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1.
Genes (Basel) ; 10(8)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31443318

RESUMO

Cryptomeria fortunei, also known as the Chinese cedar, is an important timber species in southern China. The primary component of its woody tissues is lignin, mainly present in secondary cell walls. Therefore, continuous lignin synthesis is crucial for wood formation. In this study, we aimed to discover key genes involved in lignin synthesis expressed in the vascular cambium of C. fortunei. Through transcriptome sequencing, we detected expression of two genes, 4CL and CCoAOMT, known to be homologous to enzymes involved in the lignin synthesis pathway. We studied the function of these genes through bioinformatics analysis, cloning, vascular cambium expression analysis, and transgenic cross-species functional validation studies. Our results show that Cf4CL and CfCCoAOMT do indeed function in the pathway of lignin synthesis and likely perform this function in C. fortunei. They are prime candidates for future (gene-editing) studies aimed at optimizing C. fortunei wood production.


Assuntos
Cryptomeria/genética , Lignina/biossíntese , Metiltransferases/genética , Proteínas de Plantas/genética , Transcinamato 4-Mono-Oxigenase/genética , Câmbio/genética , Câmbio/metabolismo , Cryptomeria/enzimologia , Cryptomeria/metabolismo , Lignina/genética , Metiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Transcinamato 4-Mono-Oxigenase/metabolismo
2.
Plant Mol Biol ; 101(3): 235-255, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31254267

RESUMO

KEY MESSAGE: The core set of biosynthetic genes potentially involved in developmental lignification was identified in the model C4 grass Setaria viridis. Lignin has been recognized as a major recalcitrant factor negatively affecting the processing of plant biomass into bioproducts. However, the efficient manipulation of lignin deposition in order to generate optimized crops for the biorefinery requires a fundamental knowledge of several aspects of lignin metabolism, including regulation, biosynthesis and polymerization. The current availability of an annotated genome for the model grass Setaria viridis allows the genome-wide characterization of genes involved in the metabolic pathway leading to the production of monolignols, the main building blocks of lignin. Here we performed a comprehensive study of monolignol biosynthetic genes as an initial step into the characterization of lignin metabolism in S. viridis. A total of 56 genes encoding bona fide enzymes catalyzing the consecutive ten steps of the monolignol biosynthetic pathway were identified in the S. viridis genome. A combination of comparative phylogenetic studies, high-throughput expression analysis and quantitative RT-PCR analysis was further employed to identify the family members potentially involved in developmental lignification. Accordingly, 14 genes clustered with genes from closely related species with a known function in lignification and showed an expression pattern that correlates with lignin deposition. These genes were considered the "core lignin toolbox" responsible for the constitutive, developmental lignification in S. viridis. These results provide the basis for further understanding lignin deposition in C4 grasses and will ultimately allow the validation of biotechnological strategies to produce crops with enhanced processing properties.


Assuntos
Lignina/metabolismo , Poaceae/metabolismo , Biomassa , Vias Biossintéticas , Coenzima A Ligases/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Funções Verossimilhança , Metiltransferases/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Filogenia , Plantas Geneticamente Modificadas/metabolismo , Transcinamato 4-Mono-Oxigenase/metabolismo
3.
J Agric Food Chem ; 67(24): 6725-6735, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31117506

RESUMO

Jasmonic acid (JA)- and ethylene-mediated signaling pathways are reported to have synergistic effects on inhibiting gray mold. The present study aimed to explain the role of ethylene perception in methyl jasmonate (MeJA)-mediated immune responses. Results showed that exogenous MeJA enhanced disease resistance, accompanied by the induction of endogenous JA biosynthesis and ethylene production, which led to the activation of the phenolic metabolism pathway. Blocking ethylene perception using 1-methylcyclopropene (1-MCP) either before or after MeJA treatment could differently weaken the disease responses induced by MeJA, including suppressing the induction of ethylene production and JA contents and reducing activities of lipoxygenase and allene oxide synthase compared to MeJA treatment alone. Consequently, MeJA-induced elevations in the total phenolic content and the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:coenzyme A ligase, and peroxidase were impaired by 1-MCP. These results suggested that ethylene perception participated in MeJA-mediated immune responses in tomato fruit.


