hydroxycinnamoyl-CoA Quinate Hydroxycinnamoyl Transferase Research Articles

Partial coding sequence identification, gene expression analysis, and content of anticancer phenolic compounds in Sonchus arvensis L. under drought stress conditions

Field sowthistle, a plant in the Asteraceae family, contains high levels of chlorogenic and chicoric acids, which have anticancer properties. In the current investigation, the expression of genes responsible for chlorogenic acid biosynthesis was studied in S. arvensis in drought stress conditions. The following genes were investigated: phenylalanine ammonia-lyase (PAL), cinnamate 4 hydroxylase (C4H), 4-hydroxycinnamoyl CoA ligase (4CL), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) and p-coumaroyl ester 3'-hydroxylase (C3'H). As part of a research study, the levels of several anticancer compounds and their correlation with the transcriptional activity of the surveyed genes were investigated under drought stress conditions. The study also involved the identification and submission of the partial coding sequences of the C4H, HCT, C3'H, and actin genes in S. arvensis to the GenBank database. The plants’ middle leaves exposed to 50% field capacity (FC) exhibited the highest expression of all genes. The highest amount of chicoric, chlorogenic, and 2,5 dihydroxybenzoic (2,5 DHB) acids was observed in the middle leaves under 50% FC. In contrast, the highest content of gallic acid and catechin was found in the lower leaves at 75% FC. A strong association between HQT expression and chlorogenic acid content proposed the HQT-dependent metabolic pathway as the primary route for its production in S. arvensis. Furthermore, the consistency between the patterns of gene expression and the levels of compounds found in the middle leaves at 50% FC, except for catechin, showed that the production of chlorogenic acid in S. arvensis could potentially be enhanced by stimulating the expression of the genes that are responsible for its biosynthesis.

High-resolution genome mapping and functional dissection of chlorogenic acid production in Lonicera maackii.

Amur honeysuckle (Lonicera maackii) is a widely used medicinal plant of the Caprifoliaceae family that produces chlorogenic acid. Research on this plant mainly focuses on its ornamental value and medicinal compounds, but a reference genome sequence and molecular resources for accelerated breeding are currently lacking. Herein, Nanopore sequencing and high-throughput chromosome conformation capture (Hi-C) allowed a chromosome-level genome assembly of Lonicera maackii (2n = 18). A global view of the gene regulatory network involved in the biosynthesis of chlorogenic acid and the dynamics of fruit coloration in L. maackii was established through metabolite profiling and transcriptome analyses. Moreover, we identified the genes encoding hydroxycinnamoyl-CoA quinate transferase (LmHQT) and hydroxycinnamoyl-CoA shikimic/quinate transferase (LmHCT), which localized to the cytosol and nucleus. Heterologous overexpression of these genes in Nicotiana benthamiana leaves resulted in elevated chlorogenic acid contents. Importantly, HPLC analyses revealed that LmHCT and LmHQTs recombinant proteins modulate the accumulation of chlorogenic acid (CGA) using quinic acid and caffeoyl CoA as substrates, highlighting the importance of LmHQT and LmHCT in chlorogenic acid biosynthesis. These results confirmed that LmHQTs and LmHCT catalyze the biosynthesis of CGA in vitro. The genomic data presented in this study will offer a valuable resource for the elucidation of CGA biosynthesis and facilitating selective molecular breeding.

Cross-Talk and Physiological Role of Jasmonic Acid, Ethylene, and Reactive Oxygen Species in Wound-Induced Phenolic Biosynthesis in Broccoli.

