago sativa L.). In bean cells, homoglutathione (HGSH) was the predominant soluble thiol and treatment of cells with fungal elicitor resulted in the accumulation of HGSH but not GSH. In contrast, GSH was more abundant than HGSH in unelicited alfalfa cells, and the intracellular levels of both thiols increased in response to fungal elicitor. Treatment of bean or alfalfa cells with L-oxothiazolidine-4-carboxylate, an artificial precursor for GSH biosynthesis, increased intracellular thiols in an analogous manner to that observed following treatment with fungal elicitor, but did not result in elicitation of the cultures. Differences were observed in the initial metabolic fates of exogenously supplied [(35)S]GSH in bean and alfalfa, but our data do not yet provide a basis for explaining how GSH acts as an elicitor. We conclude that the changes observed in intracellular thiols following exposure of cells to fungal elicitor are a consequence rather than a cause of the initial elicitation signal(s).