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1.
Methods Enzymol ; 676: 133-158, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36280348

RESUMO

Lignin is a complex heterogenous polymer derived from oxidative radical polymerization of three monolignols, i.e., p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These lignin monomeric precursors structurally differ in their methoxy groups of the benzene rings. In phenylpropanoid-monolignol biosynthetic pathway, the endoplasmic reticulum (ER)-resident cytochrome P450 monooxygenases, cinnamate 4-hydroxylase, coumaroyl ester 3'-hydroxylase and ferulate 5-hydroxylase, establish the key structural characteristics of monolignols. The catalysis of cytochrome P450 monooxygenase requires reducing power, which is supplied by the ER electron transfer chains, composed of cytochrome P450 oxidoreductase (CPR), cytochrome b5 reductase (CBR) and/or cytochrome b5 protein (CB5), from cofactor NADPH or NADH. While NADPH-dependent CPR serves as the typical electron donor for most P450 enzymes, in some cases, the CBR-CB5 or CPR-CB5 electron transfer system also transfers electrons to the terminal P450 enzymes. There are tremendous studies focusing on the discovery and characterization of cytochrome P450 monooxygenases. However, very limited attention has been paid to the versatility and the roles of electron transfer components in the P450 catalytic system. Due to the membrane-residence property of both P450 enzymes and electron transfer components, it is challenging to establish an effective experimental system to evaluate the functional association of P450s with their redox partners. This chapter describes a yeast cell biocatalytic system and the related experimental procedures for comparatively assessing the functional relationship of monolignol biosynthetic P450 enzymes and different redox partners in their catalysis.


Assuntos
Citocromo-B(5) Redutase , Lignina , Citocromo-B(5) Redutase/metabolismo , Lignina/metabolismo , NADP , Transcinamato 4-Mono-Oxigenase/metabolismo , Citocromos b/metabolismo , Benzeno , NAD/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredução , Ésteres
2.
Int J Mol Sci ; 23(19)2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36233324

RESUMO

Feeding ramie cultivars (Boehmaria nivea L.) are an important feedstock for livestock. Increasing their biomass and improving their nutritional values are essential for animal feeding. Gibberellin (GA3) and ethylene (ETH) are two plant hormones that regulate the growth, development, and metabolism of plants. Herein, we report effects of the GA3 and ETH application on the growth and plant metabolism of feeding ramie in the field. A combination of GA3 and ETH was designed to spray new plants. The two hormones enhanced the growth of plants to produce more biomass. Meanwhile, the two hormones reduced the contents of lignin in leaves and stems, while increased the content of flavonoids in leaves. To understand the potential mechanisms behind these results, we used RNA-seq-based transcriptomics and UPLC-MS/MS-based metabolomics to characterize gene expression and metabolite profiles associated with the treatment of GA3 and ETH. 1562 and 2364 differentially expressed genes (DEGs) were obtained from leaves and stems (treated versus control), respectively. Meanwhile, 99 and 88 differentially accumulated metabolites (DAMs) were annotated from treated versus control leaves and treated versus control stems, respectively. Data mining revealed that both DEGs and DAMs were associated with multiple plant metabolisms, especially plant secondary metabolism. A specific focus on the plant phenylpropanoid pathway identified candidates of DEGs and DEMs that were associated with lignin and flavonoid biosynthesis. Shikimate hydroxycinnamoyl transferase (HCT) is a key enzyme that is involved in the lignin biosynthesis. The gene encoding B. nivea HCT was downregulated in the treated leaves and stems. In addition, genes encoding 4-coumaryl CoA ligase (4CL) and trans-cinnamate 4-monooxygenase (CYP73A), two lignin pathway enzymes, were downregulated in the treated stems. Meanwhile, the reduction in lignin in the treated leaves led to an increase in cinnamic acid and p-coumaryl CoA, two shared substrates of flavonoids that are enhanced in contents. Taken together, these findings indicated that an appropriate combination of GA3 and ETH is an effective strategy to enhance plant growth via altering gene expression and plant secondary metabolism for biomass-enhanced and value-improved feeding ramie.