Wounding induces phenolic biosynthesis in broccoli. However, there is scarce information about the physiological and molecular mechanisms governing this stress response. In the present study, a chemical-genetics approach was used to elucidate the role of reactive oxygen species (ROS), jasmonic acid (JA), and ethylene (ET) as stress-signaling molecules in the wound-induced phenolic biosynthesis in broccoli. Wounding activated the biosynthesis of ET and JA. Likewise, the wound-induced biosynthesis of ET and JA was regulated by ROS. JA activated primary metabolism, whereas the three signaling molecules activated phenylpropanoid metabolism. The signaling molecules inhibited the wound-induced activation of the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene, which is involved in caffeoylquinic acids biosynthesis, and the main phenolics accumulated in wounded broccoli, suggesting that an alternative caffeoylquinic biosynthesis pathway is activated in the tissue due to wounding. ROS mediated the biosynthesis of most individual phenolic compounds evaluated. In conclusion, ROS, ET, and JA are essential in activating broccoli's primary and secondary metabolism, resulting in phenolic accumulation.

Differential metabolites and their transcriptional regulation in seven major tea cultivars (Camellia sinensis) in China

Various genetic and biochemical characteristics exist in tea plant cultivars, and they largely determine production suitability and tea quality. Here, we performed transcriptomic and metabolomic analyses of young shoots of seven tea cultivars and identified major regulatory transcription factors (TFs) for the characteristic metabolites in different cultivars based on weighted gene co-expression network analysis (WGCNA). Phenotypically, we found that ‘Tieguanyin’ (TGY) and ‘Fujian Shuixian’ (FJSX), which are suitable for oolong tea, had higher catechin contents. The metabolites of ‘Jinxuan’ (JX) were more prominent, especially the contents of phenolic acids, flavonoids, terpenes, and tannins, which were higher than those of the other six cultivars. Moreover, ‘Fudingdabai’ (FDDB), which is suitable for white tea, was rich in amino acids, linolenic acid, and saccharides. At the molecular level, hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HCT) (CsTGY12G0001876, and CsTGY06G0003042) led to the accumulation of chlorogenic acid in TGY. The main reason for the higher l-ascorbic acid content in FJSX was the high expression levels of L-galactono-1,4-lactone hydrogenase (GalLDH) (CsTGY13G0000389) and Myo-inositol oxygenase (MIOX) (CsTGY14G0001769, and CsTGY14G0001770), which were regulated by WRKY (CsTGY11G0001197). Furthermore, FDDB, ‘Longjing 43’ (LJ43), ‘Shuchazao’ (SCZ) and ‘Baihaozao’ (BHZ) had higher free fatty acid contents, among which MYB (CsTGY14G0002344) may be a hub gene for the regulation of palmitoleic acid accumulation. More importantly, we found that the shoots of TGY were green with purple, mainly due to the accumulation of anthocyanins and the downregulation of the Mg-protoporphyrin IX nonomethyl ester cyclase (MPEC) (CsTGY10G0001989) gene that affects chlorophyll synthesis. These results will provide a theoretical reference for tea cultivar breeding and suitability.

Molecular Mechanisms through Which Short-Term Cold Storage Improves the Nutritional Quality and Sensory Characteristics of Postharvest Sweet Potato Tuberous Roots: A Transcriptomic Study.

Sweet potato (Ipomoea batatas (L.) Lam.) is a commercially relevant food crop with high demand worldwide. This species belongs to the Convolvulaceae family and is native to tropical and subtropical regions. Storage temperature and time can adversely affect tuberous roots’ quality and nutritional profile. Therefore, this study evaluates the effect of storage parameters using physicochemical and transcriptome analyses. Freshly harvested tuberous roots (Xingxiang) were stored at 13 °C (control) or 5 °C (cold storage, CS) for 21 d. The results from chilling injury (CI) evaluation demonstrated that there was no significant difference in appearance, internal color, weight, and relative conductivity between tuberous roots stored at 13 and 5 °C for 14 d and indicated that short-term CS for 14 d promoted the accumulation of sucrose, chlorogenic acid, and amino acids with no CI symptoms development. This, in turn, improved sweetness, antioxidant capacity, and nutritional value of the tuberous roots. Transcriptome analyses revealed that several key genes associated with sucrose, chlorogenic acid, and amino acid biosynthesis were upregulated during short-term CS, including sucrose synthase, sucrose phosphate synthase, phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase, serine hydroxymethyltransferase, alanine aminotransferase, arogenate dehydrogenase, and prephenate dehydratase. These results indicated that storage at 5 °C for 14 d could improve the nutritional quality and palatability of sweet potato tuberous roots without compromising their freshness.

