Chlorogenic Acid Biosynthesis Research Articles

Transcriptional regulation of chlorogenic acid biosynthesis in carrot root slices exposed to UV-B light

Orange carrots are well known for their nutritional value as producers of β-carotene, a vitamin A precursor. Lesser known, is their ability to accumulate antioxidants such as chlorogenic acid. Chlorogenic acid is produced through the same biosynthetic pathway that produces lignins, anthocyanins, flavonols and isoflavonoids, the phenylpropanoid pathway. This pathway can be induced by UV-B light and wounding to produce large amounts of chlorogenic acid. We identified a number of the carrot structural genes in this pathway and their proposed regulators in carrot root slices and analyzed their expression in response to UV-B light exposure using real-time PCR. Our results indicate that not only does the expression of the structural genes correlate with their proposed regulators, including DcHY5 and others, but also correlate with chlorogenic acid production. Our results indicate that the HY5 — UVR8 signaling network may exist and function in carrot roots.

Different Gene Expression Patterns between Leaves and Flowers in Lonicera japonica Revealed by Transcriptome Analysis.

The perennial and evergreen twining vine, Lonicera japonica is an important herbal medicine with great economic value. However, gene expression information for flowers and leaves of L. japonica remains elusive, which greatly impedes functional genomics research on this species. In this study, transcriptome profiles from leaves and flowers of L. japonica were examined using next-generation sequencing technology. A total of 239.41 million clean reads were used for de novo assembly with Trinity software, which generated 150,523 unigenes with N50 containing 947 bp. All the unigenes were annotated using Nr, SwissProt, COGs (Clusters of Orthologous Groups), GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. A total of 35,327 differentially expressed genes (DEGs, P ≤ 0.05) between leaves and flowers were detected. Among them, a total of 6602 DEGs were assigned with important biological processes including “Metabolic process,” “Response to stimulus,” “Cellular process,” and etc. KEGG analysis showed that three possible enzymes involved in the biosynthesis of chlorogenic acid were up-regulated in flowers. Furthermore, the TF-based regulation network in L. japonica identified three differentially expressed transcription factors between leaves and flowers, suggesting distinct regulatory roles in L. japonica. Taken together, this study has provided a global picture of differential gene expression patterns between leaves and flowers in L japonica, providing a useful genomic resource that can also be used for functional genomics research on L. japonica in the future.

Potato tuber herbivory increases resistance to aboveground lepidopteran herbivores.

Plants mediate interactions between aboveground and belowground herbivores. Although effects of root herbivory on foliar herbivores have been documented in several plant species, interactions between tuber-feeding herbivores and foliar herbivores are rarely investigated. We report that localized tuber damage by Tecia solanivora (Guatemalan tuber moth) larvae reduced aboveground Spodoptera exigua (beet armyworm) and Spodoptera frugiperda (fall armyworm) performance on Solanum tuberosum (potato). Conversely, S. exigua leaf damage had no noticeable effect on belowground T. solanivora performance. Tuber infestation by T. solanivora induced systemic plant defenses and elevated resistance to aboveground herbivores. Lipoxygenase 3 (Lox3), which contributes to the synthesis of plant defense signaling molecules, had higher transcript abundance in T. solanivora-infested leaves and tubers than in equivalent control samples. Foliar expression of the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and 3-hydroxy-3-methylglutaryl CoA reductase I (HMGR1) genes, which are involved in chlorogenic acid and steroidal glycoalkaloid biosynthesis,respectively, also increased in response to tuber herbivory. Leaf metabolite profiling demonstrated the accumulation of unknown metabolites as well as the known potato defense compounds chlorogenic acid, α-solanine, and α-chaconine. When added to insect diet at concentrations similar to those found in potato leaves, chlorogenic acid, α-solanine, and α-chaconine all reduced S. exigua larval growth. Thus, despite the fact that tubers are a metabolic sink tissue, T. solanivora feeding elicits a systemic signal that induces aboveground resistance against S. exigua and S. frugiperda by increasing foliar abundance of defensive metabolites.

