General Phenylpropanoid Metabolism Research Articles

Genome-wide analysis of general phenylpropanoid and monolignol-specific metabolism genes in sugarcane.

Lignin is the main component of secondary cell walls and is essential for plant development and defense. However, lignin is recognized as a major recalcitrant factor for efficiency of industrial biomass processing. Genes involved in general phenylpropanoid and monolignol-specific metabolism in sugarcane have been previously analyzed at the transcriptomic level. Nevertheless, the number of genes identified in this species is still very low. The recently released sugarcane genome sequence has allowed the genome-wide characterization of the 11 gene families involved in the monolignol biosynthesis branch of the phenylpropanoid pathway. After an exhaustive analysis of sugarcane genomes, 438 haplotypes derived from 175 candidate genes from Saccharum spontaneum and 144 from Saccharum hybrid R570 were identified as associated with this biosynthetic route. The phylogenetic analyses, combined with the search for protein conserved residues involved in the catalytic activity of the encoded enzymes, were employed to identify the family members potentially involved in developmental lignification. Accordingly, 15 candidates were identified as bona fide lignin biosynthesis genes: PTAL1, PAL2, C4H4, 4CL1, HCT1, HCT2, C3'H1, C3'H2, CCoAOMT1, COMT1, F5H1, CCR1, CCR2, CAD2, and CAD7. For this core set of lignin biosynthetic genes, we searched for the chromosomal location, the gene expression pattern, the promoter cis-acting elements, and microRNA targets. Altogether, our results present a comprehensive characterization of sugarcane general phenylpropanoid and monolignol-specific genes, providing the basis for further functional studies focusing on lignin biosynthesis manipulation and biotechnological strategies to improve sugarcane biomass utilization.

Identification of genes from the general phenylpropanoid and monolignol-specific metabolism in two sugarcane lignin-contrasting genotypes.

The phenylpropanoid pathway is an important route of secondary metabolism involved in the synthesis of different phenolic compounds such as phenylpropenes, anthocyanins, stilbenoids, flavonoids, and monolignols. The flux toward monolignol biosynthesis through the phenylpropanoid pathway is controlled by specific genes from at least ten families. Lignin polymer is one of the major components of the plant cell wall and is mainly responsible for recalcitrance to saccharification in ethanol production from lignocellulosic biomass. Here, we identified and characterized sugarcane candidate genes from the general phenylpropanoid and monolignol-specific metabolism through a search of the sugarcane EST databases, phylogenetic analysis, a search for conserved amino acid residues important for enzymatic function, and analysis of expression patterns during culm development in two lignin-contrasting genotypes. Of these genes, 15 were cloned and, when available, their loci were identified using the recently released sugarcane genomes from Saccharum hybrid R570 and Saccharum spontaneum cultivars. Our analysis points out that ShPAL1, ShPAL2, ShC4H4, Sh4CL1, ShHCT1, ShC3H1, ShC3H2, ShCCoAOMT1, ShCOMT1, ShF5H1, ShCCR1, ShCAD2, and ShCAD7 are strong candidates to be bona fide lignin biosynthesis genes. Together, the results provide information about the candidate genes involved in monolignol biosynthesis in sugarcane and may provide useful information for further molecular genetic studies in sugarcane.

Modeling the Impact of the Type of Cutting and Storage Temperature on the Bioactive Compound Content, Phenylpropanoid Metabolism Enzymes and Quality Attributes of Fresh-Cut Strawberries

The aim of this work was to model the effect of the type of cutting (whole without hull, halved, and quartered), storage temperature (2, 6, and 13 °C) and time on the changes of bioactive compounds, phenylpropanoid metabolism enzyme activities, and quality attributes of fresh-cut strawberries. The effect of increasing the intensity of wounding revealed an activation of the phenylalanine ammonia lyase (PAL) enzyme, with the consequent synthesis of phenolic compounds. Results revealed that quartered strawberries stored at 2 °C for 15 days accumulated up to 22% more phenolics than whole strawberries. The changes on quality parameters (soluble solids, pH, and color), total anthocyanins, and polyphenol oxidase were adequately fitted with zero order kinetic. All rate constants of these attributes, except for anthocyanins, fitted appropriately with Arrhenius equation. Changes on total phenolic contents and on PAL activity were fitted with a consecutive reaction mechanistic kinetic model. The rate constants of phenolics kinetic showed no dependence with temperature. However, rate constants of PAL activity fitted appropriately with Arrhenius equation. This global study offers a better understanding of the effects of processing and storage conditions on general quality, bioactive compounds, and phenylpropanoid metabolism enzymes of fresh-cut strawberries.

