Chlorogenic Acid Biosynthesis Research Articles

Transcriptomic analysis of key genes involved in chlorogenic acid biosynthetic pathway and characterization of MaHCT from Morus alba L.

Mulberry leaves (Morus alba L.) are of high medicinal value in traditional Chinese medicine with chlorogenic acid (CGA) as its major biologically active constituent. Mulberry leaves require that they be harvested after frost; previous studies have shown CGA accumulation significantly increased after frost. However, the molecular mechanism of how frost changes the CGA content in mulberry leaves is unclear. Additionally, the mechanism of CGA biosynthesis and key genes in mulberry leaves are not well-understood. In this study, transcriptome sequencing was performed on two mulberry leaf samples with different CGA contents (before and after frost). Fifty-eight genes were annotated in the CGA biosynthetic pathway. Compared to those in pre-frost mulberry leaves, 12 and 5 genes were upregulated and downregulated, respectively, in post-frost leaves. Correlation analysis showed that the expression levels of four genes were significantly positively correlated with CGA content, including those encoding phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), and coumaroyl quinate/shikimate 3′-hydroxylase, and may be key genes in the CGA biosynthetic pathway. We cloned MaHCT4 (GenBank accession no. MH476577) from mulberry leaves. Multiple sequence alignment suggested that MaHCT4 contains the conserved domains HXXXD and DFGWG. Enzymatic assays indicated that MaHCT4 catalyzes the formation of p-coumaroyl shikimic acid, p-coumaroyl quinic acid, and CGA. The Km values of quinic acid and shikimic acid were 10 ± 1.0 and 31 ± 1.7 μM, respectively, suggesting that MaHCT4 favored quinic acid over shikimic acid as its acyl acceptor. Using quinic acid as an acyl acceptor, MaHCT4 showed a preference for p-coumaroyl-CoA over caffeoyl-CoA. Our results provide insight into the molecular mechanism of how frost alters the CGA content and roles of key genes involved in the CGA biosynthetic pathway in mulberry leaves.

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.

Structural and biochemical approaches uncover multiple evolutionary trajectories of plant quinate dehydrogenases.

Quinate is produced and used by many plants in the biosynthesis of chlorogenic acids (CGAs). Chlorogenic acids are astringent and serve to deter herbivory. They also function as antifungal agents and have potent antioxidant properties. Quinate is produced at a branch point of shikimate biosynthesis by the enzyme quinate dehydrogenase (QDH). However, little information exists on the identity and biochemical properties of plant QDHs. In this study, we utilized structural and bioinformatics approaches to establish a QDH-specific primary sequence motif. Using this motif, we identified QDHs from diverse plants and confirmed their activity by recombinant protein production and kinetic assays. Through a detailed phylogenetic analysis, we show that plant QDHs arose directly from bifunctional dehydroquinate dehydratase-shikimate dehydrogenases (DHQD-SDHs) through different convergent evolutionary events, illustrated by our findings that eudicot and conifer QDHs arose early in vascular plant evolution whereas Brassicaceae QDHs emerged later. This process of recurrent evolution of QDH is further demonstrated by the fact that this family of proteins independently evolved NAD+ and NADP+ specificity in eudicots. The acquisition of QDH activity by these proteins was accompanied by the inactivation or functional evolution of the DHQD domain, as verified by enzyme activity assays and as reflected in the loss of key DHQD active site residues. The implications of QDH activity and evolution are discussed in terms of plant growth and development.

Cloning and functional characterization of a p-coumaroyl quinate/shikimate 3′-hydroxylase from potato (Solanum tuberosum)

Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3′-hydroxylase (C3′H) for CGA biosynthesis has not been isolated and functionally characterized in potato. In this work, we cloned StC3′H from potato and showed that it catalyzed the formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the expression of StC3′H proteins was blocked through antisense (AS) inhibition under the control of a tuber-specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest that StC3′H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in the core tissue of potato tubers is an intricately controlled process and that additional C3′H activity may also be involved in CGA biosynthesis in potato.

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.

What happens in the pith stays in the pith: tissue-localized defense responses facilitate chemical niche differentiation between two spatially separated herbivores.