Integrated Analysis of the Transcriptome and Metabolome of Cecropia obtusifolia: A Plant with High Chlorogenic Acid Content Traditionally Used to Treat Diabetes Mellitus.

This investigation cultured Cecropia obtusifolia cells in suspension to evaluate the effect of nitrate deficiency on the growth and production of chlorogenic acid (CGA), a secondary metabolite with hypoglycemic and hypolipidemic activity that acts directly on type 2 diabetes mellitus. Using cell cultures in suspension, a kinetics time course was established with six time points and four total nitrate concentrations. The metabolites of interest were quantified by high-performance liquid chromatography (HPLC), and the metabolome was analyzed using directed and nondirected approaches. Finally, using RNA-seq methodology, the first transcript collection for C. obtusifolia was generated. HPLC analysis detected CGA at all sampling points, while metabolomic analysis confirmed the identity of CGA and of precursors involved in its biosynthesis. Transcriptome analysis identified differentially expressed genes and enzymes involved in the biosynthetic pathway of CGA. C. obtusifolia probably expresses a key enzyme with bifunctional activity, the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HQT/HCT), which recognizes shikimic acid or quinic acid as a substrate and incorporates either into one of the two routes responsible for CGA biosynthesis.

Nitric oxide and selenium nanoparticles confer changes in growth, metabolism, antioxidant machinery, gene expression, and flowering in chicory (Cichorium intybus L.): potential benefits and risk assessment.

This experiment was conducted to provide a better insight into the plant responses to nitric oxide (NO) and selenium nanoparticle (nSe). Chicory seedlings were sprayed with nSe (0, 4, and 40 mg l-1), and/or NO (0 and 25 μM). NO and/or nSe4 improved shoot and root biomass by an average of 32%. The nSe40 adversely influenced shoot and root biomass (mean = 26%), exhibiting moderate toxicity partly relieved by NO. The nSe and NO treatments transcriptionally stimulated the dehydration response element B1A (DREB1A) gene (mean = 29.6-fold). At the transcriptional level, nSe4 or NO moderately upregulated phenylalanine ammonia-lyase (PAL) and hydroxycinnamoyl-CoA quinate transferase (HCT1) genes (mean = sevenfold). The nSe4 + NO, nSe40, and nSe40 + NO groups drastically induced the expression of PAL and HCT1 genes (mean = 30-fold). With a similar trend, hydroxycinnamoyl-CoA Quinate/shikimate hydroxycinnamoyl transferase (HQT1) gene was also upregulated in response to nSe and/or NO (mean = 25-fold). The activities of nitrate reductase and catalase enzymes were also induced in the nSe- and/or NO-treated seedlings. Likewise, the application of these supplements associated with an increase in ascorbate concentration (mean = 31.5%) reduced glutathione (mean = 35%). NO and/or nSe enhanced the PAL activity (mean = 36.4%) and soluble phenols (mean = 40%). The flowering was also influenced by the supplements in dose and compound dependent manner exhibiting the long-time responses. It appears that the nSe-triggered signaling can associate with a plethora of developmental, physiological, and molecular responses at least in part via the fundamental regulatory roles of transcription factors, like DREB1A as one the most significant genes for conferring tolerance in crops.

The Phytochemical, Antifungal, and First Report of the Antiviral Properties of Egyptian Haplophyllum tuberculatum Extract.