A Proteolytic Regulator Controlling Chalcone Synthase Stability and Flavonoid Biosynthesis in Arabidopsis.

Flavonoids represent a large family of specialized metabolites involved in plant growth, development, and adaptation. Chalcone synthase (CHS) catalyzes the first step of flavonoid biosynthesis by directing carbon flux from general phenylpropanoid metabolism to flavonoid pathway. Despite extensive characterization of its function and transcriptional regulation, the molecular basis governing its posttranslational modification is enigmatic. Here, we report the discovery of a proteolytic regulator of CHS, namely, KFBCHS, a Kelch domain-containing F-box protein in Arabidopsis thaliana KFBCHS physically interacts with CHS and specifically mediates its ubiquitination and degradation. KFBCHS exhibits developmental expression patterns in Arabidopsis leaves, stems, and siliques and strongly responds to the dark-to-light (or the light-to-dark) switch, the blue, red, and far-red light signals, and UV-B irradiation. Alteration of KFBCHS expression negatively correlates to the cellular concentration of CHS and the production of flavonoids. Our study suggests that KFBCHS serves as a crucial negative regulator, via mediating CHS degradation, coordinately controlling flavonoid biosynthesis in response to the developmental cues and environmental stimuli.

Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.

Coumarins are the main bioactive compounds in Peucedanum praeruptorum Dunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin in P. praeruptorum have not been identified yet. 4-Coumarate: CoA ligase (4CL) catalyzes the formation of hydroxycinnamates CoA esters, and plays an essential role at the divergence point from general phenylpropanoid metabolism to major branch pathway of coumarin. Here, three novel putative 4CL genes (Pp4CL1, Pp4CL7, and Pp4CL10) were isolated from P. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilized p-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis in P. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, and o-coumaric acids and represented a bona fide 4CL. Pp4CL7 and Pp4CL10 had no catalytic activity toward hydroxycinnamic acid compounds. But they had close phylogenetic relationship to true 4CLs and were defined as 4CL-like genes. Among all putative 4CLs, Pp4CL1 was the most highly expressed gene in roots, and its expression level was significantly up-regulated in mature roots compared with seedlings. Subcellular localization studies showed that Pp4CL1 and Pp4CL10 proteins were localized in the cytosol. In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins in P. praeruptorum and provides further insights in understanding the structure–function relationships of this important family of proteins.

Positive selection drives adaptive diversification of the 4-coumarate: CoA ligase (4CL) gene in angiosperms.

Lignin and flavonoids play a vital role in the adaption of plants to a terrestrial environment. 4-Coumarate: coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for both lignin and flavonoids biosynthesis. However, very little is known about how such essential enzymatic functions evolve and diversify. Here, we analyze 4CL sequence variation patterns in a phylogenetic framework to further identify the evolutionary forces that lead to functional divergence. The results reveal that lignin-biosynthetic 4CLs are under positive selection. The majority of the positively selected sites are located in the substrate-binding pocket and the catalytic center, indicating that nonsynonymous substitutions might contribute to the functional evolution of 4CLs for lignin biosynthesis. The evolution of 4CLs involved in flavonoid biosynthesis is constrained by purifying selection and maintains the ancestral role of the protein in response to biotic and abiotic factors. Overall, our results demonstrate that protein sequence evolution via positive selection is an important evolutionary force driving adaptive diversification in 4CL proteins in angiosperms. This diversification is associated with adaption to a terrestrial environment.

Contrasting nitrogen fertilization treatments impact xylem gene expression and secondary cell wall lignification in Eucalyptus.

