An ancient role for CYP73 monooxygenases in phenylpropanoid biosynthesis and embryophyte development.

The phenylpropanoid pathway is one of the plant metabolic pathways most prominently linked to the transition to terrestrial life, but its evolution and early functions remain elusive. Here, we show that activity of the t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific step in the pathway, emerged concomitantly with the CYP73 gene family in a common ancestor of embryophytes. Through structural studies, we identify conserved CYP73 residues, including a crucial arginine, that have supported C4H activity since the early stages of its evolution. We further demonstrate that impairing C4H function via CYP73 gene inactivation or inhibitor treatment in three bryophyte species-the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis-consistently resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event in the development of the plant phenylpropanoid pathway, and underscore the deep-rooted function of the C4H enzyme in embryophyte biology.

Exogenous γ-aminobutyric acid strengthens phenylpropanoid and nitrogen metabolism to enhance the contents of flavonoids, amino acids, and the derivatives in edamame.

γ-aminobutyric acid (GABA) has been reported to improve stress resistance in plants. Nonetheless, little is known about the effects of GABA on the nutritional quality and regulatory mechanisms of edamame. Therefore, we analyzed the flavonoid and amino acid (AA) metabolism and the effects of GABA on the nutrient content of edamame seeds through physiological and metabolomic analyses. Exogenous GABA increased endogenous GABA metabolism and GABA transaminase activity and enhanced the oxoglutarate content, which entered into nitrogen metabolism and increased the activity and expression of nitrogen metabolism-related enzymes, to accumulate AAs and bioactive peptides. Meanwhile, exogenous GABA induced the metabolism of flavonoids, including total flavonoids, anthocyanins, 6''-o-malonyglycitin, glycitin, ononin, cyanin, and ginkgetin, by increasing the activity and expression of flavonoid biosynthetic enzymes. This is the first study to reveal that GABA effectively improves the nutritional quality of edamame through the accumulation of AAs, bioactive peptides, isoflavones, anthocyanins, sugars, and organic acids.

Functional characterization of NADPH-cytochrome P450 reductase and cinnamic acid 4-hydroxylase encoding genes from Scoparia dulcis L.

BACKGROUND: Most plant cytochrome P450 (P450) proteins need to be supplied with electrons from a redox partner, e.g. an NADPH-cytochrome P450 reductase (CPR), for the activation of oxygen molecules via heme. CPR is a flavoprotein with an N-terminal transmembrane domain, which transfers electrons from NADPH to the P450 via coenzymes flavin adenine dinucleotide and flavin mononucleotide. RESULTS: In this study, a novel CPR (SdCPR) was isolated from a tropical medicinal plant Scoparia dulcis L. The deduced amino acid of SdCPR showed high homology of > 76% with CPR from higher plants and belonged to the class II CPRs of dicots. Recombinant SdCPR protein reduced cytochrome c, ferricyanide (K3Fe(CN)6), and dichlorophenolindophenol in an NADPH-dependent manner. To elucidate the P450 monooxygenase activity of SdCPR, we isolated a cinnamic acid 4-hydroxylase (SdC4H, CYP73A111) gene from S. dulcis. Biochemical characterization of SdCPR/SdC4H demonstrated that SdCPR supports the oxidation step of SdC4H. Real-time qPCR results showed that expression levels of SdCPR and SdC4H were inducible by mechanical wounding treatment and phytohormone elicitation (methyl jasmonate, salicylic acid), which were consistent with the results of promotor analyses. CONCLUSIONS: Our results showed that the SdCPR and SdC4H are related to defense reactions, including the biosynthesis of secondary metabolites.

Functional expression and characterization of cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis in Physcomitrella patens.

KEY MESSAGE: Cinnamic acid 4-hydroxylase from the hornwort Anthoceros agrestis (AaC4H) was functionally expressed in the moss Physcomitrella patens and characterized at biochemical and molecular levels. Cinnamic acid 4-hydroxylase (C4H), a cytochrome P450-dependent hydroxylase, catalyzes the formation of 4-coumaric acid (=4-hydroxycinnamic acid) from trans-cinnamic acid. In the hornwort Anthoceros agrestis (Aa), this enzyme is supposed to be involved in the biosynthesis of rosmarinic acid (a caffeic acid ester of 3-(3,4-dihydroxyphenyl)lactic acid) and other related compounds. The coding sequence of AaC4H (CYP73A260) was expressed in the moss Physcomitrella patens (Pp_AaC4H). Protein extracts from the transformed moss showed considerably increased C4H activity driven by NADPH:cytochrome P450 reductase of the moss. Since Physcomitrella has own putative cinnamic acid 4-hydroxylases, enzyme characterization was carried out in parallel with the untransformed Physcomitrella wild type (Pp_WT). Apparent Km-values for cinnamic acid and NADPH were determined to be at 17.3 µM and 88.0 µM for Pp_AaC4H and 25.1 µM and 92.3 µM for Pp_WT, respectively. Expression levels of AaC4H as well as two Physcomitrella patens C4H isoforms were analyzed by quantitative real-time PCR. While PpC4H_1 displayed constantly low levels of expression during the whole 21-day culture period, AaC4H and PpC4H_2 increased their expression during the first 6-8 days of the culture period and then decreased again. This work describes the biochemical in vitro characterization of a cytochrome P450-dependent enzyme, namely C4H, heterologously expressed in the haploid model plant Physcomitrella patens.