In this study, ethanol whole plant extract (WPE) of Haplophyllum tuberculatum was characterized and tested for its antifungal and antiviral activities against Fusarium culmorum, Rhizoctonia solani and tobacco mosaic virus (TMV). High Performance Liquid Chromatography (HPLC) analysis showed that the main phytochemical constituents of H. tuberculatum WPE were resveratrol (5178.58 mg/kg), kaempferol (1735.23 mg/kg), myricetin (561.18 mg/kg), rutin (487.04 mg/kg), quercetin (401.04 mg/kg), and rosmarinic acid (387.33 mg/kg). By increasing H. tuberculatum WPE at concentrations of 1%, 2%, and 3%, all of the fungal isolates were suppressed compared to the two positive and negative controls. Under greenhouse conditions, WPE-treated Chenopodium amaranticolor plants strongly inhibited TMV infection and significantly reduced TMV accumulation levels when compared to non-treated plants. Moreover, the induction of systemic resistance with significant increases in the transcriptional levels of the pathogenesis-related protein-1 (PR-1), chalcone synthase (CHS), and hydroxycinnamoyl-CoA quinate transferase (HQT) genes for treated plants were noticed at 3 and 5 days post-inoculation (dpi) for both assays. To the best of our knowledge, this is the first reported observation of the antiviral activity of H. tuberculatum extract against plant viral infections. Finally, the results obtained suggest that H. tuberculatum WPE can be considered a promising source of both antifungal and antiviral substances for practical use and for developing plant-derived compounds for the effective management of plant diseases.

Correlation of the temporal and spatial expression patterns of HQT with the biosynthesis and accumulation of chlorogenic acid in Lonicera japonica flowers

Hydroxycinnamoyl-CoA quinate transferase (HQT) is one of the key enzymes in the biosynthesis of chlorogenic acid (CGA) in the flowers of Lonicera japonica. However, the spatiotemporal expression patterns of HQT and its relationship to the dynamics of CGA biosynthesis, transport, and storage remain largely unknown. In this study, we collected L. japonica flower samples at different growth stages (S1–S6) and examined the spatiotemporal expression pattern of HQT and the dynamic accumulation patterns of CGA using a combination of molecular and cytological techniques. Our results suggest that the spatiotemporal expression pattern of HQT is directly correlated with dynamic changes in CGA accumulation and distribution in L. japonica flowers. We further show that CGA is synthesized primarily in the cytoplasm and chloroplasts. CGA synthesized in the cytoplasm first accumulates in specialized vesicles and is then transferred to large central vacuoles for storage by fusion of CGA-containing vesicles with vacuoles. Furthermore, CGA synthesized in the chloroplasts appears to be transferred into the vacuoles for storage by direct membrane fusion between the tonoplast and the disrupted chloroplast membranes. Collectively, our results suggest that CGA is synthesized in chloroplasts and cytoplasm and finally transferred to the vacuole for long-term storage.

Caffeic Acid and α-Chaconine Influence the Resistance of Potato Tuber to Phthorimaea operculella (Lepidoptera: Gelechiidae)

Host plant quality can influence insect larval performance and subsequent adult fitness. Advanced hybrid potato clones (donor species: Solanum berthaultii Hawkes) and their parents were evaluated for resistance to the potato tuber moth (PTM) and examined for several biochemical, molecular and physical traits in order to investigate the mechanisms which underlie PTM-resistance. Resistant parent recorded 57% of mortality of pupae in comparison to the 23% observed in the susceptible parent. Expression patterns of specific genes involved in the phenolic biosynthetic pathway show that hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and cinnamate-4-hydroxylase (C4H) reflect chlorogenic and caffeic acid levels in the skin of all the genotypes (0.7 ≤ r ≤ 1.0). Skin α-chaconine and skin caffeic acid strongly and significantly related with PTM larval mortality (r2 = 0.99 and r = 0.90, respectively). This study suggests that skin α-chaconine and skin caffeic acid could play a defensive role against PTM and proposes a PTM resistant clone with high level of these bio-compounds, useful in breeding projects for plant defense and nutritional value purposes.