Alkaloid biosynthesis and metabolic profiling responses to jasmonic acid elicitation in Hamelia patens plants by NMR-based metabolomics

Hamelia patens is a perennial shrub native from Mexico which is used in traditional medicine to treat chronic wounds and tumors. Bioactive monoterpenoid indole alkaloids (MIAs) and monoterpenoid oxindole alkaloids (MOAs) have been isolated from this species, associated with the ethnomedical use. In order to study the biosynthesis and production of these alkaloids, H. patens plants were treated with the stress hormone jasmonic acid. Nuclear magnetic resonance-based metabolic profiles and multivariate data analysis were applied to explore changes or variability in elicited and control plants. According to the partial least square-discriminant analysis from MeOH/H2O fractions, sucrose, tryptophan, glutamic acid, glutamine, strictosidine, p-coumaric acid, chlorogenic acid, and the MOAs isopteropodine, palmirine and pteropodine were predominant in the treated plants, while glucose, aspartic acid, acetic acid, and loganic acid concentrations were higher in control plants. HPLC analysis showed that accumulation of pteropodine, isopteropodine, speciophylline, rumberine, hameline and palmirine in elicited plants was preceded by an increase in the transcription level of 1-deoxy-d-xylulose-5-phosphate synthase (DXS, EC 2.2.1.7) and strictosidine synthase (STR, EC 4.3.3.2), and STR activity during a time course. However, no inductions of transcription and enzyme activity of strictosidine β-glucosidase (SGD, EC 3.2.1.105) were observed in elicited plants. Correlations between primary and secondary metabolism biosynthetic pathways, such as the alkaloid and chlorogenic acid biosynthesis were found. Also, their implications in the plant fitness to stress are proposed. Confirmation of tryptophan as a precursor of MIA and MOA, via shikimate pathway, was done by L-[3′-14C]-tryptophan feeding.

Chlorogenic Acids Biosynthesis in Centella asiatica Cells Is not Stimulated by Salicylic Acid Manipulation.

Exogenous application of synthetic and natural elicitors of plant defence has been shown to result in mass production of secondary metabolites with nutraceuticals properties in cultured cells. In particular, salicylic acid (SA) treatment has been reported to induce the production of phenylpropanoids, including cinnamic acid derivatives bound to quinic acid (chlorogenic acids). Centella asiatica is an important medicinal plant with several therapeutic properties owing to its wide spectrum of secondary metabolites. We investigated the effect of SA on C. asiatica cells by monitoring perturbation of chlorogenic acids in particular. Different concentrations of SA were used to treat C. asiatica cells, and extracts from both treated and untreated cells were analysed using an optimised UHPLC-QTOF-MS/MS method. Semi-targeted multivariate data analyses with the aid of principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed a concentration-dependent metabolic response. Surprisingly, a range of chlorogenic acid derivatives were found to be downregulated as a consequence of SA treatment. Moreover, irbic acid (3,5-O-dicaffeoyl-4-O-malonilquinic acid) was found to be a dominant CGA in C. asiatica cells, although the SA treatment also had a negative effect on its concentration. Overall SA treatment was found to be an ineffective elicitor of CGA production in cultured C. asiatica cells.

Phenylpropanoids Accumulation in Eggplant Fruit: Characterization of Biosynthetic Genes and Regulation by a MYB Transcription Factor.

Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena) fruits. Chlorogenic acid (CGA) accounts for 70–90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena. Higher contents of CGA, Delphinidin 3-rutinoside, and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR, and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group six MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties. In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation. Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9) resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of the C-terminal region of SmMyb1 does not limit its capability to regulate CGA accumulation, but impairs anthocyanin biosynthesis. To our knowledge, this is the first study reporting a functional elucidation of the role of the C-term conserved domain in MYB activator proteins.

Transcriptome Analysis Reveals the Mechanism Underlying the Production of a High Quantity of Chlorogenic Acid in Young Leaves of Lonicera macranthoides Hand.-Mazz.

Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in southern China. The biosynthetic and metabolic pathways for a core secondary metabolite in L. macranthoides, chlorogenic acid (CGA), have been elucidated in many species. However, the mechanisms of CGA biosynthesis and the related gene regulatory network in L. macranthoides are still not well understood. In this study, CGA content was quantified by high performance liquid chromatography (HPLC), and CGA levels differed significantly among three tissues; specifically, the CGA content in young leaves (YL) was greater than that in young stems (YS), which was greater than that in mature flowers (MF). Transcriptome analysis of L. macranthoides yielded a total of 53,533,014 clean reads (average length 90 bp) and 76,453 unigenes (average length 703 bp). A total of 3,767 unigenes were involved in biosynthesis pathways of secondary metabolites. Of these unigenes, 80 were possibly related to CGA biosynthesis. Furthermore, differentially expressed genes (DEGs) were screened in different tissues including YL, MF and YS. In these tissues, 24 DEGs were found to be associated with CGA biosynthesis, including six phenylalanine ammonia lyase (PAL) genes, six 4-coumarate coenzyme A ligase (4CL) genes, four cinnamate 4-Hydroxylase (C4H) genes, seven hydroxycinnamoyl transferase/hydroxycinnamoyl-CoA quinate transferase HCT/HQT genes and one coumarate 3-hydroxylase (C3H) gene.These results further the understanding of CGA biosynthesis and the related regulatory network in L. macranthoides.

Altered metabolomic profile of selected metabolites and improved resistance of Cicer arietinum (L.) against Meloidogyne incognita (Kofoid & White) Chitwood following seed soaking with salicylic acid, benzothiadiazole or nicotinic acid

The seeds of chickpea Cicer arietinum (L.) were soaked in 0.00, 10.0 or 20.0 μg/mL of salicylic acid (SA), benzothiadiazole (BTH) or nicotinic acid (NA) solutions for 1 h. Treated seeds were sown in field and metabolite profiling of free reduced/oxidized glutathione (GSH/GSSG), SA, acetyl salicylic acid (ASA), jasmonic acid (JA), and chlorogenic acid (CGA) in chickpea crop was carried out at regular intervals (15, 30, 60, 90 and 120 days of sowing) using liquid chromatography (LC). These defense activators increased the levels of GSH, SA, and CGA in leaf; GSSG, ASA, and JA in root; and decreased the levels of GSSG and JA in leaf; GSH, SA, and CGA in root. In leaf, the biosynthesis of SA increased concomitantly with that of GSH. ASA and JA levels declined with increase of SA concentration. CGA biosynthesis increased with decline of JA level. In root, CGA and SA levels declined with increase of JA concentration. The SA level was directly related to CGA and inversely to ASA and JA concentrations. The CGA and SA concentrations increased with treatment dosage up to 120 days. The concentration of leaf GSSG was linked with JA level in root. Chickpea was a SA-rich crop. GSH in leaf was the key metabolite for SA and JA signaling. The reduction in root infection and reproduction of root-knot nematode (RKN), Meloidogyne incognita was highest in BTH followed by SA and NA treatments. This was attributed to increased biosynthesis of CGA. The study firstly linked GSH and SA metabolism enhancing CGA biosynthesis in chickpea. The increase in CGA improved resistance against RKN. Seed treatment at 10.0 µg/mL BTH 1 h before sowing is recommended as strategic management decision to contain RKN on chickpea.

Constitutive expression of Arabidopsis MYB transcription factor, AtMYB11, in tobacco modulates flavonoid biosynthesis in favor of flavonol accumulation

