Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke,and its expression upon UV-C stress

Globe artichoke represents a natural source of phenolic compounds with dicaffeoylquinic acids along with their biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the predominant molecules. We report the isolation and characterization of a full-length cDNA and promoter of a globe artichoke p-coumaroyl ester 3′-hydroxylase (CYP98A49), which is involved in both chlorogenic acid and lignin biosynthesis. Phylogenetic analyses demonstrated that this gene belongs to the CYP98 family. CYP98A49 was also heterologously expressed in yeast, in order to perform an enzymatic assay with p-coumaroylshikimate and p-coumaroylquinate as substrates. Real Time quantitative PCR analysis revealed that CYP98A49 expression is induced upon exposure to UV-C radiation. A single nucleotide polymorphism in the CYP98A49 gene sequence of two globe artichoke varieties used for genetic mapping allowed the localization of this gene to linkage group 10 within the previously developed maps. Nucleotide sequence data reported are available in the GenBank database under accession number: FJ225121     

Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3′-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3′H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide–binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP⁺ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering.     

Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.     

Flower colour and cytochromes P450

Flavonoids are major constituents of flower colour. Plants accumulate specific flavonoids and thus every species often exhibits a limited flower colour range. Three cytochromes P450 play critical roles in the flavonoid biosynthetic pathway. Flavonoid 3′-hydroxylase (F3′H, CYP75B) and flavonoid 3′,5′-hydroxylase (F3′5′H, CYP75A) catalyze the hydroxylation of the B-ring of flavonoids and are necessary to biosynthesize cyanidin-(red to magenta) and delphinidin-(violet to blue) based anthocyanins, respectively. Pelargonidin-based anthocyanins (orange to red) are synthesized in their absence. Some species such as roses, carnations and chrysanthemums do not have violet/blue flower colour due to deficiency of F3′5′H. Successful expression of heterologous F3′5′H genes in roses and carnations results in delphinidin production, causing a novel blue/violet flower colour. Down-regulation of F3′H and F3′5′H genes has yielded orange petunia and pink torenia colour that accumulate pelargonidin-based anthocyanins. Flavone synthase II (CYP93B) catalyzes the synthesis of flavones that contribute to the bluing of flower colour, and modulation of FNSII gene expression in petunia and tobacco changes their flower colour. Extensive engineering of the anthocyanin pathway is therefore now possible, and can be expected to enhance the range of flower colours.     

CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway.

The 4- and 5-hydroxylations of phenolic compounds in plants are catalyzed by cytochrome P450 enzymes. The 3-hydroxylation step leading to the formation of caffeic acid from p-coumaric acid remained elusive, however, alternatively described as a phenol oxidase, a dioxygenase, or a P450 enzyme, with no decisive evidence for the involvement of any in the reaction in planta. In this study, we show that the gene encoding CYP98A3, which was the best possible P450 candidate for a 3-hydroxylase in the Arabidopsis genome, is highly expressed in inflorescence stems and wounded tissues. Recombinant CYP98A3 expressed in yeast did not metabolize free p-coumaric acid or its glucose or CoA esters, p-coumaraldehyde, or p-coumaryl alcohol, but very actively converted the 5-O-shikimate and 5-O-d-quinate esters of trans-p-coumaric acid into the corresponding caffeic acid conjugates. The shikimate ester was converted four times faster than the quinate derivative. Antibodies directed against recombinant CYP98A3 specifically revealed differentiating vascular tissues in stem and root. Taken together, these data show that CYP98A3 catalyzes the synthesis of chlorogenic acid and very likely also the 3-hydroxylation of lignin monomers. This hydroxylation occurs on depsides, the function of which was so far not understood, revealing an additional and unexpected level of networking in lignin biosynthesis.     

cDNA cloning and sequence analysis of the rice Cinnamate-4-Hydroxylase gene, a cytochrome P450-dependent monooxygenase involved in the general phenylpropanoid pathway

Plant cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of phenylpropanoids, terpenes, lipids, and alkaloids. We isolated a cDNA clone for cinnamate-4-hydroxylase (C4H) from a Japonica type rice(Oryza sativa L. cv. llpumbyeo). This C4H has a deduced amino acid sequence that is 85% identical tothat ofSorghum bicolor. Our phylogenetic analysis also showed that theOsC4HL gene is closely related toC4H fromS. bicolor. A putative genomic DNA sequence corresponding toOsC4HL contained cis-elements (boxes P, A, L, and TCA motifs), AT-rich elements, and wound-response elements that control gene expression in its promoter region.OsC4HL expression was detected in all the tissue types, with the highest level being measured in the roots. It was also apparently up-regulated by wounding stress. These data suggest that theOsC4HL gene isC4H member in theCYP73 subfamily.     