In vitro evaluation of hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase expression and regulation in Taraxacum antungense in relation to 5-caffeoylquinic acid production

Chlorogenic acids (CGA; including 5-caffeoylquinic acid and its regio-isomers) in Taraxacum antungense Kitag. have antioxidant and anti-inflammatory properties and exert other pharmacological effects. T. antungense hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (TaHQT)1 and TaHQT2, which belong to the BAHD acyltransferase family, are candidates for synthesizing 5-caffeoylquinic acid and that have not been extensively characterized. In this study, we cloned the TaHQT1 and TaHQT2 genes and analysed the properties of the expressed enzymes both in vitro and in vivo. Quantitative reverse transcription PCR analysis revealed that TaHQT1 was highly expressed in the root, whereas the strongest TaHQT2 expression was observed in T. antungense leaves. In Nicotiana benthamiana leaf cells, TaHQT1 and TaHQT2 were localized at the cell periphery as well as in the cytoplasm and nucleus. The 5-caffeoylquinic acid concentrations in T. antungense calli were reduced by TaHQT1 and TaHQT2 knockdown relative to the control. Conversely, inoculation of T. antungense plants tissues with recombinant TaHQT1 and TaHQT2 increased 5-caffeoylquinic acid levels in situ. These in vitro and in vivo findings demonstrate that both HQTs are involved in regulating 5-caffeoylquinic acid biosynthesis in T. antungense, which can be exploited to increase 5-caffeoylquinic acid production in plants for medicinal or other beneficial purposes.

Pruning induced yield and quality variations and the correlated gene expression and phytohormone changes in Lonicera japonica

Lonicera japonica Thunb. is widely used in China to make teas, aromatic oils or medicines. The demand for L. japonica is large in China. Pruning measures are used to increase the yield of L. japonica in practice. However, there remains a question whether the quality of L. japonica is increased as well. Our study focused on the accumulations of the primary bioactive components and their molecular mechanisms, as well as the roles of endogenous plant hormones on L. japonica after pruning. To achieve this aim, the flower buds of L. japonica were randomly collected and divided into four groups for agronomic traits investigation, bioactive compound changes detection by liquid chromatography coupled with tandem mass spectrometry (LCMS/MS), the related gene expression levels quantification by quantitative real-time reverse transcription-PCR (qRT-PCR), and endogenous phytohormone changes determination by indirect enzyme-linked immunosorbent assay (ELISA), respectively. Results showed that pruning produced superior flower bud yield but inferior quality in L. japonica, especially at the first harvest time. More specifically, pruning significantly increased the yield (1.4-fold) but significantly decreased contents of chlorogenic acid (1.5-fold), isochlorogenic acid A (1.3-fold), luteoloside (1.4-fold), and isoquercetrin (1.3-fold) at the first harvest time. The decreased hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and chalcone isomerase (CHI) gene expression levels were probably the main reasons for L. japonica quality decrease. Pruning also induced flexible phytohormone changes in L. japonica. Indole-3-acetic acid (IAA), cytokines (CKs), jasmonic acid (JA), and abscisic acid (ABA) were the main hormones involving in building shoot architecture, flower development, and plant secondary metabolism. In conclusion, we think the pruning technique should be used carefully or with other agronomic practices to obtain the well balance between yield and quality in L. japonica.

Differential gene expressions and phytohormone changes altered Lonicera japonica quality after plant introduction