Assuntos
Boehmeria , Giberelinas , Boehmeria/metabolismo , Cromatografia Líquida , Coenzima A/metabolismo , Etilenos , Flavonoides , Regulação da Expressão Gênica de Plantas , Giberelinas/farmacologia , Hormônios , Ligases/metabolismo , Lignina/metabolismo , Compostos Organofosforados , Reguladores de Crescimento de Plantas/farmacologia , Plantas/metabolismo , Espectrometria de Massas em Tandem , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo , Transferases/metabolismo
3.
Plant Physiol Biochem ; 190: 174-183, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36116226

RESUMO

'Docteur Jules Guyot' pears were immersed in acibenzolar-S-methyl (ASM) and 0.01 mol L-1 ethyl glycol tetra acetic acid (EGTA) to investigate the changes of Ca2+ receptor proteins and phenylpropanoid pathway. Results showed that ASM treatment increased the activities of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL), polyphenol oxidase (PPO), and cinnamyl alcohol dehydrogenase (CAD) in the exocarp of pears, whereas EGTA pre-treatment inhibited the activities of these enzymes. ASM treatment also enhanced the transcription of PcPAL, PcC4H, Pc4CL, PcC3H, PcCOMT, PcCCoAOMT, PcCCR, PcPOD, PcCDPK1, PcCDPK2, PcCDPK5, PcCDPK11, PcCDPK13, PcCBL1, PcCBL9, PcCIPK14, and PcCML27 in pears. EGTA + ASM treatments inhibited the transcription of PcPAL, PcC4H, Pc4CL, PcC3H, PcCCR, PcF5H, PcCAD, PcCDPK11, PcCDPK26, PcCDPK32, PcCBL1, PcCIPK14, PcCIPK23, and PcCaM in the fruit. All these results indicated that ASM induced the gene expressions of Ca2+ receptor proteins, the key enzyme activities and gene expressions in phenylpropanoid pathway; Ca2+ mediated phenylpropane metabolism in pears after ASM treatment.


Assuntos
Pyrus , Cálcio , Catecol Oxidase , Cinamatos , Coenzima A Ligases/metabolismo , Ácido Egtázico , Glicóis , Lignina/genética , Fenilalanina Amônia-Liase/metabolismo , Pyrus/metabolismo , Tiadiazóis , Transcinamato 4-Mono-Oxigenase/metabolismo
4.
Sci Rep ; 12(1): 8128, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35581313

RESUMO

The phenylpropanoid pathway serves as a rich source of metabolites in plants, and it is considered as a starting point for the production of many other important compounds such as the flavonoids, flavonols, coumarins, and lignans. Scrophularia striata is a member of the Lamiaceae family with some biological activities similar to flavonoid compounds such as antioxidant, antibacterial, anti-inflammatory and analgesic activities. Cinnamate 4-hydroxylase (C4H) and Chalcone synthase (CHS) are key enzymes of the phenylpropanoid pathway, leading to the biosynthesis of several secondary metabolites. In this study, two S. striata CHS and C4H were isolated and then analyzed. The investigation of the expression of these genes was performed under the effects of three salicylic acid (SA), jasmonic acid (JA), and gibberellic acid (GA) at concentrations of 100 and 300 ppm with a completely randomized design at the transcript level using Real Time PCR method. These have different expression patterns at developmental stages. Moreover, these genes present different sensitivities to hormonal treatment. Considering the total results, it was found that the amount of expression of these genes during the reproductive phase is higher than that of the vegetative phase. Additionally, the treatment of 300 ppm SA in the reproductive phase is the most effective treatment on increasing the corresponding phenylpropanoid compounds. A correlation analysis was performed between the phenylpropanoid compounds content and both CHS and C4H expression values at different phenological development stages. The results indicate that the expression variations of both CHS and C4H are significantly related to the changes in total phenolic content. We believe that the isolation of CHS and C4H can be helpful in better understanding phenylpropanoid metabolis.