Flower colour and cytochromes P450

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.     

Geranylhydroquinone 3′′-hydroxylase, a cytochrome P-450 monooxygenase from Lithospermum erythrorhizon cell suspension cultures

Geranylhydroquinone 3′′-hydroxylase, which is likely to be involved in shikonin and dihydroechinofuran biosynthesis, was identified in cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae). The enzyme hydroxylates the isoprenoid side chain of geranylhydroquinone (GHQ), a known precursor of shikonin. Proton/proton correlation spectroscopic and proton/proton long-range correlation spectroscopic studies confirmed that hydroxylation takes place specifically at position 3′′, i.e. at the methyl group involved in the cyclization reaction. The enzyme is membrane-bound and was found in the microsomal fraction. It requires NADPH and molecular oxygen as cofactors, and is inhibited by cytochrome P-450 inhibitors such as cytochrome c and CO. The inhibitory effect of CO is reversed by illumination. These data suggest that the enzyme is a cytochrome P-450-dependent monooxygenase. The optimum pH of GHQ 3′′-hydroxylase is 7.4, and the apparent K _m value for GHQ is 1.5 μM. The reaction velocity obtained with 3-geranyl-4-hydroxybenzoic acid was more than 100 times lower than that obtained with geranylhydroquinone. Received: 20 March 1999 / Accepted: 20 July 1999     

Structural characterization of the gene and corresponding cDNA for the cytochrome P450rm from Rhodotorula minuta which catalyzes formation of isobutene and 4-hydroxylation of benzoate

Cytochrome P450rm was previously isolated from the basidiomycete yeast Rhodotorula minuta as a bifunctional enzyme with isobutene-forming and benzoate 4-hydroxylase activities. We cloned the gene and corresponding cDNA for P450rm in order to characterize the enzyme in the context of fungal phylogeny and physiology. From the cDNA sequence, P450rm was deduced to have 527 amino acids with a calculated molecular weight of 59 136. P450rm shared 48% amino acid sequence identity with CYP53A1 from Aspergillus niger, indicating that the gene belongs to a novel subfamily of CYP53, CYP53B. However, the organization of the P450rm gene, which has eight exons and seven introns, differed completely to that of CYP53A1. Northern analysis demonstrated that the level of P450rm mRNA expression increased when L-phenylalanine was used as sole carbon source. These results suggest that P450rm has been well conserved during the evolution of fungi as a benzoate 4-hydroxylase in the dissimilation pathway starting from L-phenylalanine Received: 18 February 1997 / Accepted: 18 May 1997     

Catalytic activity,duplication and evolution of the CYP98 cytochrome P450 family in wheat

A burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Isolation and Analysis of Cinnamic Acid 4-Hydroxylase Homologous Genes from a Hybrid Aspen,Populus kitakamiensis

Cinnamic acid 4-hydroxylase (CA4H) is the second enzyme involved in phenylpropanoid biosynthesis and is a member of the cytochrome P-450 superfamily. Three CA4H homologous genes, cyp73a, cyp73b, and cyp73c, and a cDNA clone of cyp73a were isolated from a genomic library and a cDNA library of a hybrid aspen; Populus kitakamiensis, and were characterized. They might be interrupted by two introns each. cyp73a and cyp73b were very similar to each other not only in coding regions but also in non-coding regions. Southern blot analysis showed that four homologous genes for CA4H constructed a small gene family in the diploid genome of P. kitakamiensis. In the promoter regions, there were many common m-element-like sequences in phenylpropanoid biosynthesis genes.     

Cytochrome P450s in flavonoid metabolism

In this review, cytochrome P450s characterized at the molecular level catalyzing aromatic hydroxylations, aliphatic hydroxylations and skeleton formation in the flavonoid metabolism are surveyed. They are involved in the biosynthesis of anthocyanin pigments and condensed tannin (CYP75, flavonoid 3′,5′-hydroxylase and 3′-hydroxylase), flavones [CYP93B, (2S)-flavanone 2-hydroxylase and flavone synthase II], and leguminous isoflavonoid phytoalexins [CYP71D9, flavonoid 6-hydroxylase; CYP81E, isoflavone 2′-hydroxylase and 3′-hydroxylase; CYP93A, 3,9-dihydroxypterocarpan 6a-hydroxylase; CYP93C, 2-hydroxyisoflavanone synthase (IFS)]. Other P450s of the flavonoid metabolism include methylenedioxy bridge forming enzyme, cyclases producing glyceollins, flavonol 6-hydroxylase and 8-dimethylallylnaringenin 2′-hydroxylase. Mechanistic studies on the unusual aryl migration by CYP93C, regulation of IFS expression in plant organs and its biotechnological applications are introduced, and flavonoid metabolisms by non-plant P450s are also briefly discussed.     

