Search

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 b

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.

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. Abstract 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.

'Docteur Jules Guyot' pears were immersed in acibenzolar-S-methyl (ASM) and 0.01 mol L

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.

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.

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.

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.

• 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 auxin transport. The upstream accumulation in cis‐cinnamic acid was found to likely cause polar auxin transport inhibition. Conversely, a downstream depletion in lignin perturbed phloem‐mediated auxin transport. Restoring lignin deposition effectively reestablished phloem transport and, accordingly, auxin 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 auxin distribution in seedlings. Our data position the phenylpropanoid pathway and lignin in a new physiological framework, consolidating their importance in plant growth and development.

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.

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsisreduced epidermal fluorescence 3(ref3) mutants, which carry hypomorphic mutations inC4H, 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-typeC4His expressed in theref3-2background. Expression ofC4Hin 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 ofC4Hexpression inref3-2pOpC4H, 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 toref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in theref3-2mutant root endodermis, which is restored by induction ofC4Hexpression.HighlightThe work presented this paper provides evidence of metabolite turnover, plasticity of the developmental window for lignification, and the impact of reduced and restored cinnamate-4-hydroxylase (C4H)expression on the Casparian strip.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. .

Summary Isoflavonoids are specialized plant metabolites, almost exclusive to legumes, and their biosynthesis forms a branch of the diverse phenylpropanoid pathway. Plant metabolism may be coordinated at many levels, including formation of protein complexes, or ‘metabolons’, which represent the molecular level of organization. Here, we have confirmed the existence of the long-postulated isoflavonoid metabolon by identifying elements of the complex, their subcellular localizations and their interactions. Isoflavone synthase (IFS) and cinnamate 4–hydroxylase (C4H) have been shown to be tandem P450 enzymes that are anchored in the ER, interacting with soluble enzymes of the phenylpropanoid and isoflavonoid pathways (chalcone synthase, chalcone reductase and chalcone isomerase). The soluble enzymes of these pathways, whether localized to the cytoplasm or nucleus, are tethered to the ER through interaction with these P450s. The complex is also held together by interactions between the soluble elements. We provide evidence for IFS interaction with upstream and non-consecutive enzymes. The existence of such a protein complex suggests a possible mechanism for flux of metabolites into the isoflavonoid pathway. Further, through interaction studies, we identified several candidates that are associated with GmIFS2, an isoform of IFS, in soybean hairy roots. This list provides additional candidates for various biosynthetic and structural elements that are involved in isoflavonoid production. Our interaction studies provide valuable information about isoform specificity among isoflavonoid enzymes, which may guide future engineering of the pathway in legumes or help overcome bottlenecks in heterologous expression.

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&rsquo, H&eacute, r.) 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 &mu, M 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.

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. Electronic supplementary material The online version of this article (10.1186/s12870-018-1477-0) contains supplementary material, which is available to authorized users.

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.

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), β-glucuronidase (GUS), baicalein-7

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.

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.

Kenaf (Hibiscus cannabinus) is cultivated worldwide for its fiber, however, the medicinal properties of this plant are currently attracting increasing attention. In this study, we investigated the expression levels of genes involved in the biosynthesis of kaempferitrin, a compound with many biological functions, in different kenaf organs. We found that phenylalanine ammonia lyase (HcPAL) was more highly expressed in stems than in other organs. Expression levels of cinnamate 4-hydroxylase (HcC4H) and 4-coumarate-CoA ligase (Hc4CL) were highest in mature leaves, followed by stems and young leaves, and lowest in roots and mature flowers. The expression of chalcone synthase (HcCHS), chalcone isomerase (HcCHI), and flavone 3-hydroxylase (HcF3H) was highest in young flowers, whereas that of flavone synthase (HcFLS) was highest in leaves. An analysis of kaempferitrin accumulation in the different organs of kenaf revealed that the accumulation of this compound was considerably higher (&gt, 10-fold) in leaves than in other organs. On the basis of a comparison of kaempferitrin contents with the expression levels of different genes in different organs, we speculate that HcFLS plays an important regulatory role in the kaempferitrin biosynthetic pathway in kenaf.