Herbivore attack is known to elicit systemic defense responses that spread throughout the host plant and influence the performance of other herbivores. While these plant-mediated indirect competitive interactions are well described, and the co-existence of herbivores from different feeding guilds is common, the mechanisms of co-existence are poorly understood. In both field and glasshouse experiments with a native tobacco, Nicotiana attenuata, we found no evidence of negative interactions when plants were simultaneously attacked by two spatially separated herbivores: a leaf chewer Manduca sexta and a stem borer Trichobaris mucorea. T.mucorea attack elicited jasmonic acid (JA) and jasmonoyl-l-isoleucine bursts in the pith of attacked stems similar to those that occur in leaves when M. sexta attacks N. attenuata leaves. Pith chlorogenic acid (CGA) levels increased 1000-fold to levels 6-fold higher than leaf levels after T.mucorea attack; these increases in pith CGA levels, which did not occur in M.sexta-attacked leaves, required JA signaling. With plants silenced in CGA biosynthesis (irHQT plants), CGA, as well as other caffeic acid conjugates, was demonstrated in both glasshouse and field experiments to function as a direct defense protecting piths against T.mucorea attack, but not against leaf chewers or sucking insects. T.mucorea attack does not systemically activate JA signaling in leaves, while M.sexta leaf-attack transiently induces detectable but minor pith JA levels that are dwarfed by local responses. We conclude that tissue-localized defense responses allow tissue-specialized herbivores to share the same host and occupy different chemical defense niches in the same hostplant.

Identification and analysis of CYP450 genes from transcriptome of Lonicera japonica and expression analysis of chlorogenic acid biosynthesis related CYP450s.

BackgroundLonicera japonica is an important medicinal plant that has been widely used in traditional Chinese medicine for thousands of years. The pharmacological activities of L. japonica are mainly due to its rich natural active ingredients, most of which are secondary metabolites. CYP450s are a large, complex, and widespread superfamily of proteins that participate in many endogenous and exogenous metabolic reactions, especially secondary metabolism. Here, we identified CYP450s in L. japonica transcriptome and analyzed CYP450s that may be involved in chlorogenic acid (CGA) biosynthesis.MethodsThe recent availability of L. japonica transcriptome provided opportunity to identify CYP450s in this herb. BLAST based method and HMM based method were used to identify CYP450s in L. japonica transcriptome. Then, phylogenetic analysis, conserved motifs analysis, GO annotation, and KEGG annotation analyses were conducted to characterize the identified CYP450s. qRT-PCR was used to explore expression patterns of five CGA biosynthesis related CYP450s.ResultsIn this study, 151 putative CYP450s with complete cytochrome P450 domain, which belonged to 10 clans, 45 families and 76 subfamilies, were identified in L. japonica transcriptome. Phylogenetic analysis classified these CYP450s into two major branches, A-type (47%) and non-A type (53%). Both types of CYP450s had conserved motifs in L. japonica. The differences of typical motif sequences between A-type and non-A type CYP450s in L. japonica were similar with other plants. GO classification indicated that non-A type CYP450s participated in more molecular functions and biological processes than A-type. KEGG pathway annotation totally assigned 47 CYP450s to 25 KEGG pathways. From these data, we cloned two LjC3Hs (CYP98A subfamily) and three LjC4Hs (CYP73A subfamily) that may be involved in biosynthesis of CGA, the major ingredient for pharmacological activities of L. japonica. qRT-PCR results indicated that two LjC3Hs exhibited oppositing expression patterns during the flower development and LjC3H2 exhibited a similar expression pattern with CGA concentration measured by HPLC. The expression patterns of three LjC4Hs were quite different and the expression pattern of LjC4H3 was quite similar with that of LjC3H1.DiscussionOur results provide a comprehensive identification and characterization of CYP450s in L. japonica. Five CGA biosynthesis related CYP450s were cloned and their expression patterns were explored. The different expression patterns of two LjC3Hs and three LjC4Hs may be due to functional divergence of both substrate and catalytic specificity during plant evolution. The co-expression pattern of LjC3H1 and LjC4H3 strongly suggested that they were under coordinated regulation by the same transcription factors due to same cis elements in their promoters. In conclusion, this study provides insight into CYP450s and will effectively facilitate the research of biosynthesis of CGA in L. japonica.