Heterologous expression of AtMYB11 , a flavonol-specific transcription factor from Arabidopsis , in tobacco modulates flavonoid biosynthesis, however, with a lower efficiency as compared to its paralogs AtMYB12 and AtMYB111. Transcriptional regulation is the most important means for controlling flavonoid biosynthesis under temporal and spatial cues. In Arabidopsis, three functionally redundant MYB transcription factors (AtMYB11, AtMYB111 and AtMYB12) have been characterized as flavonol-specific regulators which positively modulate expression of biosynthetic genes involved in flavonol biosynthesis. Based on expression of AtMYB111 and AtMYB12 in heterologous systems, studies suggest that these transcription factors can be used to develop plants with enhanced flavonol biosynthesis. The potential of AtMYB11 to activate flavonol biosynthesis in a heterologous system has not yet been studied. In this study, the regulatory potential of AtMYB11 has been studied in Nicotiana tabacum by developing transgenic plants constitutively expressing AtMYB11. Our analysis using leaf and petal tissues of the transgenic plants indicates that AtMYB11 enhances flavonol and chlorogenic acid (CGA) biosynthesis in tobacco through up-regulation of the biosynthetic genes. Activation of flavonol biosynthesis in tobacco by AtMYB11 is not as pronounced as with AtMYB12 or AtMYB111. Taken together, these results reveal a differential regulatory mechanism in plants for modulating flavonol biosynthesis. This study demonstrated that AtMYB11 can be strategically used for enhancing the health beneficial flavonols in species other than Arabidopsis.

Chlorogenic Acid Biosynthesis Appears Linked with Suberin Production in Potato Tuber (Solanum tuberosum).

Potato (Solanum tuberosum L.) is a good source of dietary antioxidants. Chlorogenic acid (CGA) and caffeic acid (CA) are the most abundant phenolic acid antioxidants in potato and are formed by the phenylpropanoid pathway. A number of CGA biosynthetic routes that involve hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and/or hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) have been proposed, but little is known about their path in potato. CA production requires a caffeoyl shikimate esterase (CSE), and CA serves as a substrate of lignin precursor ferulic acid via the action of caffeic/5-hydroxyferulic acid O-methyltransferase (COMTI). CGA is precursor of caffeoyl-CoA and, via caffeoyl-CoA O-methyltransferase (CCoAOMT), of feruloyl-CoA. Feruloyl-CoA is required for lignin and suberin biosynthesis, crucial for tuber development. Here, metabolite and transcript levels of the mentioned and related enzymes, such as cinnamate 4-hydroxylase (C4H), were determined in the flesh and skin of fresh and stored tubers. Metabolite and transcript levels were higher in skin than in flesh, irrespective of storage. CGA and CA production appear to occur via p-coumaroyl-CoA, using HQT and CSE, respectively. HCT is likely involved in CGA remobilization toward suberin. The strong correlation between CGA and CA, the correspondence with C4H, HQT, CCoAOMT2, and CSE, and the negative correlation of HCT and COMTI in potato tubers suggest a major flux toward suberin.

Gene cloning and bioinformatics analysis of new gene for chlorogenic acid biosynthesis of Lonicera hypoglauca

To obtain the key genes for chlorogenic acid biosynthesis of Lonicera hypoglauca, four new genes ware obtained from the our dataset of L. hypoglauca. And we also predicted the structure and function of LHPAL4, LHHCT1 , LHHCT2 and LHHCT3 proteins. The phylogenetic tree showed that LHPAL4 was closely related with LHPAL1, LHHCT1 was closely related with LHHCT3, LHHCT2 clustered into a single group. By Real-time PCR to detect the gene expressed level in different organs of L. hypoglauca, we found that the transcripted level of LHPAL4, LHHCT1 and LHHCT3 was the highest in defeat flowers, and the transcripted level of LHHCT2 was the highest in leaves. These result provided a basis to further analysis the mechanism of active ingredients in different organs, as well as the element for in vitro biosynthesis of active ingredients.

Location of chlorogenic acid biosynthesis pathway and polyphenol oxidase genes in a new interspecific anchored linkage map of eggplant.