Evolution of coumaroyl conjugate 3‐hydroxylases in land plants: lignin biosynthesis and defense

Multiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long‐distance water transport. Cytochrome P450 monooxygenase 98 (CYP98) catalyzes a rate‐limiting step in phenylpropanoid biosynthesis. Phylogenetic reconstructions suggest that a single copy of CYP98 founded each major land plant lineage (bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms), and was maintained as a single copy in all lineages but the angiosperms. In angiosperms, a series of independent gene duplications and losses occurred. Biochemical assays in four angiosperm species tested showed that 4‐coumaroyl‐shikimate, a known intermediate in lignin biosynthesis, was the preferred substrate of one member in each species, while independent duplicates in Populus trichocarpa and Amborella trichopoda each showed broad substrate ranges, accepting numerous 4‐coumaroyl‐esters and ‐amines, and were thus capable of producing a wide range of hydroxycinnamoyl conjugates. The gymnosperm CYP98 from Pinus taeda showed a broad substrate range, but preferred 4‐coumaroyl‐shikimate as its best substrate. In contrast, CYP98s from the lycophyte Selaginella moellendorffii and the fern Pteris vittata converted 4‐coumaroyl‐shikimate poorly in vitro, but were able to use alternative substrates, in particular 4‐coumaroyl‐anthranilate. Thus, caffeoyl‐shikimate appears unlikely to be an intermediate in monolignol biosynthesis in non‐seed vascular plants, including ferns. The best substrate for CYP98A34 from the moss Physcomitrella patens was also 4‐coumaroyl‐anthranilate, while 4‐coumaroyl‐shikimate was converted to lower extents. Despite having in vitro activity with 4‐coumaroyl‐shikimate, CYP98A34 was unable to complement the Arabidopsis thaliana cyp98a3 loss‐of‐function phenotype, suggesting distinct properties also in vivo.     

Cloning, Functional Identification and Sequence Analysis of Flavonoid 3′-hydroxylase and Flavonoid 3′,5′-hydroxylase cDNAs Reveals Independent Evolution of Flavonoid 3′,5′-hydroxylase in the Asteraceae Family

Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Phylogenetic analysis of known sequences of F3′H and F3′5′H indicated that F3′5′H was recruited from F3′H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3′Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3′H activity but surprisingly, three recombinant proteins showed F3′5′H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3′5′H. Furthermore, sequence analysis of these unusual F3′5′H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3′5′H is necessary for the synthesis of 3′,4′,5′-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3′5′H probably reflects the adaptive value of efficient attraction of insect pollinators.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

A new gene coding for <Emphasis Type="Italic">p</Emphasis>-coumarate 3-hydroxylase from <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

p-Coumarate 3-hydroxylase (C3H) is a rate-limiting enzyme involved in monolignol biosynthesis. The full-length cDNA from Ginkgo biloba and genomic DNA sequence encoding C3H (designated as GbC3H) were cloned and characterized for the first time by rapid amplification of cDNA ends technique. The full-length cDNA of GbC3H was of 1860 bp containing a 1527 bp open reading frame encoding a cytochrome P450 protein of 508 amino acids with a calculated mol wt of 57.46 kD and an isoelectric point of 7.09. Two introns were present in the GbC3H gene. Comparative and bioinformatic analyses revealed that GbC3H had close similarity with C3Hs from other species and contained a conserved cytochrome P450 cysteine heme-iron ligand signature. Phylogenetic analysis indicated that GbC3H shared a common evolutionary origin based on sequence and had the closest relationship to C3H from gymnosperm species. Southern blot analysis indicated that GbC3H belonged to a small-gene family. Tissue expression pattern analysis revealed the highest expression of GbC3H in roots followed by leaves, and no expression was detected in stems. Only a few proteins of this class have been found, so the cloning and characterization of GbC3H will be useful in understanding the role of C3Hs in the lignin biosynthesis at the molecular level. This text was submitted by the authors in English.     

cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from <Emphasis Type="Italic">Coleus blumei</Emphasis> involved in rosmarinic acid biosynthesis

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K _m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and 40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.     

Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco.

A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.     

A coumaroyl-ester-3-hydroxylase insertion mutant reveals the existence of nonredundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth

Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.