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi\u2013SlPH tomato line with reduced fruit acidity and higher pH

BackgroundThe destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi–SlPH tomato lines was sequenced and gene-expression patterns were compared.ResultsC. gloeosporioides inoculation of the RNAi–SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi–SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi–SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi–SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi–SlPH tomato, it mainly upregulates the nitrate metabolic process.ConclusionsModulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi–SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi–SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

DNA Methylation Influences Chlorogenic Acid Biosynthesis in Lonicera japonica by Mediating LjbZIP8 to Regulate Phenylalanine Ammonia-Lyase 2 Expression.

The content of active compounds differ in buds and flowers of Lonicera japonica (FLJ) and L. japonica var. chinensis (rFLJ). Chlorogenic acid (CGAs) were major active compounds of L. japonica and regarded as measurements for quality evaluation. However, little is known concerning the formation of active compounds at the molecular level. We quantified the major CGAs in FLJ and rFLJ, and found the concentrations of CGAs were higher in the buds of rFLJ than those of FLJ. Further analysis of CpG methylation of CGAs biosynthesis genes showed differences between FLJ and rFLJ in the 5′-UTR of phenylalanine ammonia-lyase 2 (PAL2). We identified 11 LjbZIP proteins and 24 rLjbZIP proteins with conserved basic leucine zipper domains, subcellular localization, and electrophoretic mobility shift assay showed that the transcription factor LjbZIP8 is a nuclear-localized protein that specifically binds to the G-box element of the LjPAL2 5′-UTR. Additionally, a transactivation assay and LjbZIP8 overexpression in transgenic tobacco indicated that LjbZIP8 could function as a repressor of transcription. Finally, treatment with 5-azacytidine decreased the transcription level of LjPAL2 and CGAs content in FLJ leaves. These results raise the possibility that DNA methylation might influence the recruitment of LjbZIP8, regulating PAL2 expression level and CGAs content in L. japonica.

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.

Overexpression of the LmHQT1 gene increases chlorogenic acid production in Lonicera macranthoides Hand-Mazz

Lonicera macranthoides Hand-Mazz contains high levels of chlorogenic acid (CGA). The CGA is synthesized via different biosynthetic pathways in various plant species, and hydroxycinnamoyl-coenzyme A quinate transferases (HQTs) are key enzymes in these routes. In this study, we isolated the LmHQT1 gene, which encodes a protein of 447 amino acid residues with conserved HXXXD and DFGWG motifs. It is very closely homologous to HQT genes in Lonicera japonica (LjHQT), Solanum lycopersicum (SlHQT) and Nicotiana sylvestris (NsHQT). Quantitative reverse-transcription polymerase chain reaction showed that LmHQT1 gene expression decreased following leaf senescence. The CGA contents displayed similar trends, suggesting a potential role of LmHQT1 in CGA biosynthesis. To characterize its function, LmHQT1 overexpressing plants were generated via Agrobacterium transformation methods established previously. Upregulation of LmHQT1 in L. macranthoides was observed to elevate the CGA levels up to 60% in leaves. These findings indicated that LmHQT1was devoted to CGA biosynthesis in L. macranthoides.

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.

Stimulatory Effects of Acibenzolar-S-Methyl on Chlorogenic Acids Biosynthesis in Centella asiatica Cells.

Centella asiatica is a perrenial herb that grows in tropical regions with numerous medicinal properties mostly attributed to the presence of pentacyclic triterpenoids. Interestingly, this plant also possess a significant amount of phenylpropanoid-derived chlorogenic acids (CGAs) that have recently been reported to confer neuroprotective properties. In a biotechnological attempt to increase the biosynthesis of CGA-derivatives in cultured Centella cells, acibenzolar-S-methyl was applied as a xenobiotic inducer in combination with quinic acid and shikimic acid as precursor molecules. Applying a semi-targeted metabolomics-based approach, time and concentration studies were undertaken to evaluate the effect of the manipulation on cellular metabolism leading to CGA production. Phytochemical extracts were prepared using methanol and analyzed using a UHPLC-qTOF-MS platform. Data was processed and analyzed using multivariate data models. A total of four CGA-derivatives, annotated as trans-5-feruloylquinic acid, 3,5 di-caffeoylquinic acid, 3,5-O-dicaffeoyl-4-O-malonylquinic acid (irbic acid) and 3-caffeoyl, 5-feruloylquinic acid, were found to be upregulated by the acibenzolar-S-methyl treatment. To the best of our knowledge, this is the first report on the induction of CGA derivatives in this species. Contrary to expectations, the effects of precursor molecules on the levels of the CGAs were insignificant. However, a total of 16 metabolites, including CGA derivatives, were up-regulated by precursor treatment. Therefore, this study shows potential to biotechnologically manipulate C. asiatica cells to increase the production of these health beneficial CGAs.

Genome-Wide Identification of BAHD Acyltransferases and In vivo Characterization of HQT-like Enzymes Involved in Caffeoylquinic Acid Synthesis in Globe Artichoke.

Globe artichoke (Cynara cardunculus L. var. scolymus) is a rich source of compounds promoting human health (phytonutrients), among them caffeoylquinic acids (CQAs), mainly represented by chlorogenic acid (CGA), and dicaffeoylquinic acids (diCQAs). The enzymes involved in their biosynthesis belong to the large family of BAHD acyltransferases. Following a survey of the globe artichoke genome, we identified 69 BAHD proteins carrying the catalytic site (HXXXD). Their phylogenetic analysis together with another 43 proteins, from 21 species, representative of the BAHD family, highlighted their grouping in seven major clades. Nine globe artichoke acyltransferases clustered in a sub-group of Clade V, with 3 belonging to hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) and 2 to hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) like proteins. We focused our attention on the former, HQT1, HQT2, and HQT3, as they are known to play a key role in CGA biosynthesis. The expression of genes coding for the three HQTs and correlation of expression with the CQA content is reported for different globe artichoke tissues. For the first time in the globe artichoke, we developed and applied the virus-induced gene silencing approach with the goal of assessing in vivo the effect of HQT1 silencing, which resulted in a marked reduction of both CGA and diCQAs. On the other hand, when the role of the three HQTs was assessed in leaves of Nicotiana benthamiana through their transient overexpression, significant increases in mono- and diCQAs content were observed. Using transient GFP fusion proteins expressed in N. benthamiana leaves we also established the sub-cellular localization of these three enzymes.

De novo transcriptome assembly and characterization of nine tissues of Lonicera japonica to identify potential candidate genes involved in chlorogenic acid, luteolosides, and secoiridoid biosynthesis pathways

Lonicera japonica is one of the most important medicinal plants with applications in traditional Chinese and Japanese medicine for thousands of years. Extensive studies on the constituents of L. japonica extracts have revealed an accumulation of pharmaceutically active metabolite classes, such as chlorogenic acid, luteolin and other flavonoids, and secoiridoids, which impart characteristic medicinal properties. Despite being a rich source of pharmaceutically active metabolites, little is known about the biosynthetic enzymes involved, and their expression profile across different tissues of L. japonica. In this study, we performed de novo transcriptome assembly for L. japonica, representing transcripts from nine different tissues. A total of 22 Gbps clean RNA-seq reads from nine tissues of L. japonica were used, resulting in 243,185 unigenes, with 99,938 unigenes annotated based on a homology search using blastx against the NCBI-nr protein database. Unsupervised principal component analysis and correlation studies using transcript expression data from all nine tissues of L. japonica showed relationships between tissues, explaining their association at different developmental stages. Homologs for all genes associated with chlorogenic acid, luteolin, and secoiridoid biosynthesis pathways were identified in the L. japonica transcriptome assembly. Expression of unigenes associated with chlorogenic acid was enriched in stems and leaf-2, unigenes from luteolin were enriched in stems and flowers, while unigenes from secoiridoid metabolic pathways were enriched in leaf-1 and shoot apex. Our results showed that different tissues of L. japonica are enriched with sets of unigenes associated with specific pharmaceutically important metabolic pathways and, therefore, possess unique medicinal properties. The present study will serve as a resource for future attempts for functional characterization of enzyme coding genes within key metabolic processes.

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.