BackgroundEggplant is a powerful source of polyphenols which seems to play a key role in the prevention of several human diseases, such as cancer and diabetes. Chlorogenic acid is the polyphenol most present in eggplant, comprising between the 70% and 90% of the total polyphenol content. Introduction of the high chlorogenic acid content of wild relatives, such as S. incanum, into eggplant varieties will be of great interest. A potential side effect of the increased level polyphenols could be a decrease on apparent quality due to browning caused by the polyphenol oxidase enzymes mediated oxidation of polyphenols. We report the development of a new interspecific S. melongena × S. incanum linkage map based on a first backcross generation (BC1) towards the cultivated S. melongena as a tool for introgressing S. incanum alleles involved in the biosynthesis of chlorogenic acid in the genetic background of S. melongena.ResultsThe interspecific genetic linkage map of eggplant developed in this work anchor the most informative previously published genetic maps of eggplant using common markers. The 91 BC1 plants of the mapping population were genotyped with 42 COSII, 99 SSRs, 88 AFLPs, 9 CAPS, 4 SNPs and one morphological polymorphic markers. Segregation marker data resulted in a map encompassing 1085 cM distributed in 12 linkage groups. Based on the syntheny with tomato, the candidate genes involved in the core chlorogenic acid synthesis pathway in eggplant (PAL, C4H, 4CL, HCT, C3′H, HQT) as well as five polyphenol oxidase (PPO1, PPO2, PPO3, PPO4, PPO5) were mapped. Except for 4CL and HCT chlorogenic acid genes were not linked. On the contrary, all PPO genes clustered together. Candidate genes important in domestication such as fruit shape (OVATE, SISUN1) and prickliness were also located.ConclusionsThe achievements in location of candidate genes will allow the search of favorable alleles employing marker-assisted selection in order to develop new varieties with higher chlorogenic content alongside a lower polyphenol oxidase activity. This will result into an enhanced product showing a lower fruit flesh browning with improved human health properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0350-z) contains supplementary material, which is available to authorized users.

Synthesis and regulation of chlorogenic acid in potato: Rerouting phenylpropanoid flux in HQT-silenced lines.

Chlorogenic acid (CGA) is the major phenolic sink in potato tubers and can constitute over 90% of total phenylpropanoids. The regulation of CGA biosynthesis in potato and the role of the CGA biosynthetic gene hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) was characterized. A sucrose induced accumulation of CGA correlated with the increased expression of phenylalanine ammonia-lyase (PAL) rather than HQT. Transient expression of the potato MYB transcription factor StAN1 (anthocyanin 1) in tobacco increased CGA. RNAi suppression of HQT resulted in over a 90% reduction in CGA and resulted in early flowering. The reduction in total phenolics and antioxidant capacity was less than the reduction in CGA, suggesting flux was rerouted into other phenylpropanoids. Network analysis showed distinct patterns in different organs, with anthocyanins and phenolic acids showing negative correlations in leaves and flowers and positive in tubers. Some flavonols increased in flowers, but not in leaves or tubers. Anthocyanins increased in flowers and showed a trend to increase in leaves, but not tubers. HQT suppression increased biosynthesis of caffeoyl polyamines, some of which are not previously reported in potato. Decreased PAL expression and enzyme activity was observed in HQT suppressed lines, suggesting the existence of a regulatory loop between CGA and PAL. Electrophysiology detected no effect of CGA suppression on potato psyllid feeding. Collectively, this research showed that CGA in potatoes is synthesized through HQT and HQT suppression altered phenotype and redirected phenylpropanoid flux.

Inhibitory effect of chlorogenic acid on fruit russeting in ‘Golden Delicious’ apple

Analysis of the secondary metabolites in apple peels showed that the levels of total flavonoids and chlorogenic acid (CA) during 15–60 days after full bloom (DAFB) were significantly higher in cultivar ‘Feng Shuai’, a spontaneous non-russeting mutant of ‘Golden Delicious’ apple, than in its maternal plant, which indicated higher level of endogenous CA may be associated with the inhibition of russet formation. Application of 0.5mmolL−1 exogenous CA solution to ‘Golden Delicious’ fruits at 30 DAFB effectively inhibited fruit russet formation. It also increased the levels of CA and total flavonoids in fruit peels at the 7th day after treatment, but depressed the lignin content. Furthermore, CA treatment induced the activity of phenylalnine ammonialyase (PAL) and cinnamate-4-hydrolase (C4H), but depressed activity of polyphenol oxidase (PPO) and peroxidase (POD). These effects were further supported by RT-PCR, where the expressions of PAL, C4H, 4CL, CHS, and CHI were up-regulated while those of CAD, POD and PPO were down-regulated by CA treatment. Interestingly, the up-regulated genes are involved in the biosynthesis of chlorogenic acid and flavonoids while the down-regulated ones are involved in lignin biosynthesis. Therefore, it can be deduced that the inhibition of russet formation in ‘Golden Delicious’ apple by CA is associated with the inhibition of lignin biosynthesis, not the enhancement of total flavonoids biosynthesis.