Assuntos
Acetatos/imunologia , Botrytis/fisiologia , Ciclopentanos/imunologia , Etilenos/imunologia , Lycopersicon esculentum/imunologia , Oxilipinas/imunologia , Doenças das Plantas/imunologia , Reguladores de Crescimento de Planta/imunologia , Resistência à Doença , Frutas/imunologia , Frutas/microbiologia , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/imunologia
4.
Food Chem ; 289: 112-120, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955592

RESUMO

Sucrose acts as a vital signal that modulates fruit ripening. In current study, 50 mM sucrose was applied in strawberry fruit to investigate the regulation of sucrose in anthocyanin synthesis after harvest. The results showed that sucrose treatment increased the contents of glucose, fructose and sucrose, which were 19.76%, 15.83% and 16.50% higher, respectively, compared with control at the end of storage. The increase of glucose and fructose contents resulted from the activation of acid invertase by sucrose treatment. In addition, sucrose treatment specifically increased four pelargonidin derivatives, pelargonidin 3-glucoside, pelargonidin 3-rutinoside, pelargonidin 3-malonylglucoside and pelargonidin 3-methylmalonyglucoside, during the storage. Further, transcriptional profiles and enzyme activities analysis revealed that the accumulation of pelargonidin derivatives was related to the activation of the pentose phosphate pathway, shikimate pathway, phenylpropanoid pathway, and flavonoid pathway. These results provided new insights into the regulation of sucrose on the accumulation of individual anthocyanins.


Assuntos
Antocianinas/biossíntese , Fragaria/metabolismo , Antocianinas/análise , Antocianinas/metabolismo , Flavonoides/metabolismo , Fragaria/química , Frutas/química , Frutas/metabolismo , Glucose/análise , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Espectrometria de Massas , Via de Pentose Fosfato/efeitos dos fármacos , Fenilalanina Amônia-Liase/metabolismo , Sacarose/farmacologia , Transcinamato 4-Mono-Oxigenase/metabolismo
5.
Food Chem ; 286: 226-233, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30827600

RESUMO

Gray mold caused by Botrytis cinerea is a major postharvest disease of table grapes that leads to enormous economic losses during storage and transportation. The objective of this study was to evaluate the effectiveness of fulvic acid on controlling gray mold of table grapes and explore its mechanism of action. The results showed that fulvic acid application significantly reduced downy blight severity in table grapes without exhibiting any antifungal activity in vitro. Fulvic acid induced phenylpropanoid metabolism, as evidenced by accumulation of phenolic compounds and flavonoids, higher activities of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL), up-regulation of genes related to phenylpropanoid biosynthesis (PAL, C4H, 4CL, STS, ROMT and CHS). Our results suggested that fulvic acid induces resistance to B. cinerea mainly through the activation of phenylpropanoid pathway and can be used as a new activator of plant defense responses to control postharvest gray mold in table grapes.


Assuntos
Benzopiranos/farmacologia , Botrytis/patogenicidade , Frutas/microbiologia , Vitis/metabolismo , Vitis/microbiologia , Botrytis/efeitos dos fármacos , Coenzima A Ligases/metabolismo , Resistência à Doença , Flavonoides/metabolismo , Microbiologia de Alimentos , Frutas/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Fenilalanina Amônia-Liase/metabolismo , Fenilpropionatos/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Transcinamato 4-Mono-Oxigenase/metabolismo , Vitis/efeitos dos fármacos
6.
Molecules ; 23(12)2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30513965

RESUMO

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.