De Novo Biosynthesis of p-Coumaric Acid in E. coli with a trans-Cinnamic Acid 4-Hydroxylase from the Amaryllidaceae Plant Lycoris aurea.

p-Coumaric acid is a commercially available phenolcarboxylic acid with a great number of important applications in the nutraceutical, pharmaceutical, material and chemical industries. p-Coumaric acid has been biosynthesized in some engineered microbes, but the potential of the plant CYP450-involved biosynthetic route has not investigated in Escherichia coli. In the present study, a novel trans-cinnamic acid 4-hydroxylase (C4H) encoding the LauC4H gene was isolated from Lycoris aurea (L' Hér.) Herb via rapid amplification of cDNA ends. Then, N-terminal 28 amino acids of LauC4H were characterized, for the subcellular localization, at the endoplasmic reticulum membrane in protoplasts of Arabidopsis thaliana. In E. coli, LauC4H without the N-terminal membrane anchor region was functionally expressed when fused with the redox partner of A. thaliana cytochrome P450 enzyme (CYP450), and was verified to catalyze the trans-cinnamic acid to p-coumaric acid transformation by whole-cell bioconversion, HPLC detection and LC-MS analysis as well. Further, with phenylalanine ammonia-lyase 1 of A. thaliana, p-coumaric acid was de novo biosynthesized from glucose as the sole carbon source via the phenylalanine route in the recombinant E. coli cells. By regulating the level of intracellular NADPH, the production of p-coumaric acid was dramatically improved by 9.18-fold, and achieved with a titer of 156.09 µM in shake flasks. The recombinant cells harboring functional LauC4H afforded a promising chassis for biological production of p-coumaric acid, even other derivatives, via a plant CYP450-involved pathway.

The Plant Growth-Promoting Fungus (PGPF) Alternaria sp. A13 Markedly Enhances Salvia miltiorrhiza Root Growth and Active Ingredient Accumulation under Greenhouse and Field Conditions.

Plant growth-promoting fungi (PGPF) have attracted considerable interest as bio-fertilisers due to their multiple beneficial effects on plant quantity and quality and their positive relationship with the ecological environment. Advancements in the development of PGPF for crops and economic plant cultivation applications have been achieved, but such improvements for the use of PGPF with popular medicinal herbs, such as Salvia miltiorrhiza, are rare. In this study, we collected S. miltiorrhiza specimens inhabiting wild, semi-wild, farmland and pot-cultured areas in the Henan province of China and isolated endophytes from the roots, shoots and leaves of these samples. Twenty-eight strains of the dominant genus Alternaria were identified and selected as candidate PGPF. Under greenhouse conditions, Alternaria sp. A13 simultaneously enhanced the dry root biomass and secondary metabolite accumulation of S. miltiorrhiza as the optimal PGPF of the 28 candidate isolates. To further assess the interaction between S. miltiorrhiza and Alternaria sp. A13, the effects on seedlings growth, active ingredient accumulation, and the activity of key enzymes for effective biosynthetic pathways were investigated over a period of six months under field conditions. Compared to uninoculated seedlings, S. miltiorrhiza seedlings colonised by Alternaria sp. A13 showed significant increment of 140% in fresh weight, 138% in dry weight, and enhancement in the contents of total phenolic acid, lithospermic acids A and B (LAA and LAB, respectively) of 210%, 128% and 213%, respectively. Examination of the related enzyme activities showed that the elicitation effect of A13 on LAB accumulation correlated with cinnamic acid 4-hydroxylase (C4H) activity in the phenylpropanoid pathway under field conditions. Our results confirmed that Alternaria sp. A13 not only contributes to the stimulation of S. miltiorrhiza root growth, but also boosts the secondary metabolism, thus demonstrating its application potential as a bio-fertiliser for S. miltiorrhiza cultivation, especially in areas outside of its native growth regions.

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants.

Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity.

Molecular cloning and expression analysis of cinnamic acid 4-hydroxylase gene from Isatis indigotica / 中草药

Objective: To clone cinnamate 4-hydroxylase (C4H) gene from Isatis indigotica and to analyze its bioinformatics and expression. Methods: The full-length cDNA of C4H was 1 674 bp (GenBank accession No. GU014562) long with an open reading frame (ORF) of 1 530 bp encoding a polypeptide of 509 amino acid residues. The comparison of C4H genomic DNA sequences and C4H cDNA sequence revealed that the C4H genomic DNA contained two introns. Southern-blotting analysis indicated that C4H was a multiple copy gene. C4H expression could be detected in all tissues at different expression levels, with the strongest expression in the roots. Further expression analysis revealed that the signaling components of defense/stress pathways, such as ultraviolet-B radiation (UV-B), methyl jasmonate (MeJA), abscisic acid (ABA), and Gibberellins (GA3) could up-regulate the C4H transcript levels compared with the control. Conclusion: We have first extracted the full length cDNA of C4H gene from I. indigotica, and its structural and bioinformatic analyses are carried out, which will help us to further illuminate this pathway. The research also provides a possibility to study the antiviral active constiuents in I. indigotica by plant secondary metabolic engineering.