Penylalanine ammonia-lyase (PAL), cinnamic-4-hydroxylase (C4H), capsaicin synthase (CS), and peroxidase (POD) are involved in the capsaicinoid biosynthesis pathway and may be altered in cultivars with different pungency levels. This study clarified the action of these enzymes under drought stress for hot Capsicum cultivars with low, medium,and high pungency levels. At the flowering stage, control plants were watered at field capacity, whereas drought-induced plants were subjected to gradual drought stress. Under drought stress, PAL, C4H, CS, and POD enzyme activities increased as compared to the non-drought-stressed plants. A novel discovery was that PAL was the critical enzyme in capsaicinoid biosynthesis under drought stress because its activities and capsaicinoid increased across the different pungency levels of hot pepper cultivars examined.

To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO₄, and 0.2 g/L CaSO₄ and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 μg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously.

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, β-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 .

12 páginas.-- 5 figuras.-- 2 tablas.-- 65 referencias.-- Supplementary information.-- Farhana Shafrin et al... Scientific Reports 7, 39984 (2017), 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., The research was supported by a financial grant received from the BAS-USDA-PALS Program

The study of gene expression in plants is fundamental, and understanding the molecular mechanisms involved in important biological processes, such as biochemical pathways or signaling that are used or manipulated in improvement programs, are key for the production of high-quality soybean seeds. Reports related to gene expression of lignin in seeds are scarce in the literature. We studied the expression of the phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase, 4-hydroxycinnamate 3-hydroxylase, and cinnamyl alcohol dehydrogenase genes involved in lignin biosynthesis during the development of soybean (Glycine max L. Merrill) seeds. As the endogenous control, the eukaryotic elongation factor 1-beta gene was used in two biological replicates performed in triplicate. Relative quantitative expression of these genes during the R4, R5, R6, and R7 development stages was analyzed. Real-time polymerase chain reaction was used for the gene expression study. The analyses were carried out in an ABI PRISM 7500 thermocycler using the comparative Ct method and SYBR Green to detect amplification. The seed samples at the R4 stage were chosen as calibrators. Increased expression of the cinnamate-4-hydroxylase and PAL genes occurred in soybean seeds at the R5 and R6 development stages. The cinnamyl alcohol dehydrogenase gene was expressed during the final development phases of soybean seeds. In low-lignin soybean cultivars, the higher expression of the PAL gene occurs at development stages R6 and R7. Activation of the genes involved in the lignin biosynthesis pathway occurs at the beginning of soybean seed development.

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.

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.

To clarify the mechanisms of selenium (Se) tolerance in peanut seedlings, we grew peanut seedlings with sodium selenite (0, 3, and 6 mg/L), and investigated the phenylpropanoids metabolism in seedling roots. The results showed that selenite up-regulated the expression of genes and related enzyme activities involving in the phenylpropanoids biosynthesis cascade, such as phenylalanine ammonia-lyase, trans-cinnamate-4-hydroxylase, chalcone synthase, chalcone isomerase, and cinnamyl-alcohol dehydrogenase. Selenite significantly increased phenolic acids and flavonoids, which contributed to the alleviation of selenite-induced stress. Moreover, selenite enhanced the formation of endodermis in roots, which may be attributed to the up-regulation of lignin biosynthesis mediated by the selenite-induced changes of H2O2 and NO, which probably regulated the selenite uptake from an external medium. Accumulation of polyphenolic compounds via the phenylpropanoid pathway may be one of the mechanisms of the increasing selenite tolerance in plants, by which peanut seedlings survived in seleniferous soil, accompanied by accumulation of Se.

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.