Assuntos
Escherichia coli/metabolismo , Lycoris/enzimologia , Propionatos/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo , Arabidopsis/genética , Clonagem Molecular , Escherichia coli/genética , Glucose/metabolismo , Lycoris/genética , NADP/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
7.
BMC Plant Biol ; 18(1): 278, 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30419822

RESUMO

BACKGROUND: The phenylpropanoid pathway is responsible for the synthesis of numerous compounds important for plant growth and responses to the environment. In the first committed step of phenylpropanoid biosynthesis, the enzyme phenylalanine ammonia-lyase (PAL) deaminates L-phenylalanine into trans-cinnamic acid that is then converted into p-coumaric acid by cinnamate-4-hydroxylase (C4H). Recent studies showed that the Kelch repeat F-box (KFB) protein family of ubiquitin ligases control phenylpropanoid biosynthesis by promoting the proteolysis of PAL. However, this ubiquitin ligase family, alternatively named Kiss Me Deadly (KMD), was also implicated in cytokinin signaling as it was shown to promote the degradation of type-B ARRs, including the key response activator ARR1. Considering that ubiquitin ligases typically have narrow target specificity, this dual targeting of structurally and functionally unrelated proteins appeared unusual. RESULTS: Here we show that the KFBs indeed target PAL but not ARR1. Moreover, we show that changes in early phenylpropanoid biosynthesis alter cytokinin sensitivity - as reported earlier - but that the previously documented cytokinin growth response changes are primarily the result of altered auxin signaling. We found that reduced PAL accumulation decreased, whereas the loss of C4H function increased the strength of the auxin response. The combined loss of function of both enzymes led to a decrease in auxin sensitivity, indicating that metabolic events upstream of C4H control auxin sensitivity. This auxin/phenylpropanoid interaction impacts both shoot and root development and revealed an auxin-dependent stimulatory effect of trans-cinnamic acid feeding on leaf expansion and thus biomass accumulation. CONCLUSIONS: Collectively, our results show that auxin-regulated plant growth is fine-tuned by early steps in phenylpropanoid biosynthesis and suggest that metabolites accumulating upstream of the C4H step impact the auxin response mechanism.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Fenilpropionatos/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Vias Biossintéticas , Cinamatos/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Genes Reporter , Repetição Kelch , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
8.
Zhongguo Zhong Yao Za Zhi ; 43(13): 2670-2675, 2018 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-30111015

RESUMO

The study is aimed to explore the effect of different temperature on the content of baicalin and gene expression in the growth of Scutellaria baicalensis. Four culture temperatures were used to establish the callus culture of S. baicalensis under dark conditions for 40 days and once every 5 days. The growth and baicalin contents were determined. 18S RNA was used as a reference gene to analyze the five key factors in baicalin biosynthesis pathway (PAL), cinnamic acid 4-hydroxylase (C4H), chalcone synthase (CHS), ß-glucuronidase (GUS), baicalein-7-O-glucuronosyltransferase (UBGAT) gene expression levels. The results showed that biomass, baicalin content and accumulation increased with the increase of temperature. 25 °C and 30 °C were more suitable for the growth of S. baicalensis. The content and accumulation of baicalin at 25 °C reached the highest level at 30 days, reaching 2.75% and 12.44 mg, respectively. The relative expression levels of C4H, CHS, GUS and UGBAT genes at 15 °C were higher than those at other treatments. The correlation between the relative expression levels of each key enzyme and the content of baicalin was negatively correlated with the increase of incubation temperature. The relative expression levels of PAL, C4H and CHS genes at 25 °C and 30 °C were significantly correlated with the contents of baicalin and reached a highly significant or significant level. Relative low temperature conditions were not conducive to the growth of S. baicalensis and the accumulation of baicalin. The accumulation of baicalin by PAL and C4H in the upstream of the synthetic pathway was significant.


Assuntos
Scutellaria baicalensis , Flavonoides , Temperatura , Transcinamato 4-Mono-Oxigenase
9.
Int J Biol Macromol ; 117: 1264-1279, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29894786