Analysis of critical genes expression of chlorogenic acid and luteolin biosyntheses in Lonicera confusa

This study analysed the tissue specific expression of critical genes involved in chlorogenic acid and luteolin biosynthesis, for exploiting the molecular mechanism of components biosynthesis in Lonicera confusa. Expression of PAL, 4CL, C4H, CHS, CHI, FNS and HQT gene families of chlorogenic acid and luteolin biosynthesis-related genes in buds and leaves of L. confusa were analyed by Real-time PCR. Expressions of PAL1, C4H1, 4CL1, CHS1, CHI3 and HQT2 in buds were lower than that in leaves, and expressions of PAL3, 4CL2, CHI2 and FNS2 in buds were higher than that in leaves. The results indicated that that PAL3 and 4CL2 may be associated with accumulation of chlorogenic acid, and the expression patterns of PAL1, CHS1, CHI3 and HQT2 in buds and leaves of L. confusa were different with L. japonica. This study provided some theoretical basis for the further research on genetic mechanism of active components differences in L. confusa and L. japonica.

Analysis of the Transcriptome of Erigeron breviscapus Uncovers Putative Scutellarin and Chlorogenic Acids Biosynthetic Genes and Genetic Markers

Background Erigeron breviscapus (Vant.) Hand-Mazz. is a famous medicinal plant. Scutellarin and chlorogenic acids are the primary active components in this herb. However, the mechanisms of biosynthesis and regulation for scutellarin and chlorogenic acids in E. breviscapus are considerably unknown. In addition, genomic information of this herb is also unavailable.Principal FindingsUsing Illumina sequencing on GAIIx platform, a total of 64,605,972 raw sequencing reads were generated and assembled into 73,092 non-redundant unigenes. Among them, 44,855 unigenes (61.37%) were annotated in the public databases Nr, Swiss-Prot, KEGG, and COG. The transcripts encoding the known enzymes involved in flavonoids and in chlorogenic acids biosynthesis were discovered in the Illumina dataset. Three candidate cytochrome P450 genes were discovered which might encode flavone 6-hydroase converting apigenin to scutellarein. Furthermore, 4 unigenes encoding the homologues of maize P1 (R2R3-MYB transcription factors) were defined, which might regulate the biosynthesis of scutellarin. Additionally, a total of 11,077 simple sequence repeat (SSR) were identified from 9,255 unigenes. Of SSRs, tri-nucleotide motifs were the most abundant motif. Thirty-six primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism. The result revealed that 34 (94.40%) primer pairs were successfully amplified and 19 (52.78%) primer pairs exhibited polymorphisms.ConclusionUsing next generation sequencing (NGS) technology, this study firstly provides abundant genomic data for E. breviscapus. The candidate genes involved in the biosynthesis and transcriptional regulation of scutellarin and chlorogenic acids were obtained in this study. Additionally, a plenty of genetic makers were generated by identification of SSRs, which is a powerful tool for molecular breeding and genetics applications in this herb.