Assuntos
Scrophularia , Aciltransferases/genética , Aciltransferases/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica de Plantas , Ácido Salicílico/metabolismo , Scrophularia/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
5.
New Phytol ; 230(6): 2275-2291, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33728703

RESUMO

The phenylpropanoid pathway serves a central role in plant metabolism, providing numerous compounds involved in diverse physiological processes. Most carbon entering the pathway is incorporated into lignin. Although several phenylpropanoid pathway mutants show seedling growth arrest, the role for lignin in seedling growth and development is unexplored. We use complementary pharmacological and genetic approaches to block CINNAMATE-4-HYDROXYLASE (C4H) functionality in Arabidopsis seedlings and a set of molecular and biochemical techniques to investigate the underlying phenotypes. Blocking C4H resulted in reduced lateral rooting and increased adventitious rooting apically in the hypocotyl. These phenotypes coincided with an inhibition in AUX transport. The upstream accumulation in cis-cinnamic acid was found to be likely to cause polar AUX transport inhibition. Conversely, a downstream depletion in lignin perturbed phloem-mediated AUX transport. Restoring lignin deposition effectively reestablished phloem transport and, accordingly, AUX homeostasis. Our results show that the accumulation of bioactive intermediates and depletion in lignin jointly cause the aberrant phenotypes upon blocking C4H, and demonstrate that proper deposition of lignin is essential for the establishment of AUX distribution in seedlings. Our data position the phenylpropanoid pathway and lignin in a new physiological framework, consolidating their importance in plant growth and development.


Assuntos
Cinamatos , Plântula , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plântula/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
6.
J Exp Bot ; 72(8): 3061-3073, 2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33585900

RESUMO

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Propanóis/metabolismo , Transcinamato 4-Mono-Oxigenase , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
7.
Food Chem ; 347: 129009, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444889

RESUMO

Litchis are tasty fruit with economic importance. However, the extreme susceptibility of harvested litchis to litchi downy blight caused by Peronophythora litchii leads to compromised quality. This study aimed to study the effects of melatonin on postharvest resistance to P. litchii in 'Feizixiao' litchis. Results showed that melatonin restricted lesion expansion in litchis after P. litchi inoculation. Melatonin enhanced the activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase and 4-hydroxycinnamate CoA ligase while promoting the accumulations of phenolics and flavonoids. Nicotinamide adenine dinucleotide phosphate content and glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities were higher in treated fruit than control fruit. Higher energy status along with elevated H+-ATPase, Ca2+-ATPase, succinate dehydrogenase and cytochrome C oxidase activities were observed in treated fruit. Ultrastructural observation showed reduced damage in mitochondria in treated fruit. The results suggest that melatonin induced resistance in litchis by modulating the phenylpropanoid and pentose phosphate pathways as well as energy metabolism. .


Assuntos
Metabolismo Energético , Litchi/metabolismo , Melatonina/farmacologia , Micoses/tratamento farmacológico , Doenças das Plantas , Flavonoides/análise , Flavonoides/metabolismo , Frutas/química , Litchi/química , Litchi/efeitos dos fármacos , Litchi/microbiologia , Melatonina/química , Fenóis/análise , Fenóis/metabolismo , Fenilalanina Amônia-Liase/análise , Fenilalanina Amônia-Liase/metabolismo , Transcinamato 4-Mono-Oxigenase/análise , Transcinamato 4-Mono-Oxigenase/metabolismo
8.
Plant Cell Rep ; 40(2): 375-391, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33392729