RESUMO

Galanthamine (GAL), the well-known Amaryllidaceae alkaloid, is a clinically used drug for the treatment of Alzheimer's disease. L-Phenylalanine (Phe) and trans-cinnamic acid (CA) were enzymatically transformed into the catechol portion of GAL. Herein, a Phe ammonia-lyase-encoding gene LrPAL3 and a cinnamate 4-hydroxylase-encoding gene LrC4H were cloned from Lycoris radiata, a GAL-producing plant. LrPAL3 was overexpressed in Escherichia coli and purified to homogeneity. LrPAL3 catalyzes the forward deamination conversion of L-Phe into trans-CA. The 3-chloro- and 4-fluoro-L-Phe were deaminated to generate the corresponding 3-chloro- and 4-fluoro-trans-CA by LrPAL3. LrPAL3-catalyzed reverse hydroamination was confirmed by the conversion of trans-CA into L-Phe with exceptional regio- and stereo-selectivity. LrC4H was overexpressed in E. coli with tCamCPR, a cytochrome P450 reductase-encoding gene. LrC4H catalyzes the regioselective para-hydroxylation on trans-CA to form p-coumaric acid. The transcriptional levels of both LrPAL3 and LrC4H were positively associated with the GAL contents within the leaves and flowers of L. radiata, which suggested that their expression and function are co-regulated and involved in the biosynthesis of GAL. The present investigations on the biosynthetic genes of GAL will promote the development of synthetic biology platforms for this kind of important drug via metabolic engineering.


Assuntos
Lycoris/enzimologia , Lycoris/genética , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Catálise , Clonagem Molecular , Galantamina/biossíntese , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Fenilalanina Amônia-Liase/química , Conformação Proteica , Análise de Sequência de DNA , Relação Estrutura-Atividade , Transcinamato 4-Mono-Oxigenase/química
10.
Enzyme Microb Technol ; 112: 59-64, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29499781

RESUMO

In this study, we engineered E. coli cells to express l-tyrosine converting enzymes, including tyrosine ammonia-lyase (TAL), p-coumarate 3-hydroxylase (C3H), feruloyl-CoA synthetase (FCS), and enoyl-CoA hydratase/aldolase (ECH). A catabolic circuit, which consisted of the protocatechualdehyde and p-hydroxybenzaldehyde production pathways, was reconstituted through combinatorial production of discrete enzymes. First, cells expressing FCS and ECH could convert each 5mM of caffeic acid and ferulic acid into protocatechualdehyde (70.5%) and vanillin (96.5%), respectively. Second, TAL and C3H were co-expressed with FCS and ECH. This strain converted l-tyrosine into caffeic acid, which was then converted into protocatechualdehyde. Ascorbic acid was used as an inhibitor of catechol aldehyde-based melanin formation, and the production yields of protocatechualdehyde and p-hydroxybenzaldehyde were 31.0±5.6 and 24.0±4.2mg/L, respectively. Finally, caffeic acid-based melanin formation was observed with higher production rate of 40.9±6.2mg/L/h by co-expressing FCS and ECH in the presence of caffeic acid.


Assuntos
Coenzima A Ligases/metabolismo , Enoil-CoA Hidratase/metabolismo , Escherichia coli/metabolismo , Melaninas/biossíntese , Tirosina/metabolismo , Amônia-Liases/genética , Amônia-Liases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzaldeídos/metabolismo , Burkholderia/enzimologia , Burkholderia/genética , Coenzima A Ligases/genética , Enoil-CoA Hidratase/genética , Escherichia coli/genética , Cinética , Engenharia Metabólica , Redes e Vias Metabólicas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
11.
Physiol Plant ; 162(3): 274-289, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28940509

RESUMO

Lignification of the plant cell wall could serve as the first line of defense against pathogen attack, but the molecular mechanisms of virulence and disease between oil palm and Ganoderma boninense are poorly understood. This study presents the biochemical, histochemical, enzymology and gene expression evidences of enhanced lignin biosynthesis in young oil palm as a response to G. boninense (GBLS strain). Comparative studies with control (T1), wounded (T2) and infected (T3) oil palm plantlets showed significant accumulation of total lignin content and monolignol derivatives (syringaldehyde and vanillin). These derivatives were deposited on the epidermal cell wall of infected plants. Moreover, substantial differences were detected in the activities of enzyme and relative expressions of genes encoding phenylalanine ammonia lyase (EC 4.3.1.24), cinnamate 4-hydroxylase (EC 1.14.13.11), caffeic acid O-methyltransferase (EC 2.1.1.68) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195). These enzymes are key intermediates dedicated to the biosynthesis of lignin monomers, the guaicyl (G), syringyl (S) and ρ-hydroxyphenyl (H) subunits. Results confirmed an early, biphasic and transient positive induction of all gene intermediates, except for CAD enzyme activities. These differences were visualized by anatomical and metabolic changes in the profile of lignin in the oil palm plantlets such as low G lignin, indicating a potential mechanism for enhanced susceptibility toward G. boninense infection.


Assuntos
Arecaceae/genética , Regulação da Expressão Gênica de Plantas , Lignina/biossíntese , Doenças das Plantas/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Arecaceae/metabolismo , Arecaceae/microbiologia , Benzaldeídos/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/microbiologia , Ganoderma/fisiologia , Interações Hospedeiro-Patógeno , Metiltransferases/genética , Metiltransferases/metabolismo , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Doenças das Plantas/microbiologia , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismo , Epiderme Vegetal/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
12.
J Agric Food Chem ; 65(36): 8003-8010, 2017 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-28813608

RESUMO

To explore the effects of hot air (HA, 38 °C for 12 h) treatment on the phenylpropanoid metabolism in cherry tomatoes, phenylpropanoid metabolite levels and the activities and expression of key enzymes were analyzed in HA-treated fruit. HA treatment enhanced phenylpropanoid metabolism, as evidenced by elevated levels of phenolics and flavonoids, higher activities of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase, and upregulated expression of LeCHS, LeCHI, LeF3H, and LeFLS. Levels of several phenylpropanoid metabolites were higher after HA treatment, including p-coumaric acid, caffeic acid, chlorogenic acid, isoquercitrin, quercetin, and rutin. These metabolic changes may be related to the reduced disease incidence and smaller lesion diameters observed in HA-treated fruit inoculated with Alternaria alternata (black mold) or Botrytis cinerea (gray mold). The results suggest that HA treatment induces disease resistance by activating the phenylpropanoid pathway in cherry tomato fruit.


Assuntos
Conservação de Alimentos/métodos , Frutas/química , Lycopersicon esculentum/metabolismo , Alternaria/fisiologia , Botrytis/fisiologia , Ácidos Cumáricos/análise , Ácidos Cumáricos/metabolismo , Flavonoides/análise , Flavonoides/metabolismo , Conservação de Alimentos/instrumentação , Frutas/genética , Frutas/metabolismo , Frutas/microbiologia , Lycopersicon esculentum/química , Lycopersicon esculentum/genética , Lycopersicon esculentum/microbiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Propionatos , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
13.
Plant Physiol Biochem ; 117: 42-50, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28587992

RESUMO

The plant phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolites. The second step in phenylpropanoid synthesis is carried out by the cytochrome P450 monooxygenase enzyme cinnamate 4-hydroxylase (C4H), which catalyzes the p-hydroxylation of trans-cinnamic acid to p-coumarate. Genes encoding C4H have been characterized in many vascular plant species, but as yet not in any bryophyte species. Here, a survey of the transcriptome sequences of four liverwort species was able to identify eight putative C4Hs. The three liverwort C4H genes taken forward for isolation and functional characterization were harbored by Plagiochasma appendiculatum (PaC4H) and Marchantia paleacea (MpC4H1 and MpC4H2). When the genes were heterologously expressed in yeast culture, an assay of enzyme activity indicated that PaC4H and MpC4H1 had a higher level of activity than MpC4H2. The favored substrate (trans-cinnamic acid) of all three liverwort C4Hs was the same as that of higher plant C4Hs. The co-expression of PaC4H in yeast cells harboring PaPAL (a P. appendiculatum ene encoding phenylalanine ammonia lyase) allowed the conversion of L-phenylalanine to p-coumaric acid. Furthermore, the expression level of PaC4H was enhanced after treatment with abiotic stress inducers UV irradiation or salicylic acid in the thallus of P. appendiculatum. The likelihood is that high activity C4Hs evolved in the liverworts and have remained highly conserved across the plant kingdom.


Assuntos
Hepatófitas/enzimologia , Hepatófitas/genética , Transcinamato 4-Mono-Oxigenase/genética , Regulação da Expressão Gênica de Plantas , Hepatófitas/metabolismo , Marchantia/enzimologia , Marchantia/genética , Marchantia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Transcinamato 4-Mono-Oxigenase/metabolismo , Leveduras/genética
14.
Mol Biol Evol ; 34(8): 2041-2056, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28505373

RESUMO

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.


Assuntos
Brachypodium/genética , Transcinamato 4-Mono-Oxigenase/genética , Sequência de Aminoácidos , Brachypodium/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Retículo Endoplasmático/metabolismo , Evolução Molecular , Duplicação Gênica/genética , Genes Duplicados/genética , Lignina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxirredução , Filogenia , Domínios Proteicos/genética , Sementes/metabolismo , Transcinamato 4-Mono-Oxigenase/metabolismo
15.
Phytochemistry ; 139: 1-7, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28366608

RESUMO

Basil (Ocimum basilicum L.), a medicinal plant of the Lamiaceae family, is used in traditional medicine; its essential oil is a rich source of phenylpropanoids. Methylchavicol and methyleugenol are the most important constituents of basil essential oil. Drought stress is proposed to enhance the essential oil composition and expression levels of the genes involved in its biosynthesis. In the current investigation, an experiment based on a completely randomized design (CRD) with three replications was conducted in the greenhouse to study the effect of drought stress on the expression level of four genes involved in the phenylpropanoid biosynthesis pathway in O. basilicum c.v. Keshkeni luvelou. The genes studied were chavicol O-methyl transferase (CVOMT), eugenol O-methyl transferase (EOMT), cinnamate 4-hydroxylase (C4H), 4-coumarate coA ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD). The effect of drought stress on the essential oil compounds and their relationship with the expression levels of the studied genes were also investigated. Plants were subjected to levels of 100%, 75%, and 50% of field capacity (FC) at the 6-8 leaf stage. Essential oil compounds were identified by gas chromatography/mass spectrometry (GC-MS) at flowering stage and the levels of gene expression were determind by real time PCR in plant leaves at the same stage. Results showed that drought stress increased the amount of methylchavicol, methyleugenol, ß-Myrcene and α-bergamotene. The maximum amount of these compounds was observed at 50% FC. Real-time PCR analysis revealed that severe drought stress (50% FC) increased the expression level of CVOMT and EOMT by about 6.46 and 46.33 times, respectively, whereas those of CAD relatively remained unchanged. The expression level of 4CL and C4H reduced under drought stress conditions. Our results also demonstrated that changes in the expression levels of CVOMT and EOMT are significantly correlated with methylchavicol (r = 0.94, P ≤ 0.05) and methyleugenol (r = 0.98, P ≤ 0.05) content. Thus, drought stress probably increases the methylchavicol and methyleugenol content, in part, through increasing the expression levels of CVOMT and EOMT.


Assuntos
Secas , Ocimum basilicum/química , Óleos Voláteis/química , Plantas Medicinais/química , Monoterpenos Acíclicos , Anisóis/química , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/isolamento & purificação , Eugenol/análogos & derivados , Eugenol/química , Eugenol/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Expressão Gênica , Interação Gene-Ambiente , Estrutura Molecular , Monoterpenos/química , Monoterpenos/isolamento & purificação , Ocimum basilicum/genética , Folhas de Planta/química , Óleos Vegetais/química , Plantas Medicinais/genética , Reação em Cadeia da Polimerase em Tempo Real , Sementes/química , Estresse Fisiológico/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
16.
Sci Rep ; 7: 39984, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28051165

RESUMO

Lignin, a cross-linked macromolecule of hydrophobic aromatic structure, provides additional rigidity to a plant cell wall. Although it is an integral part of the plant cell, presence of lignin considerably reduces the quality of the fiber of fiber-yielding plants. Decreasing lignin in such plants holds significant commercial and environmental potential. This study aimed at reducing the lignin content in jute-a fiber crop, by introducing hpRNA-based vectors for downregulation of two monolignoid biosynthetic genes- cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT). Transgenic generations, analyzed through Southern, RT-PCR and northern assays showed downregulation of the selected genes. Transgenic lines exhibited reduced level of gene expression with ~ 16-25% reduction in acid insoluble lignin for the whole stem and ~13-14% reduction in fiber lignin content compared to the control lines. Among the two transgenic plant types one exhibited an increase in cellulose content and concomitant improvement of glucose release. Composition of the lignin building blocks was found to alter and this alteration resulted in a pattern, different from other plants where the same genes were manipulated. It is expected that successful COMT-hpRNA and C4H-hpRNA transgenesis in jute will have far-reaching commercial implications leading to product diversification and value addition.


Assuntos
Corchorus/genética , Corchorus/metabolismo , Lignina/biossíntese , Regulação da Expressão Gênica de Plantas , Lignina/genética , Metiltransferases/genética , Plantas Geneticamente Modificadas , Transcinamato 4-Mono-Oxigenase/genética
17.
Acta Biol Hung ; 67(4): 379-392, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28000512

RESUMO

Phenylalanine-ammonia-lyase and cinnamate-4-hydroxylase play important role in the phenylpropanoid pathway, which produces many biologically important secondary metabolites participating in normal plant development. Flavonol quercetin is the main representant of these compounds that has been identified in numerous Juglans spp. In this survey, the developmental expression patterns of PAL and C4H genes during in vitro rooting of two walnut cultivars 'Sunland' and 'Howard' was examined by RT-PCR. To understand the potential role in rooting, the changing pattern of endogenous content of quercetin was also analyzed by HPLC. The 'Sunland' with better capacity to root had more quercetin content during the "inductive phase" of rooting than 'Howard'. In each cultivar, the level of PAL transcripts showed the same behavior with the changing patterns of quercetin during root formation of microshoots. The positive correlation between the changes of quercetin and PAL-mRNA indicated that PAL gene may have an immediate effect on flavonoid pathway metabolites including quercetin. Although the behavioral change of C4H expression was similar in both cultivars during root formation (with significantly more level for 'Howard'), it was not coincide with the changes of quercerin concentrations. Our results showed that C4H function is important for the normal development, but its transcriptional regulation does not correlate with quercetin as an efficient phenolic compound for walnut rhizogenesis.


Assuntos
Juglans/genética , Fenilalanina Amônia-Liase/genética , Desenvolvimento Vegetal/genética , Raízes de Plantas/crescimento & desenvolvimento , Quercetina/metabolismo , RNA Mensageiro/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Brotos de Planta , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Plant Physiol ; 172(1): 198-220, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27485881

RESUMO

Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization.


Assuntos
Arabidopsis/metabolismo , Iodobenzoatos/farmacologia , Lignina/metabolismo , Transcinamato 4-Mono-Oxigenase/antagonistas & inibidores , Arabidopsis/citologia , Arabidopsis/genética , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Análise por Conglomerados , Inibidores Enzimáticos/química , Inibidores Enzimáticos/classificação , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Iodobenzoatos/química , Espectrometria de Massas , Estrutura Molecular , Propanóis/metabolismo , Plântula/enzimologia , Plântula/genética , Plântula/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
19.
Plant Physiol ; 172(2): 874-888, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27506238

RESUMO

The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA.


Assuntos
Cinamatos/farmacologia , Homeostase/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Lignina/biossíntese , Fenilpropionatos/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Benzoatos/metabolismo , Benzoatos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Cinamatos/química , Cinamatos/metabolismo , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Relação Dose-Resposta a Droga , Espectrometria de Massas , Microscopia Confocal , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Transcinamato 4-Mono-Oxigenase/antagonistas & inibidores , Transcinamato 4-Mono-Oxigenase/metabolismo
20.
Sci Rep ; 6: 26458, 2016 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-27220407

RESUMO

Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H.


Assuntos
Artemisia annua/enzimologia , Artemisininas/metabolismo , Proteínas de Plantas/genética , Transcinamato 4-Mono-Oxigenase/genética , Artemisia annua/genética , Regulação para Baixo , Técnicas de Silenciamento de Genes , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , Estresse Fisiológico , Transcinamato 4-Mono-Oxigenase/metabolismo
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