Background: Lonicera japonica Thunb. (LJ) is a famous traditional Chinese medicinal plant. For great viability and important medical value, LJ was introduced from the geo-authentic areas to nonauthentic production areas. However, the quality changes in LJ after plant production remained unclear. Objective: The objective of the study is to analyze the bioactive compounds, the corresponding gene expressions, and the endogenous plant hormone changes after 3-year introduction. Materials and Methods: In September 2014, LJ cuttings were planted in the two sites. In May 2017, LJ flower buds were randomly harvested and divided into three groups for liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), quantitative real-time reverse transcription-polymerase chain reaction, and indirect enzyme-linked immunosorbent assay, respectively. Results: The results showed that chlorogenic acid significantly decreased while luteoloside and luteolin significantly increased after plant introduction. Gene expression profiles related to biosynthesis pathways showed differentially expressed patterns although most of them were not significantly different. The down-regulated expression of hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes resulted in a decrease of chlorogenic acid content while the up-regulated expression of key genes related to synthesizing the universal precursor p-Coumaroyl-CoA and the branched luteoloside biosynthetic pathway led to an increase of luteoloside content. Altered endogenous phytohormone levels, especially jasmonic acid, regulate LJ quality changes. Conclusion: The data demonstrate that irrespective of the decrease of chlorogenic acid, the considerable increase of flavonoids demonstrated the benefit of LJ introduction. Abbreviations used: LJ: Lonicera japonica Thunb.; PAL: Phenylalanine ammonia-lyase (EC 4.3.1.24); C4H: Cinnamate 4-hydroxylase (EC 1.14.13.11); 4CL: 4-Hydroxycinnamoyl-CoA ligase/4-coumarate-CoA ligase (EC 6.2.1.12); HQT: Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (EC 2.3.1.99); CHS: Chalcone synthase (EC 2.3.1.74); CHI: Chalcone isomerase (EC 5.5.1.6); FNS: Flavone synthase (EC 1.14.11.22); GL: Glycosyltransferase (flavone 7-O-beta-glucosyltransferase, EC 2.4.1.81).

Overexpression of and RNA interference with hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase affect the chlorogenic acid metabolic pathway and enhance salt tolerance in Taraxacum antungense Kitag

Dandelions (Taraxacum spp.) are wild plants with a long history of use as a functional food and as medicine. Chlorogenic acid (CGA) and caffeic acid (CA) are organic acid components of Taraxacum antungense Kitag. that exhibit antioxidant, anti-inflammatory, and other pharmacological effects. This study aimed to determine the regulatory mechanism of CGA biosynthesis and the role of the CGA biosynthetic gene (HQT), encoding hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase, in T. antungense. Quantitative real-time polymerase chain reaction revealed significant changes in the tissue distribution of HQT expression in T. antungense transgenic lines overexpressing HQT. CGA but not CA concentrations differed significantly between the wild-type T. antungense and transgenic lines overexpressing HQT. Further, the CGA concentration was significantly higher in the leaves than in the roots. The upregulation of TaHQT elevated the CGA levels by up to 82.49% in the leaves, whereas RNA interference (RNAi) resulted in 51.48% reduction. Comparison of physiological parameters (malondialdehyde, proline, and total chlorophyll concentrations) under salt stress conditions revealed that the overexpression lines were more salt resistant than the wild-type and RNAi lines. These findings indicate that TaHQT positively regulates the CGA biosynthesis and enhances salt tolerance in the overexpression lines.

Similar, but different: structurally related azelaic acid and hexanoic acid trigger differential metabolomic and transcriptomic responses in tobacco cells