Cytochromes P450 from plants have the potential to be valuable catalysts for industrial hydroxylation reactions, but their application is hindered by poor solubility, the lack of suitable expression systems and the requirement of P450s for auxiliary redox-transport proteins for the delivery of reducing equivalents from NAD(P)H. In the interests of enabling useful P450 activity from plants, we have developed a suite of vectors for the expression of plant P450s as non-natural genetic fusions with reductase proteins. First, we have fused the P450 isoflavone synthase (IFS) from Glycine max with the bacterial P450 reductase domain (Rhf-RED) from Rhodococcus sp., by using our LICRED vector developed previously (F. Sabbadin, R. Hyde, A. Robin, E.-M. Hilgarth, M. Delenne, S. Flitsch, N. Turner, G. Grogan, N. C. Bruce, ChemBioChem 2010, 11, 987-994) creating the first active bacterial-plant fusion P450 enzyme. We have then created a complementary vector, ACRyLIC for the fusion of selected plant P450 enzymes to the P450 reductase ATR2 from Arabidopsis thaliana. The applicability of this vector to the creation of active P450 fusion enzymes was demonstrated using both IFS1 and the cinnamate-4-hydroxylase (C4H) from A. thaliana. Overall the fusion vector systems will allow the rapid creation of libraries of plant P450s with the aim of identifying enzyme activities with possible applications in industrial biocatalysis.

Leucaena leucocephala is a leguminous tree species accounting for one-fourth of raw material supplied to paper and pulp industry in India. Cinnamate 4-Hydroxylase (C4H, EC 1.14.13.11) is the second gene of phenylpropanoid pathway and a member of cytochrome P450 family. There is currently intense interest to alter or modify lignin content of L. leucocephala. Three highly similar C4H alleles of LlC4H1 gene were isolated and characterized. The alleles shared more than 98 % sequence identity at amino acid level to each other. Binding of partial promoter of another C4H gene LlC4H2, to varying amounts of crude nuclear proteins isolated from leaf and stem tissues of L. leucocephala formed two loose and one strong complex, respectively, suggesting that the abundance of proteins that bind with the partial C4H promoter is higher in stem tissue than in leaf tissue. Quantitative Real Time PCR study suggested that among tissues of same age, root tissues had highest level of C4H transcripts. Maximum transcript level was observed in 30 day old root tissue. Among the tissues investigated, C4H activity was highest in 60 day old root tissues. Tissue specific quantitative comparison of lignin from developing seedling stage to 1 year old tree stage indicated that Klason lignin increased in tissues with age.

Sorghum, a species able to produce a high yield of biomass and tolerate both drought and poor soil fertility, is considered to be a potential bioenergy crop candidate. The reduced lignin content characteristic of brown midrib (bmr) mutants improves the efficiency of bioethanol conversion from biomass. Suppression subtractive hybridization combined with cDNA microarray profiling was performed to characterize differential gene expression in a set of 13 bmr mutants, which accumulate significantly less lignin than the wild-type plant BTx623. Among the 153 differentially expressed genes identified, 43 were upregulated and 110 downregulated in the mutants. A semi-quantitative RT-PCR analysis applied to 12 of these genes largely validated the microarray analysis data. The transcript abundance of genes encoding l-phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase was less in the mutants than in the wild type, consistent with the expectation that both enzymes are associated with lignin synthesis. However, the gene responsible for the lignin synthesis enzyme cinnamic acid 4-hydroxylase was upregulated in the mutants, indicating that the production of monolignol from l-phenylalanine may involve more than one pathway. The identity of the differentially expressed genes could be useful for breeding sorghum with improved efficiency of bioethanol conversion from lignocellulosic biomass.

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

The allelopathic effect of caffeic acid was tested on root growth, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, hydrogen peroxide (H(2)O(2)) accumulation, lignin content and monomeric composition of soybean (Glycine max) roots. We found that exogenously applied caffeic acid inhibited root growth, decreased the PAL activity and H(2)O(2) content and increased the soluble and cell wall-bound POD activities. The p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomers and total lignin (H+G+S) increased in the caffeic acid-exposed roots. When applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), caffeic acid equalized the inhibitory effect of PIP, whereas the application of methylene dioxocinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL) plus caffeic acid decreased lignin production. These results indicate that exogenously applied caffeic acid can be channeled into the phenylpropanoid pathway via the 4CL reaction, resulting in an increase of lignin monomers that solidify the cell wall and inhibit root growth.

Common buckwheat (Fagopyrum esculentum) is a short-season grain crop that is a source of rutin and other phenolic compounds. In this study, we isolated the cDNAs of 11 F. esculentum enzymes in the flavonoid biosynthesis pathway, namely, phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL) 1 and 2, chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonol synthase (FLS) 1 and 2, and anthocyanidin synthase (ANS). Quantitative real-time polymerase chain reaction analysis showed that these genes were most highly expressed in the stems and roots. However, high performance liquid chromatography analysis indicated that their flavonoid products, such as rutin and catechin, accumulated in the flowers and leaves. These results suggested that flavonoids may be transported within F. esculentum. In addition, light and dark growth conditions affected the expression levels of the biosynthesis genes and accumulation of phenolic compounds in F. esculentum sprouts.

The cDNAs encoding phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) were cloned from garlic (Allium sativum) using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE) PCR. Amino acid sequence alignments showed that AsPAL and AsC4H have more than 70% amino acid identity with their homologues in other plants. The expression of AsPAL and AsC4H transcripts was highest in the roots but surprisingly low in the bulbils, where phenylpropanoid compounds are most concentrated. These results suggest that some phenylpropanoids are synthesized in the roots and subsequently transported to the bulbils of A. sativum .

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) are important enzymes in the phenylpropanoid pathway and also in the accumulation of decursin (1) and decursinol angelate (2), which are major secondary metabolites in Angelica gigas. Using PCR with degenerate primers targeted to conserved regions of available orthologous PAL and C4H sequences, cDNAs encoding PAL and C4H from A. gigas were isolated. Both genes were used to show the comparative developmental and inducible accumulation of mRNAs in different organs and in suspension cells of A. gigas. PAL and C4H were induced most strongly in response to 300 microM methyl jasmonate treatment at 6 and 12 h, respectively, and were highly expressed in the fine roots of A. gigas. Similarly, the production of 1 and 2 was most prolific in the fine roots of the plant.

Several key enzymes in lignin biosynthesis of Populus have been down-regulated by transgenic approaches to investigate their role in wood lignification and to explore their potential for lignin modification. Cinnamate 4-hydroxylase is an enzyme in the early phenylpropanoid pathway that has not yet been functionally analyzed in Populus . This study shows that down-regulation of cinnamate 4-hydroxylase reduced Klason lignin content by 30% with no significant change in syringyl to guaiacyl ratio. The lignin reduction resulted in ultrastructural differences of the wood and a 10% decrease in wood density. Mechanical properties investigated by tensile tests and dynamic mechanical analysis showed a decrease in stiffness, which could be explained by the lower density. The study demonstrates that a large modification in lignin content only has minor influences on tensile properties of wood in its axial direction and highlights the usefulness of wood modified beyond its natural variation by transgene technology in exploring the impact of wood biopolymer composition and ultrastructure on its material properties.

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) plays an important role in the phenylpropanoid pathway, which produces many economically important secondary metabolites. A gene coding for C4H, designated as PhC4H (GenBank accession no. DQ211885) was isolated from Parthenocissus henryana. The full-length PhC4H cDNA is 1,747 bp long with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 40-bp 5' non-coding region and a 189-bp 3'-untranslated region. Secondary structure of the deduced PhC4H protein consists of 41.78% alpha helix, 15.64% extended strand and 42.57% random coil. The genomic DNA of PhC4H is 2,895 bp long and contains two introns; intron I is 205-bp and intron II is 1,172-bp (GenBank accession no. EU440734). DNA gel blot analysis revealed that there might be a single copy of PhC4H in Parthenocissus henryana genome. By using anchored PCR, a 963-bp promoter sequence was isolated and it contains many responsive elements conserved in the upstream region of PAL, C4H and 4CL including the P-, A-, L- and H-boxes.

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA(3)) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.