Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana

Fukasawa-Akada, Tomoko; Kung, Shain‐dow; Watson, John C.

doi:10.1007/bf00019006

Cited by 102 publications

(61 citation statements)

References 34 publications

(28 reference statements)

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“…Such putative channel complexes might be associated with specific isoforms of PAL, which is encoded by a multigene family in tobacco (Fukasawa-Akada et al, 1996) and most other species studied. Such complexes might direct Phe or cinnamate specifically into the production of G or S units, and partitioning of flux into these complexes might not be equally affected by down-regulation of PAL compared with C4H, particularly if different tobacco PAL forms are differentially sensitive to co-suppression by the bean PAL transgene.…”

Section: Genetic Manipulation Of Lignin Composition In Transgenic Tobmentioning

confidence: 99%

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

et al. 1997

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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...

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Section: Genetic Manipulation Of Lignin Composition In Transgenic Tobmentioning

confidence: 99%

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

et al. 1997

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“…The two Coupling of PAL and C4H in Tobacco 1545 families are represented by single-copy PAL genes in the two progenitor species, Nicotiana sylvestris and N. tomentosiformis (Fukasawa-Akada et al, 1996). In this study, PAL1 refers to the product encoded by the PAL gene of family I, described by Fukasawa-Akada et al (1996), and PAL2 refers to the product of the PAL gene from family II, as reported by Nagai et al (1994). To determine whether specific forms of PAL are associated with tobacco microsomes, we raised antibodies against synthetic peptide sequences specific for tobacco PAL1, tobacco PAL2, and bean PAL2 (Howles et al, 1996).…”

Section: The Nature Of Pal Associated With Microsomal Membranesmentioning

confidence: 99%

“…For raising antibodies specific to tobacco PAL proteins, the following amino acid sequences, derived from the available tobacco PAL sequences (Nagai et al, 1994;Pellegrini et al, 1994;Fukasawa-Akada et al, 1996), were used: VRDKSANG (positions 69 to 76 from tobacco PAL1) and VAQNGHQEMDFCVKV (positions 4 to 18 from tobacco PAL2). These sequences represent the few stretches that are unique between the different tobacco PAL forms.…”

Section: Generation Of Antibodies Specific For Tobacco and Bean Pal Fmentioning

confidence: 99%

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Transgene-Mediated and Elicitor-Induced Perturbation of Metabolic Channeling at the Entry Point into the Phenylpropanoid Pathway

1999

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3 H-L -Phenylalanine is incorporated into a range of phenylpropanoid compounds when fed to tobacco cell cultures. A significant proportion of 3 H-trans -cinnamic acid formed from 3 H-L -phenylalanine did not equilibrate with exogenous trans -cinnamic acid and therefore may be rapidly channeled through the cinnamate 4-hydroxylase (C4H) reaction to 4-coumaric acid. Such compartmentalization of trans -cinnamic acid was not observed after elicitation or in cell cultures constitutively expressing a bean phenylalanine ammonia-lyase ( PAL ) transgene. Channeling between PAL and C4H was confirmed in vitro in isolated microsomes from tobacco stems or cell suspension cultures. This channeling was strongly reduced in microsomes from stems or cell cultures of transgenic PAL -overexpressing plants or after elicitation of wild-type cell cultures. Protein gel blot analysis showed that tobacco PAL1 and bean PAL were localized in both soluble and microsomal fractions, whereas tobacco PAL2 was found only in the soluble fraction. We propose that metabolic channeling of trans -cinnamic acid requires the close association of specific forms of PAL with C4H on microsomal membranes. INTRODUCTIONThe phenylpropanoid pathway is involved in the biosynthesis of a wide variety of natural products from plants. Many of these products have important functions in plant development and in interactions of the plant with its environment (Hahlbrock and Grisebach, 1979;Hahlbrock and Scheel, 1989;Dixon and Paiva, 1995). Many studies have addressed the transcriptional regulation of genes encoding enzymes of the phenylpropanoid pathway and subsequent changes in extractable enzyme activities in response to developmental and environmental cues Lawton and Lamb, 1987;Hahlbrock and Scheel, 1989). Far less attention has been paid to how the cell regulates flux into different end products of the pathway once all of the enzymatic machinery is assembled.The first committed step in the biosynthesis of phenylpropanoid compounds is the conversion of L -phenylalanine (Phe) to trans -cinnamic acid by L -Phe ammonia-lyase (PAL; EC 4.3.1.5; Figure 1). PAL is a tetrameric enzyme whose subunits are encoded by a multigene family in most species that have been studied (Cramer et al., 1989;Nagai et al., 1994; Wanner et al., 1995;Fukasawa-Akada et al., 1996).PAL genes are transcriptionally activated after microbial infection or treatment of plant cells with microbial elicitors (Edwards et al., 1985;Lawton and Lamb, 1987). The second step in the phenylpropanoid pathway, the hydroxylation of trans -cinnamic acid to 4-coumaric acid, is catalyzed by a cytochrome P450 monooxygenase, cinnamic acid 4-hydroxylase (C4H; EC 1.14. 13.11;Russell and Conn, 1967;Fahrendorf and Dixon, 1993;Teutsch et al., 1993). C4H is induced by light, elicitors, and wounding (Fahrendorf and Dixon, 1993;Buell and Somerville, 1995;Batard et al., 1997;Bell-Lelong et al., 1997), and its induction often is closely coordinated with PAL induction (Mizutani et al., 1997).Enzymes of complex metabolic pathways may...

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“…They are derived from trans-cinnamic acid formed by deamination of L-Phe by L-Phe ammonia-lyase (PAL; EC 4.3.1.5). PAL is a tetrameric enzyme whose subunits are encoded by multigene families in most species studied (Cramer et al, 1989;Nagai et al, 1994;Wanner et al, 1995;Fukasawa-Akada et al, 1996). In tobacco (Nicotiana tabacum), PAL genes have a simple organization consisting of two families, each with two very closely related genes (Nagai et al, 1994;Pellegrini et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Colocalization of l-Phenylalanine Ammonia-Lyase and Cinnamate 4-Hydroxylase for Metabolic Channeling in Phenylpropanoid Biosynthesis

et al. 2004

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Metabolic channeling has been proposed to occur at the entry point into plant phenylpropanoid biosynthesis. To determine whether isoforms of L-Phe ammonia-lyase (PAL), the first enzyme in the pathway, can associate with the next enzyme, the endomembrane-bound cinnamate 4-hydroxylase (C4H), to facilitate channeling, we generated transgenic tobacco (Nicotiana tabacum) plants independently expressing epitope-tagged versions of two PAL isoforms (PAL1 and PAL2) and C4H. Subcellular fractionation and protein gel blot analysis using epitope-and PAL isoform-specific antibodies indicated both microsomal and cytosolic locations of PAL1 but only cytosolic localization of PAL2. However, both PAL isoforms were microsomally localized in plants overexpressing C4H. These results, which suggest that C4H itself may organize the complex for membrane association of PAL, were confirmed using PAL-green fluorescent protein (GFP) fusions with localization by confocal microscopy. Coexpression of unlabeled PAL1 with PAL2-GFP resulted in a shift of fluorescence localization from endomembranes to cytosol in C4H overexpressing plants, whereas coexpression of unlabeled PAL2 with PAL1-GFP did not affect PAL1-GFP localization, indicating that PAL1 has a higher affinity for its membrane localization site than does PAL2. Dual-labeling immunofluorescence and fluorescence energy resonance transfer (FRET) studies confirmed colocalization of PAL and C4H. However, FRET analysis with acceptor photobleaching suggested that the colocalization was not tight.

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Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana

Cited by 102 publications

References 34 publications

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

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

Transgene-Mediated and Elicitor-Induced Perturbation of Metabolic Channeling at the Entry Point into the Phenylpropanoid Pathway

Colocalization of l-Phenylalanine Ammonia-Lyase and Cinnamate 4-Hydroxylase for Metabolic Channeling in Phenylpropanoid Biosynthesis

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