RESUMO

KEY MESSAGE: RgC4H promotes phenolic accumulation in R. glutinosa, activating the molecular networks of its antioxidant systems, and enhancing the tolerance to oxidative stresses exposed to drought, salinity and H2O2 conditions. Rehmannia glutinosa is of great economic importance in China and increasing R. glutinosa productivity relies, in part, on understanding its tolerance to oxidative stress. Oxidative stress is a key influencing factor for crop productivity in plants exposed to harsh conditions. In the defense mechanisms of plants against stress, phenolics serve an important antioxidant function. Cinnamate 4-hydroxylase (C4H) is the first hydroxylase in the plant phenolics biosynthesis pathway, and elucidating the molecular characteristics of this gene in R. glutinosa is essential for understanding the effect of tolerance to oxidative stress tolerance on improving yield. Using in vitro and in silico methods, a C4H gene, RgC4H, from R. glutinosa was isolated and characterized. RgC4H has 86.34-93.89% amino acid sequence identity with the equivalent protein in other plants and localized to the endoplasmic reticulum. An association between the RgC4H expression and total phenolics content observed in non-transgenic and transgenic R. glutinosa plants suggests that this gene is involved in the process of phenolics biosynthesis. Furthermore, the tolerance of R. glutinosa to drought, salinity and H2O2 stresses was positively or negatively altered in plants with the overexpression or knockdown of RgC4H, respectively, as indicated by the analysis in some antioxidant physiological and molecular indices. Our study highlights the important role of RgC4H in the phenolics/phenylpropanoid pathway and reveals the involvement of phenolic-mediated regulation in oxidative stress tolerance in R. glutinosa.


Assuntos
Antioxidantes/metabolismo , Fenóis/metabolismo , Rehmannia/enzimologia , Transcinamato 4-Mono-Oxigenase/metabolismo , Sequência de Aminoácidos , China , Secas , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Propanóis/metabolismo , Rehmannia/genética , Rehmannia/fisiologia , Salinidade , Estresse Fisiológico , Transcinamato 4-Mono-Oxigenase/genética
9.
Phytochemistry ; 182: 112594, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33341029

RESUMO

The scopoletin one of the major bioactive components of Convolvulus prostratus Forssk known to have a role in acetylcholinesterase inhibitor, memory enhancer, antimicrobial, antioxidative etc. properties are investigated in the present study. The concentration of scopoletin in C. prostratus is investigated in leaf, stem and root at different growth stages of plant development viz., 30, 45, 60 and 90 days after sowing (DAS). A highly sensitive LC-MS method was developed to quantify the scopoletin even at low concentration with LOD and LOQ of 8 and 24 ng/ml, respectively. The highest quantity of scopoletin was recorded in stem (732 µg/g dry weight) and leaf (650 µg/g dry weight) collected 90 DAS whereas lowest was recorded at 45 DAS in leaf (90.00 µg/g dry weight) and Stem (110 µg/g dry weight). Based on the highest and lowest concentration of scopoletin in stem and root tissues at 45 and 90 DAS were selected for transcriptome study. Differential gene expression analysis revealed the differential expression of genes involved in scopoletin biosynthesis. Seven genes viz., phenylalanine ammonia-lyase (PAL), 4-coumarate CoA ligase (4CL), trans-cinnamate 4-monooxygenase (TCM), shikimate O- hydroxycinnamoyl transferase (C3'H), 5-O-4-coumaroyl-D-quinate 3'-monooxygenase (HCT), caffeoyl-CoA-O-methyltransferase (CCoAOMT) and feruloyl-CoA 6'-hydroxylase (F6'H) were identified in the phenyl propanoid pathway. Expression of the novel enzyme F6'H showed down regulation in both tissues at 45 DAS. Real-time PCR showed a correlation with the expression of this F6'H genes with the accumulation of scopoletin at 90 DAS. This indicated that the growth stage of plant and expression of F6'H control the scopoletin accumulation in Convolvulus. The results of present investigation may useful in pharmaceutical, drug and cosmetic industries that the harvesting of plant part especially stem of C.prostratus at 90 DAS to get maximum quantity of scopoletin. Also, the novel gene F6'H need to be further characterized to understand its expression dynamics so that scopoletin content can be increase at the highest.