Nucleotide diversity of genes related to chlorogenic acid biosynthesis of coffea

Chlorogenic acids (CGAs) are important chemical compounds of Coffea spp. related to beverage quality as they affect its astringency and can change its aroma and flavor. About 310,000 Coffea Expressed Sequence Tags (ESTs) are available and provide access to the nucleotide variability of the plant and to the development of molecular markers linked to beverage quality for the main enzymes involved in biosynthesis of the CGAs: PAL, C4H, 4CL, CQT and C3’H. In this study we identified SNP, INDELS and SSR polymorphisms within the nucleotide sequences available from the Brazilian Coffee Genome database and from the NCBI. The EST sequences for CGAs were trimmed and clustered by the program Codon Code Aligner, and polymorphisms and their validation detected (chromatogram quality). We identified six isoforms for PAL, one for C4H, six for 4CL, two for CQT and two for C3’H. The contigs formed exhibited a total of 248 polymorphisms (236 SNPs and 12 INDELs), with 201 in the coding region (127 non-synonymous and 74 synonymous). The frequency of polymorphisms was greater in the UTR regions (1pol/54pb) in relation to the coding region (1pol/81pb). The analysis of C. arabica sequences allowed identification of two different subgroups of sequences, related to their ancestral genomes (C. canephora and C. eugenioides). The presence of 67,4% of the polymorphisms between the ancestral groups and 32,6% within the groups were observed em C. arabica . The characterization of nucleotide diversity on those genes is essential for further studies on differential expression of their homeologs, as well as the use of CGAs as molecular markers related to genetic mapping.

Transcriptome analysis of buds and leaves using 454 pyrosequencing to discover genes associated with the biosynthesis of active ingredients in Lonicera japonica Thunb.

Background Lonicera japonica Thunb. is a plant used in traditional Chinese medicine known for its anti-inflammatory, anti-oxidative, anti-carcinogenic, and antiviral pharmacological properties. The major active secondary metabolites of this plant are chlorogenic acid (CGA) and luteoloside. While the biosynthetic pathways of these metabolites are relatively well known, the genetic information available for this species, especially the biosynthetic pathways of its active ingredients, is limited.Methodology/Principal FindingsWe obtained one million reads (average length of 400 bp) in a whole sequence run using a Roche/454 GS FLX titanium platform. Altogether, 85.69% of the unigenes covering the entire life cycle of the plant were annotated and 325 unigenes were assigned to secondary metabolic pathways. Moreover, 2039 unigenes were predicted as transcription factors. Nearly all of the possible enzymes involved in the biosynthesis of CGA and luteoloside were discovered in L. japonica. Three hydroxycinnamoyl transferase genes, including two hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes and one hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) gene featuring high similarity to known genes from other species, were cloned. The HCT gene was discovered for the first time in L. japonica. In addition, 188 candidate cytochrome P450 unigenes and 245 glycosyltransferase unigenes were found in the expressed sequence tag (EST) dataset.ConclusionThis study provides a high quality EST database for L. japonica by 454 pyrosequencing. Based on the EST annotation, a set of putative genes involved in CGA and luteoloside biosynthetic pathways were discovered. The database serves as an important source of public information on genetic markers, gene expression, genomics, and functional genomics in L. japonica.

Characterization of Genes for a Putative Hydroxycinnamoyl-coenzyme A Quinate Transferase and p-Coumarate 3′-Hydroxylase and Chlorogenic Acid Accumulation in Tartary Buckwheat

Tartary buckwheat ( Fagopyrum tataricum Gaertn.) contains a high level of flavonoid compounds, which have beneficial and pharmacological effects on health. In this study, we isolated full-length cDNAs encoding hydroxycinnamoyl-coenzyme A quinate hydroxycinnamoyltransferase (HQT) and p-coumarate 3'-hydroxylase (C3H), which are involved in chlorogenic acid (CGA) biosynthesis. We examined the expression levels of HQT and C3H using real-time RT-PCR in different organs and sprouts of two tartary buckwheat cultivars (Hokkai T8 and T10) and analyzed CGA content using high-performance liquid chromatography. Among the organs, the flowers in both cultivars showed the highest levels of CGA. We concluded that the expression pattern of FtHQT and FtC3H did not match the accumulation pattern of CGA in different organs of T8 and T10 cultivars. Gene expression and CGA content varied between the cultivars. We presume that FtHQT and FtC3H levels might be controlled by multiple metabolic pathways in different organs of tartary buckwheat. Probably, FtC3H might have a greater effect on CGA biosynthesis than FtHQT. Our results will be helpful for a greater understanding of CGA biosynthesis in tartary buckwheat.

Coupling Deep Transcriptome Analysis with Untargeted Metabolic Profiling in Ophiorrhiza pumila to Further the Understanding of the Biosynthesis of the Anti-Cancer Alkaloid Camptothecin and Anthraquinones

The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila.