Overexpression of L-Phenylalanine Ammonia-Lyase in Transgenic Tobacco Plants Reveals Control Points for Flux into Phenylpropanoid Biosynthesis

Howles, Paul A.; Sewalt, Vincent; Paiva, Nancy L.; Elkind, Yonatan; Bate, Nicholas J.; Lamb, Chris; Dixon, R. A.

doi:10.1104/pp.112.4.1617

Cited by 215 publications

(181 citation statements)

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“…The induction of phenolic compounds by stress might also involve changes in enzymes of speciflc branch pathways, as suggested b}^ Dixon & Paiva (1995) and Howies et al (1996). Therefore, plant secondary metabolism might respond to enhanced u.v.-radiation by increased synthesis of those compounds that are most effective as u.v.-protectants at the expense of those that either absorb u.v.-B radiation poorly or are weak antioxidants.…”

Section: Resultsmentioning

confidence: 99%

The effect of u.v.‐B radiation on u.v.‐absorbing secondary metabolites in birch seedlings grown under simulated forest soil conditions

et al. 1997

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Section: Resultsmentioning

confidence: 99%

The effect of u.v.‐B radiation on u.v.‐absorbing secondary metabolites in birch seedlings grown under simulated forest soil conditions

et al. 1997

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“…Although PAL catalyzes the first step in phenylpropanoid biosynthesis and plays a major role in regulating carbon flow into the pathway (Bate et al, 1994), additional flux control points occur at various downstream branches (Howles et al, 1996). Superimposed on regulation of the individual pathway enzymes is the allocation of chorismate to Phe biosynthesis and partitioning of Phe to PAL; chorismate and Phe are branch point intermediates that are substrates for multiple enzymes.…”

Section: Discussionmentioning

confidence: 99%

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

et al. 2004

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p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5# untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T 1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T 1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nucleartransformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.All plants normally produce p-hydroxybenzoic acid (pHBA), albeit usually in small quantities. Radioisotope studies with Lithospermum erythrorhizon suggest that this compound is derived from the CoA ester of p-hydroxycinnamic acid (pHCA-CoA) through a b-oxidation-like mechanism (Loscher and Heide, 1994). However, earlier studies with the same plant species (Yazaki et al., 1991) and carrot (Daucus carota) cell cultures (Schnitzler et al., 1992) supported a cleavage mechanism that occurs via intermediacy of p-hydroxybenzaldehyde. Notwithstanding our current ignorance of the detailed plant biosynthetic pathway, a number of studies have shown that it is possible to dramatically elevate pHBA levels in plants through metabolic engineering. Indeed, two different ''singleenzyme'' pathways have been described, both involving microbial proteins that have no known plant counterparts. One of these enzymes is chorismate pyruvate-lyase (CPL). Its substrate is chorismate, an important branch po...

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“…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). Soluble and microsomal proteins were isolated from wild-type, PAL-overexpressing (from the bean PAL2 transgene), and PAL-suppressed tobacco stem tissues, subjected to SDS-PAGE, and probed with these antibodies.…”

Section: The Nature Of Pal Associated With Microsomal Membranesmentioning

confidence: 99%

“…These results indicate that externally added unlabeled transcinnamic acid does not dilute isotope incorporation from 3 H-Phe into phenylpropanoid compounds in tobacco cell cultures, whereas externally added unlabeled 4-coumaric acid does, suggesting a specific metabolic compartmentalization of trans -cinnamic acid. This does not directly prove channeling or necessarily indicate the presence of more than one endogenous pool of trans -cinnamic acid in the absence of an externally fed compound.PAL -overexpressing tobacco lines, resulting from constitutive expression of a bean PAL2 gene, overproduce phenylpropanoid compounds (Howles et al, 1996), whereas underexpressing lines, resulting from epigenetic gene silencing (Elkind et al, 1990;Bate et al, 1994), have reduced levels of phenylpropanoid compounds. We initiated callus and then cell suspension cultures from PAL -overexpressing transgenic tobacco; constitutive PAL activities in the cell suspension cultures were at least threefold higher than in comparable cultures derived from wild-type plants.…”

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confidence: 99%

“…Tobacco represents such a system. We have generated a series of transgenic tobacco plants containing a heterologous bean PAL2 gene, in which the levels of extractable PAL activity are increased or decreased when compared with wild-type plants (Elkind et al, 1990;Howles et al, 1996;Sewalt et al, 1997). These plants allow assessment of the effects of quantitative and qualitative changes in a component of a potential metabolic channel on the operation of that channel.…”

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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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Overexpression of L-Phenylalanine Ammonia-Lyase in Transgenic Tobacco Plants Reveals Control Points for Flux into Phenylpropanoid Biosynthesis

Cited by 215 publications

References 39 publications

The effect of u.v.‐B radiation on u.v.‐absorbing secondary metabolites in birch seedlings grown under simulated forest soil conditions

The effect of u.v.‐B radiation on u.v.‐absorbing secondary metabolites in birch seedlings grown under simulated forest soil conditions

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

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

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