Assuntos
Convolvulus , Escopoletina , Regulação da Expressão Gênica de Plantas , Fenilalanina Amônia-Liase/genética , Folhas de Planta , Transcinamato 4-Mono-Oxigenase
10.
Plant Physiol Biochem ; 156: 135-145, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32937268

RESUMO

Cinnamate 4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway in plants and is involved in the biosynthesis of secondary metabolites such as lignin and flavonoids. However, the function of C4H in pear plants (Pyrus bretschneideri) has not yet been fully elucidated. By searching pear genome databases, we identified three C4H genes (PbC4H1, PbC4H2 and PbC4H3) encoding proteins that share higher identity with bonafide C4Hs from several species with typical cytochrome P450 domains, suggesting that all three PbC4Hs are also bonafide C4Hs that have close evolutionary relationships with C4Hs from other land plants. Quantitative real-time PCR (qRT-PCR) results indicated that the three PbC4Hs were specifically expressed in one or more tissues. The expression levels of PbC4H1 and PbC4H3 first increased and then decreased during pear fruit development. Treatment with exogenous hormones (ABA, MeJA, and SA) altered the expression of the three PbC4Hs to varying degrees. The expression levels of the PbC4Hs were first induced and then decreased under ABA treatment, while MeJA treatment significantly increased the expression levels of the PbC4Hs. Following treatment with SA, expression levels of PbC4H1 and PbC4H2 increased, while expression levels of PbC4H3 decreased. Enzymatic analysis of the recombinant proteins expressed in yeast indicated that PbC4H1 and PbC4H3 catalysed the conversion of trans-cinnamic acid to p-coumaric acid. Moreover, the expression of PbC4H1 and PbC4H3 in Arabidopsis resulted in an increase in both the lignin content and the thickness of cell walls for intervascular fibres and xylem cells. Taken together, the results of our study not only revealed the potential role of PbC4H1 and PbC4H3 in lignin biosynthesis but also established a foundation for future investigations of the regulation of lignin synthesis and stone cell development in pear fruit by molecular biological techniques.


Assuntos
Proteínas de Plantas/genética , Pyrus/enzimologia , Transcinamato 4-Mono-Oxigenase/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Família Multigênica , Proteínas de Plantas/metabolismo , Pyrus/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
11.
Gene ; 758: 144950, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32683078

RESUMO

Sunflower (Helianthus annuus L.) is an important oil crop, the secondary metabolites of it include many compounds such as flavonoids and lignin. However, the research on the biosynthesis of phenolic compounds in sunflowers is still scarce. Cinnamate 4-hydroxylase (C4H) belongs to the cytochrome P450-dependent monooxygenase family and is involved in the synthesis of many phenolic compounds, but C4H in sunflowers has not yet been cloned and functionally characterized. In this study, we screened three C4H genes from the sunflower transcriptome and genomic databases, named HaC4H1, HaC4H2, and, HaC4H3, respectively. In heterologous expression experiments, we had improved a method from previous studies by the addition of restriction sites to make it easier to express multiple C4H functions and suitable for in vitro activity verification. HaC4Hs without the N-terminal membrane anchor region was fused with a redox partner of Arabidopsis thaliana cytochrome P450 enzyme (CYP450) by the method and functionally expressed in E. coli and the results showed that these three enzymes catalyzed the formation of p-coumaric acid. To further investigate whether our fusion protein approach is applicable to other C4Hs, we used this method to explore the functions of C4H from Peucedanum praeruptorum and Angelica decursiva, and they can also convert trans-cinnamic acid to p-coumaric acid. The gene expression profile showed that all three HaC4H genes showed the highest transcription levels in the roots and might be up-regulated by MeJA. In summary, these results reveal the function of HaC4Hs in sunflower and provide a simpler way to explore C4H and even other cytochrome P450 enzymes in prokaryotic expression systems.


Assuntos
Helianthus/enzimologia , Propionatos/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo , Sequência de Aminoácidos , Angelica/genética , Apiaceae/genética , Arabidopsis/genética , Clonagem Molecular , Ácidos Cumáricos , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Transcriptoma/genética
12.
Food Chem ; 331: 127282, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32559597

RESUMO

Phenolics are important secondary metabolites in plants with strong antioxidant effects. Seeds germination and exogenous stimulation could activate endogenous enzymes to enhance the content of phenolic acids and flavonoids. Barley seeds geminated under NaCl (1-20 mM) treatment to evaluate the accumulation of phenolics in this study. Results showed that NaCl treatment significantly enhanced the growth of seedlings, especially bud length. NaCl treatment up-regulated genes and proteins expression of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL), resulting in the enhancement of their activities. As a result, phenolic acids and flavonoids contents increased by 11.19% and 32.54%, respectively, in which gallic acid, protocatechuic, fisetin, myricetin and quercetin were affected mostly. Moreover, NaCl treatment enhanced 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging capacity. Hence, NaCl stimulated the synthesis of phenolic components via enhancing gene, protein expression and the activity of key enzymes.


Assuntos
Hordeum/efeitos dos fármacos , Hordeum/metabolismo , Fenóis/metabolismo , Plântula/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Antioxidantes/análise , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Fenóis/análise , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Plântula/metabolismo , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo
13.
Plant Physiol ; 183(3): 957-973, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32332088

RESUMO

Cinnamate 4-hydroxylase (C4H; CYP73A) is a cytochrome P450 monooxygenase associated externally with the endoplasmic reticulum of plant cells. The enzyme uses NADPH-cytochrome P450 reductase as a donor of electrons and hydroxylates cinnamic acid to form 4-coumaric acid in phenylpropanoid metabolism. In order to better understand the structure and function of this unique class of plant P450 enzymes, we have characterized the enzyme C4H1 from lignifying tissues of sorghum (Sorghum bicolor), encoded by Sobic.002G126600 Here we report the 1.7 Å resolution crystal structure of CYP73A33. The obtained structural information, along with the results of the steady-state kinetic analysis and the absorption spectroscopy titration, displays a high degree of similarity of the structural and functional features of C4H to those of other P450 proteins. Our data also suggest the presence of a putative allosteric substrate-binding site in a hydrophobic pocket on the enzyme surface. In addition, comparing the newly resolved structure with those of well-investigated cytochromes P450 from mammals and bacteria enabled us to identify those residues of critical functional importance and revealed a unique sequence signature that is potentially responsible for substrate specificity and catalytic selectivity of C4H.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Sorghum/genética , Sorghum/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transcinamato 4-Mono-Oxigenase/metabolismo , Genes de Plantas , Estrutura Molecular
14.
Plant Cell Rep ; 39(5): 597-607, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32055924

RESUMO

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.


Assuntos
Anthocerotophyta/enzimologia , Bryopsida/metabolismo , Transcinamato 4-Mono-Oxigenase/metabolismo , Anthocerotophyta/genética , Bryopsida/genética , Clonagem Molecular , Expressão Gênica , Cinética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Fenóis/análise , Filogenia , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Transformação Genética
15.
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
16.
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
17.
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 , Oxilipinas/imunologia , Doenças das Plantas/imunologia , Reguladores de Crescimento de Plantas/imunologia , /imunologia , Resistência à Doença , Frutas/imunologia , Frutas/microbiologia , Regulação da Expressão Gênica de Plantas , /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
18.
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
19.
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
20.
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 , Ácidos Cumáricos , Escherichia coli/genética , Glucose/metabolismo , Lycoris/genética , NADP/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos
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