BackgroundNitrogen (N) is a main nutrient required for tree growth and biomass accumulation. In this study, we analyzed the effects of contrasting nitrogen fertilization treatments on the phenotypes of fast growing Eucalyptus hybrids (E. urophylla x E. grandis) with a special focus on xylem secondary cell walls and global gene expression patterns.ResultsHistological observations of the xylem secondary cell walls further confirmed by chemical analyses showed that lignin was reduced by luxuriant fertilization, whereas a consistent lignin deposition was observed in trees grown in N-limiting conditions. Also, the syringyl/guaiacyl (S/G) ratio was significantly lower in luxuriant nitrogen samples. Deep sequencing RNAseq analyses allowed us to identify a high number of differentially expressed genes (1,469) between contrasting N treatments. This number is dramatically higher than those obtained in similar studies performed in poplar but using microarrays. Remarkably, all the genes involved the general phenylpropanoid metabolism and lignin pathway were found to be down-regulated in response to high N availability. These findings further confirmed by RT-qPCR are in agreement with the reduced amount of lignin in xylem secondary cell walls of these plants.ConclusionsThis work enabled us to identify, at the whole genome level, xylem genes differentially regulated by N availability, some of which are involved in the environmental control of xylogenesis. It further illustrates that N fertilization can be used to alter the quantity and quality of lignocellulosic biomass in Eucalyptus, offering exciting prospects for the pulp and paper industry and for the use of short coppices plantations to produce second generation biofuels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0256-9) contains supplementary material, which is available to authorized users.

Divergent and Overlapping Function of Five 4-Coumarate/Coenzyme A Ligases from Populus tomentosa

4-Coumarate/coenzyme A ligases (4CLs) play key roles in the biosynthesis of plant secondary compounds and act at the divergence point from general phenylpropanoid metabolism to several major branch pathways, such as lignins and flavonoids. In this study, five 4CL genes in Populus tomentosa were cloned and characterized. Quantitative polymerase chain reaction (qPCR) and promoter-β-glucuronidase analysis showed that the expression of Pto4CLs is significantly divergent and certainly overlapping. The five Pto4CL recombinant proteins also have different substrate specificities and catalytic turnover rates. Pto4CL4 has the highest substrate affinity and catalytic turnover rate compared with other Pto4CL proteins. All five Pto4CL recombinant proteins had no detectable activity towards sinapate. Deletion of V338 residues in Pto4CL mutants resulted in activity towards sinapate. The low accumulation of sinapate and high proportion of S unit lignin implies that the ability of 4CL to catalyze the activation of sinapate is not necessary for lignin biosynthesis. Overexpression of the five Pto4CLs in transgenic tobacco using the 35S promoter revealed significantly increased lignin contents and decreased hydroxycinnamate derivative contents in phloem, xylem, root, and leaf tissues. An increased content of naringenin in Pto4CL4 transgenic tobacco roots was also observed. Our results suggest that the substrate specificity and divergent and overlapping expression patterns of Pto4CL isoforms may play important roles in regulating the lignin and flavonoid biosynthesis.

Expression analysis of key genes of phenylpropanoid pathway and phenol profiling during Ricinus communis–Fusarium oxysporum f. sp. ricini interaction

The general phenylpropanoid metabolism generates an array of secondary metabolites and phenolic acids which imparts disease resistance in plants. Expression pattern of the key genes of phenylpropanoid pathway was studied at 0, 24 and 48h after infection (h.a.i.) and phenolic acid profiling was carried out at 0, 24, 48 and 72h.a.i. in the leaves of wilt infected and non- infected of both resistant and susceptible genotypes of castor. Expression analysis of PAL (phenylalanine ammonia lyase), C4H1 (cinnamate 4-hydroxylase 1) and C4H2 (cinnamate 4-hydroxylase 2) genes using RT-PCR with gene specific primers showed appreciable increase in the expression of PAL and C4H-2 gene in resistant genotypes at 48h interval than 24h interval compared to susceptible genotype. However, gene C4H-1 was down regulated in susceptible genotypes after 24 and 48h.a.i. while up regulated in resistant genotypes. Phenol profiling using HPTLC showed the presence of three phenolic acids i.e. caffeic acid, ferulic acid and salicylic acid in non-infected and infected castor genotypes. Higher content of caffeic and ferulic acid was detected in infected and non-infected resistant genotypes at 0, 24 and 48h.a.i, whereas caffeic acid was not detected in susceptible genotypes at 0h.a.i. These results suggest the critical role of phenols in castor disease resistance.

Cloning and analysis of a new 4CL-like gene in Populus tomentosa

The phenylpropanoid enzyme 4-coumarate: coenzyme A ligase (4CL), plays a key role in general phenylpropanoid metabolism. Our investigation involves a new 4CL-like gene cloned from Populus tomentosa. This new 4CLlike gene is 1,692 bp and encodes a protein of 552 aa. After analysis of its domain, we found a highly conserved region of a 4CL gene family in this 4CL-like gene. Based on our results, we believe that this gene belongs to the family of 4CL. A recombinant vector, referred to as pET30a-4CL-like, was constructed by connecting this 4CL-like gene fragment to pET30a. After inducing it with IPTG for 3 h, the 4CL-like protein was purified by a Ni-NTA method. This 4CL-like protein has a calculated molecular weight of 60 kDa by SDS-PAGE.

Molecular cloning, expression pattern, and putative cis-acting elements of a 4-coumarate:CoA ligase gene in bamboo (Neosinocalamus affinis)

Background: 4-coumarate:CoA ligase (4CL) plays an important role at the divergence point from general phenylpropanoid metabolism to several branch pathways. Although 4CL sin higher plants have been extensively studied, little has known about the 4CL gene of bamboo. Results: In current study, a Na4CL gene putative encoding 4-coumarate:CoA ligase (4CL) and its 5'-flanking region were isolated from bamboo ( Neosinocalamus affinis ) by RACE-PCR and genomic DNA walker, respectively. Na4CL encodes a predicted protein of 557 amino acids, with conserved motifs of adenylate-forming enzymes. Phylogenetic analysis showed that Na4CL shared 62~85% identity with other known plant 4CLs, and cluster closely with some known 4CLs in monocots. Sequence analysis revealed conserved cis-acting elements (Box A and AC-II element) present in the Na4CL promoter. Additionally, a Na4CL RNAi construct was transformed into tobacco. Transgenic tobaccos displayed significant down-expression of endogenesis 4CL and reduced lignin contents. Conclusion: These results contribute to the knowledge of the presence of Na4CL gene and its possible role in phenylpropanoid metabolism.

Evolution of the 4‐coumarate:coenzyme A ligase (4CL) gene family: Conserved evolutionary pattern and two new gene classes in gymnosperms

Abstract The 4‐coumarate:coenzyme A ligase (4CL) is the branch point enzyme that channels the general phenylpropanoid metabolism into specific lignin and flavonoid biosynthesis branches. Genetic engineering experiments on the 4CL gene have been carried out in many species, but the precise functions of different gene members are still unresolved. To investigate the evolutionary relationships and functional differentiation of the 4CL gene family, we made a comprehensive evolutionary analysis of this gene family from 27 species representing the major lineages of land plants. The phylogenetic analysis indicates that both vascular and seed plant 4CL genes form monophyletic groups, and that three and two 4CL classes can be recognized in gymnosperms and angiosperms, respectively. The evolutionary rate and frequency of duplication of the 4CL gene family are much more conserved than that of the CAD/SAD (cinnamyl/sinapyl alcohol dehydrogenase) gene family, which catalyzes the last step in monolignol biosynthesis. This may be due to different selective pressures on these genes whose products catalyze different steps in the biosynthesis pathway. In addition, we found two new major classes of 4CL genes in gymnosperms.

Grapevine cell early activation of specific responses to DIMEB, a resveratrol elicitor

BackgroundIn response to pathogen attack, grapevine synthesizes phytoalexins belonging to the family of stilbenes. Grapevine cell cultures represent a good model system for studying the basic mechanisms of plant response to biotic and abiotic elicitors. Among these, modified β-cyclodextrins seem to act as true elicitors inducing strong production of the stilbene resveratrol.ResultsThe transcriptome changes of Vitis riparia × Vitis berlandieri grapevine cells in response to the modified β-cyclodextrin, DIMEB, were analyzed 2 and 6 h after treatment using a suppression subtractive hybridization experiment and a microarray analysis respectively. At both time points, we identified a specific set of induced genes belonging to the general phenylpropanoid metabolism, including stilbenes and hydroxycinnamates, and to defence proteins such as PR proteins and chitinases. At 6 h we also observed a down-regulation of the genes involved in cell division and cell-wall loosening.ConclusionsWe report the first large-scale study of the molecular effects of DIMEB, a resveratrol inducer, on grapevine cell cultures. This molecule seems to mimic a defence elicitor which enhances the physical barriers of the cell, stops cell division and induces phytoalexin synthesis.

Proanthocyanidin biosynthesis in the seed coat of yellow-seeded, canola qualityBrassica napusYN01-429 is constrained at the committed step catalyzed by dihydroflavonol 4-reductaseThis paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

The yellow seed characteristic in Brassica napus L. is desirable because of its association with higher oil content and better quality of oil-extracted meal. YN01-429 is a yellow-seeded canola-quality germplasm developed in Canada arising from several years of research. Seed-coat pigmentation is due to oxidized proanthocyanidins (PA; condensed tannins) derived from phenylpropanoids and malonyl CoA. We found PA accumulation to be most robust in young seed coats (20 d post anthesis; dpa) of a related black-seeded line N89-53 and only very little PA in YN01-429, which also contained much less extractable phenolics. The flavonol content, however, did not show as great a difference between these two lines. Furthermore, sinapine, a product of the general phenylpropanoid metabolism, was present at comparable levels in the embryos of both lines. Dihydroflavonol reductase (DFR) activity that commits phenolics to PA synthesis was lower in YN01-429 seed coats. The results of Southern blot and in silico analyses were indicative of two copies of the DFR gene in B. napus. Both copies were functional in YN01-429, ruling out homeoallelic repression or silencing, but together they showed very low expression levels (17-fold fewer transcripts) relative to DFR activity in N89-53 seed coats. These results collectively suggest that YN01-429 differs in regulatory circuits that impact the PA synthesis branch much more than the flavonol synthesis branch in the seed coats and such circuits do not impinge upon general phenylpropanoid metabolism in the embryos.

Identification of a 4-coumarate:CoA ligase gene family in the moss, Physcomitrella patens

Since the early evolution of land plants from primitive green algae, phenylpropanoid compounds have played an important role. In the biosynthesis of phenylpropanoids, 4-coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role at the divergence point from general phenylpropanoid metabolism to several major branch pathways. Although higher plant 4CLs have been extensively studied, little information is available on the enzymes from bryophytes. In Physcomitrella patens, we have identified a 4CL gene family consisting of four members, taking advantage of the available EST sequences and a draft sequence of the P. patens genome. The encoded proteins of three of the genes display similar substrate utilization profiles with highest catalytic efficiency towards 4-coumarate. Interestingly, the efficiency with cinnamate as substrate is in the same range as with caffeate and ferulate. The deduced proteins of the four genes share sequence identities between 78% and 86%. The intron/exon structures are pair wise similar. Pp4CL2 and Pp4CL3 each consists of four exons and three introns, whereas Pp4CL1 and Pp4CL4 are characterized each by five exons and four introns. Pp4CL1, Pp4CL2 and Pp4CL3 are expressed in both gametophore and protonema tissue of P. patens, unlike Pp4CL4 whose expression could not be demonstrated under the conditions employed. Phylogenetic analysis suggests an early evolutionary divergence of Pp4CL gene family members. Using Streptomyces coelicolor cinnamate:CoA ligase ( ScCCL) as an outgroup, the P. patens 4CLs are clearly separated from the spermatophyte proteins, but are intercalated between the angiosperm 4CL class I and class II. A comparison of three P. patens subspecies from diverse geographical locations shows high sequence identities for the four 4CL isoforms.

Enzymatic Synthesis of Phenolic CoAs Using 4-Coumarate:coenzyme A Ligase (4CL) from Rice

The enzyme, 4-coumarate:CoA ligase (4CL) catalyzes theconversion of 4-coumaric acid into coumaroyl-CoA and afew related substrates into their corresponding products suchas cinnamoyl-CoA, caffeoyl-CoA, and feruloyl-CoA in a pro-cess utilizing ATP and thus channels the common, phenyl-alanine-derived building block into the widely distinct branchesof general phenylpropanoid metabolism.

The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes.

4-Coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role in the biosynthesis of plant secondary compounds at the divergence point from general phenylpropanoid metabolism to several major branch pathways. In Arabidopsis thaliana, we have identified a previously undetected, fourth and final member of the At4CL gene family. The encoded enzyme, At4CL4, exhibits the rare property of efficiently activating sinapate, besides the usual 4CL substrates (4-coumarate, caffeate, and ferulate), indicating a distinct metabolic function. Phylogenetic analysis suggests an early evolutionary and functional divergence of three of the four gene family members, At4CL2-4, whereas At4CL1 appears to have originated much later by duplication of its structurally and functionally closest relative, At4CL2. Various characteristics shared by all known plant 4CL genes, as well as by the encoded proteins, define and delimit the At4CL gene family and distinguish it from the closely related family of "At4CL-like" genes.

Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant— Ruta graveolens—exhibiting low sensitivity to psoralen inactivation

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant. The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae. Mechanism-based inactivation was investigated using various psoralen derivatives. Only psoralen and 8-methoxypsoralen were found to inactivate C4H. The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration. Inactivation stoichiometry was 0.9 (± 0.2) for CYP73A1 and 1.1 (± 0.2) for CYP73A32. SDS–PAGE analysis demonstrated that [ 3H]psoralen was irreversibly bound to the C4H apoprotein. K i and k inact for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1. Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum. This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation. Cinnamic acid hydroxylation is a key step in the biosynthesis of phenylpropanoid compounds, psoralen derivatives included. Our results suggest a possible evolution of R. graveolens and P. crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism.

Benzoic acid biosynthesis in cell cultures of Hypericum androsaemum.

Biosynthesis of benzoic acid from cinnamic acid has been studied in cell cultures of Hypericum androsaemum L. The mechanism underlying side-chain shortening is CoA-dependent and non-beta-oxidative. The enzymes involved are cinnamate:CoA ligase, cinnamoyl-CoA hydratase/lyase and benzaldehyde dehydrogenase. Cinnamate:CoA ligase was separated from benzoate:CoA ligase and 4-coumarate:CoA ligase, which belong to xanthone biosynthesis and general phenylpropanoid metabolism, respectively. Cinnamoyl-CoA hydratase/lyase catalyzes hydration and cleavage of cinnamoyl-CoA to benzaldehyde and acetyl-CoA. Benzaldehyde dehydrogenase finally supplies benzoic acid. In cell cultures of H. androsaemum, benzoic acid is a precursor of xanthones, which accumulate during cell culture growth and after methyl jasmonate treatment. Both the constitutive and the induced accumulations of xanthones were preceded by increases in the activities of all benzoic acid biosynthetic enzymes. Similar changes in activity were observed for phenylalanine ammonia-lyase and the xanthone biosynthetic enzymes benzoate:CoA ligase and benzophenone synthase.

Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families.

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in general phenylpropanoid metabolism. 4CL is related to a larger class of prokaryotic and eukaryotic adenylate-forming enzymes and shares several conserved peptide motifs with these enzymes. In order to better characterize the nature of 4CL gene families in poplar, parsley, and tobacco, we used degenerate primers to amplify 4CL sequences from these species. In each species additional, divergent 4CL genes were found. Complete cDNA clones for the two new poplar 4CL genes were obtained, allowing examination of their expression patterns and determination of the substrate utilization profile of a xylem-specific isoform. Phylogenetic analysis of these genes and gene fragments confirmed previous results showing that 4CL proteins fall into two evolutionarily ancient subgroups . A comparative phylogenetic analysis of enzymes in the adenylate-forming superfamily showed that 4CLs, luciferases, and acetate CoA ligases each form distinct clades within the superfamily. According to this analysis, four Arabidopsis 4CL-like genes identified from the Arabidopsis Genome Project are only distantly related to bona fide 4CLs or are more closely related to fatty acid CoA ligases, suggesting that the three Arabidopsis 4CL genes previously characterized represent the extent of the 4CL gene family in this species.