BackgroundPlants respond to various stress stimuli by activating an enhanced broad-spectrum defensive ability. The development of novel resistance inducers represents an attractive, alternative crop protection strategy. In this regard, hexanoic acid (Hxa, a chemical elicitor) and azelaic acid (Aza, a natural signaling compound) have been proposed as inducers of plant defense, by means of a priming mechanism. Here, we investigated both the mode of action and the complementarity of Aza and Hxa as priming agents in Nicotiana tabacum cells in support of enhanced defense.ResultsMetabolomic analyses identified signatory biomarkers involved in the establishment of a pre-conditioned state following Aza and Hxa treatment. Both inducers affected the metabolomes in a similar manner and generated common biomarkers: caffeoylputrescine glycoside, cis-5-caffeoylquinic acid, feruloylglycoside, feruloyl-3-methoxytyramine glycoside and feruloyl-3-methoxytyramine conjugate. Subsequently, quantitative real time-PCR was used to investigate the expression of inducible defense response genes: phenylalanine ammonia lyase, hydroxycinnamoyl CoA quinate transferase and hydroxycinnamoyl transferase to monitor activation of the early phenylpropanoid pathway and chlorogenic acids metabolism, while ethylene response element-binding protein, small sar1 GTPase, heat shock protein 90, RAR1, SGT1, non-expressor of PR genes 1 and thioredoxin were analyzed to report on signal transduction events. Pathogenesis-related protein 1a and defensin were quantified to investigate the activation of defenses regulated by salicylic acid and jasmonic acid respectively. The qPCR results revealed differential expression kinetics and, in general (except for NPR1, Thionin and PR1a), the relative gene expression ratios observed in the Hxa-treated cells were significantly greater than the expression observed in the cells treated with Aza.ConclusionsThe results indicate that Aza and Hxa have a similar priming effect through activation of genes involved in the establishment of systemic acquired resistance, associated with enhanced synthesis of hydroxycinnamic acids and related conjugates.

Regulation of chlorogenic acid biosynthesis by hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase in Lonicera japonica

For many centuries, Lonicera japonica has been used as an effective herb for the treatment of inflammation and swelling because of the presence of bioactive components such as chlorogenic acid (CGA). To clarify the relationship between L. japonica hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) gene expression and CGA content, an HQT eukaryotic expression system was constructed using Gateway cloning. L. japonica callus transformed with HQT was obtained using Agrobacterium tumefaciens-mediated transformation. We found a positive correlation between CGA content, determined by High-Performance Liquid Chromatography (HPLC), and the expression of HQT, analyzed by semi-quantitative RT-PCR. This study demonstrates that the HQT gene positively regulates CGA synthesis and lays the foundation for further study into enhancing efficacious components of medicinal plants.

Isolation and functional characterization of a Lonicera japonica hydroxycinnamoyl transferase involved in chlorogenic acid synthesis

AbstractThe flower buds of

Impact of novel SNPs identified in Cynara cardunculus genes on functionality of proteins regulating phenylpropanoid pathway and their association with biological activities

BackgroundCynara cardunculus L. offers a natural source of phenolic compounds with the predominant molecule being chlorogenic acid. Chlorogenic acid is gaining interest due to its involvement in various biological properties such as, antibacterial, antifungal, antioxidant, hepatoprotective, and anticarcinogenic activities.ResultsIn this work we screened a Cynara cardunculus collection for new allelic variants in key genes involved in the chlorogenic acid biosynthesis pathway. The target genes encode p-coumaroyl ester 3′-hydroxylase (C3′H) and hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (HQT), both participating in the synthesis of chlorogenic acid. Using high-resolution melting, the C3′H gene proved to be highly conserved with only 4 haplotypes while, for HQT, 17 haplotypes were identified de novo. The putative influence of the identified polymorphisms in C3′H and HQT proteins was further evaluated using bioinformatics tools. We could identify some polymorphisms that may lead to protein conformational changes. Chlorogenic acid content, antioxidant and antithrombin activities were also evaluated in Cc leaf extracts and an association analysis was performed to assess a putative correlation between these traits and the identified polymorphisms.ConclusionIn this work we identified allelic variants with putative impact on C3′H and HQT proteins which are significantly associated with chlorogenic acid content and antioxidant activity. Further study of these alleles should be explored to assess putative relevance as genetic markers correlating with Cynara cardunculus biological properties with further confirmation by functional analysis.

Correlation between the dynamic accumulation of the main effective components and their associated regulatory enzyme activities at different growth stages in Lonicera japonica Thunb

Flos Lonicerae Japonicae (FLJ), the dried flower buds of Lonicera japonica Thunb (Caprifoliaceae), is of high medicinal value in traditional Chinese medicine. Chlorogenic acid (CGA) and luteoloside are used as standard compounds to evaluate the chemical quality of FLJ. The relationship between the activities of the regulatory enzymes associated with CGA and luteoloside biosynthesis during different growth stages remains unclear. In this study, we systematically examined the dynamic accumulation of both compounds at six growth stages in L. japonica [(young alabastrum (S1), green alabastrum (S2), slightly white alabastrum (S3), whole white alabastrum (S4), silvery flower (S5), and golden flower (S6)] using an HPLC-DAD-base method. Additionally, physiological and biochemical methods were used to assay the regulatory enzyme activities. The following results indicated that: (1) The accumulation of both CGA and luteoloside first increased and then decreased throughout the growth of L. japonica. The highest content of CGA was observed in S3 (44.88μg/mg), followed by S5 (41.19μg/mg). The amount of luteoloside was relatively higher in S3 (1.41μg/mg) and S4 (1.34μg/mg) compared with that at other growth stages. (2) The activity of hydroxycinnamoyl-CoA quinate transferase (HQT) and chalcone isomerase (CHI) exhibited a positive correlation with the accumulation of CGA and luteoloside at different growth stages in L. japonica, respectively. In addition, the accumulation of CGA and luteoloside at different growth stages in L. japonica was also correlated with variation in the initial activities of several other regulatory enzymes in the phenylalanine metabolic pathway, including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA ligase (4CL). (3) Based on the relative higher yields of both CGA and luteoloside in S3 and S4, these stages are thus recommended as the best harvesting time for FLJ.

Eight artichoke accessions show differences in cynarin content and Cshct and Cshqt gene expression during the growing season

In order to add-value to artichoke production for industry and fresh purposes, different artichoke accessions were evaluated in term of cynarin content (1,3-dicaffeoylquinic acid), a metabolite associated to breakdown fat, bile-stimulating effects, and reduced cholesterol production. During 2011-2012 and 2012-2013 seasons, cynarin, Cshct and Cshqt gene expression were evaluated in artichoke accessions (Cynara scolymus L. Fiori) during vegetative growth and bud yield. Eight artichoke accessions ('Francesa', 'Argentina-CAT4', 'Argentina-CAT5', 'Argentina-CAT32', 'Chilena Llay-Llay', 'Talpiot', 'Sur Temuco-3', 'Green Globe') were vegetative propagated, and cultivated under irrigation at 33°34'S and 70°38'W. Leaves and bud samples were collected in a completely randomized design, with 4 replications by accession and time course point since fall to spring for further analysis. Cynarin quantification was done by HPLC system (a quaternary pump Jasco PU-2089 PLUS with a vacuum degasser, multiple wavelength Jasco UV-2075 detector, interface modulated LC-NetII/ADC); in bud (inner bracts, outer bracts, and heart) during spring (September and October), and in leaves during fall (April and May) and also in spring (September and October). The expression of genes encoding hydroxycinnamoyl-transferase (Cshct) and hydroxycinnamoyl CoA quinate transferase (Cshqt) was also evaluated by quantitative polymerase chain reactions (qRT-PCR) in a LightCycler®96 Real-Time PCR System (Roche, Germany), using FastStart Essential DNA Green Master (Roche, Germany). All qRT-PCR data were the average of three independent biological pool samples and three technical replicates. Significant differences were observed in cynarin in leaves, among accessions and growing season (p<0.0001), leaves collected in October (spring) showed higher cynarin concentration (42.06 µg cynarin g-1 DW). In bud, the higher cynarin content was observed in 'Argentina-CAT4' accession, while bud analysis show that outer bracts have higher concentration of this metabolite (p<0.0001) with values between 9.7 to 17.9 µg cynarin g-1 DW. In relation with Cshct and Cshqt gene expression, higher hct gene expression in conjunction with higher cynarin concentration was observed in the majority of evaluated accessions (Pearson=0.896*). No significant differences were observed in hqt gene expression.