Chlorogenic Acid Biosynthesis Research Articles

Integrated transcriptomic and CGAs analysis revealed IbGLK1 is a key transcription factor for chlorogenic acid accumulation in sweetpotato (Ipomoea batatas [L.] Lam.) blades

Sweetpotato blades are rich in the functional secondary metabolite chlorogenic acid (CGA), which deepen potential for effective utilization of the blade in industry. In this study, we evaluated the type and content of CGA in the blades of 16 sweetpotato genotypes and analyzed the correlation between CGA content and antioxidant capacity. Then we isolated and characterized IbGLK1, a GARP-type transcription factor, by comparative transcriptome analysis. A subcellular localization assay indicated that IbGLK1 is located in the nucleus. Overexpression and silencing of IbGLK1 in sweetpotato blade resulted in a 0.90-fold increase and 1.84-fold decrease, respectively, in CGA content compared to the control. Yeast one-hybrid and dual-luciferase assays showed that IbGLK1 binds and activates the promoters of IbHCT, IbHQT, IbC4H, and IbUGCT, resulting in the promotion of CGA biosynthesis. In conclusion, our study provides insights into a high-quality gene for the regulation of CGA metabolism and germplasm resources for breeding sweetpotato.

Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation.

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.

High-Level Biosynthesis of Chlorogenic Acid from Mixed Carbon Sources of Xylose and Glucose through a Rationally Refactored Pathway Network.

Chlorogenic acid (CGA) has incredible potential for various pharmaceutical, nutraceutical, and agricultural applications. However, the traditional extraction approach from plants is time-consuming, further limiting its production. Herein, we design and construct the de novo biosynthesis pathway of CGA using modular coculture engineering in Escherichia coli, which is composed of MG09 and BD07 strains. To accomplish this, the phenylalanine-deficient MG09 strain was engineered to utilize xylose preferentially and to overproduce precursor caffeic acid, while the tyrosine-deficient BD07 strain was constructed to consume glucose exclusively to enhance another precursor quinic acid availability for the biosynthesis of CGA. Further pathway modularization and balancing in the context of syntrophic cocultures resulted in additional production improvement. The coculture strategy avoids metabolic flux competition in the biosynthesis of two CGA precursors, caffeic acid and quinic acid, and allows for production improvement by balancing module proportions. Finally, the optimized coculture based on the aforementioned efforts produced 131.31 ± 7.89 mg/L CGA. Overall, the modular coculture engineering strategy in this study provides a reference for constructing microbial cell factories that can efficiently biomanufacture complex natural products.

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.

NtERF4 promotes the biosynthesis of chlorogenic acid and flavonoids by targeting PAL genes in Nicotiana tabacum.

Chlorogenic acid (CGA) and flavonoids are important secondary metabolites, which modulate plant growth and development, and contribute to plant resistance to various environmental stresses. ERF4 has been shown to be a repressor of anthocyanin accumulation in grape, but its full roles in regulating the biosynthesis of other phenylpropanoid compounds still needs to be further studied. In the present study, two NtERF4 genes were identified from N. tabacum genome. The expression level of NtERF4a was higher than that of NtERF4b in all the tobacco tissues examined. Over-expression of NtERF4a significantly promoted the accumulation of CGA and flavonoids in tobacco leaves, while silencing of NtERF4a significantly repressed the biosynthesis of CGA and flavonoids. RNA-seq analysis of NtERF4a-OE and WT plants revealed 8 phenylpropanoids-related differentially expressed genes (DEGs), including 4 NtPAL genes that encode key enzymes in the phenylpropanoid pathway. Activation of NtERF4a-GR fusion protein in tobacco significantly induced the transcription of NtPAL1 and NtPAL2 in the presence of protein synthesis inhibitor. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtERF4a could bind to the GCC box presented in the promoters of NtPAL1 and NtPAL2, thereby activating their transcription. Moreover, ectopic expression of NtERF4a induced the transcription of NtGSK1, NtMYC2, and NtJAZ3 genes, and enhanced the resistance of tobacco seedlings to salt and drought stresses, indicating multiple roles of NtERF4a in plants. Our findings revealed new roles of NtERF4a in modulating the accumulation of phenylpropanoid compounds in tobacco, and provided a putative target for improving phenylpropanoids synthesis and stress resistance in plants.

Efficient Production of Chlorogenic Acid in Escherichia coli Via Modular Pathway and Cofactor Engineering.

Chlorogenic acid is a natural phenolic compound widely used in the food and daily chemical industries. Compared to plant extraction, microbial cell factories provide a green and sustainable production method for the production of chlorogenic acid. However, complex metabolic flux distribution and potential byproducts limited the biosynthesis of chlorogenic acid in microorganisms. A de novo biosynthesis pathway for chlorogenic acid was constructed in Escherichia coli via modular engineering. Increasing the shikimate pathway flux greatly promoted chlorogenic acid production, and the influence of pyruvate metabolism on chlorogenic acid synthesis was also explored. The supply of cofactors for the key enzymes quinate/shikimate 5-dehydrogenase (YdiB) and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC) was enhanced by a cofactor regeneration system. Furthermore, mutants of YdiB were verified for chlorogenic acid production in vivo. Chlorogenic acid browning occurred when the buffer pH of the buffer exceeded 6.0, but two-stage pH control achieved a chlorogenic acid titer of 2789.2 mg/L in a 5 L fermenter, the highest reported to date. This study provided a strategy for the efficient production of chlorogenic acid from simple carbon sources.

Meta-Topolin-induced mass shoot multiplication and biosynthesis of valuable secondary metabolites in Stevia rebaudiana Bertoni bioreactor culture

Stevia rebaudiana Bertoni possesses various medicinal and food industrial applications. This study is the first to explore the effect of the cytokinins meta-Topolin (mT; 6-(3-hydroxybenzylamino) purine), zeatin, kinetin, and BAP (6-benzylaminopurine) at concentrations of 0 (control), 5, 10, and 15 µM on shoot multiplication, as well as stevioside, rebaudioside A, phenolic acid, and flavonoid content in bioreactor cultures. The highest number of shoots (23.4 per explant) was obtained in the medium containing 5 μM of mT. However, 15 μM of mT was superior for fresh biomass production and dry biomass accumulation. Reversed-phase (RP)-HPLC analysis showed a beneficial effect of 5 μM mT on stevioside (11.43 mg/g dry weight [DW]) and rebaudioside A (10.74 mg/g DW) biosynthesis. In all conditions, the ratio of rebaudioside A/stevioside ranged from 0.75 to 1.12. The phenolic acids chlorogenic, neochlorogenic, isochlorogenic A, and rosmarinic were confirmed in the stevia extracts, as were the flavonoids isoquercetin, and quercitrin. The highest accumulations of chlorogenic and neochlorogenic acids and flavonoids were observed in shoot tissues derived from 5 µM mT, whereas 5 µM of BAP stimulated biosynthesis of chlorogenic, isochlorogenic A, and rosmarinic acids. This is the first report on the use of mT-cytokinin showing high potential in stevia cultures.

Integrated metabolic profiling and transcriptome analysis of Lonicera japonica flowers for chlorogenic acid, luteolin and endogenous hormone syntheses

The active ingredients of many medicinal plants are the secondary metabolites associated with the growth period. Lonicera japonica Thunb. is an important traditional Chinese medicine, and the flower development stage is an important factor that influences the quality of medicinal ingredients. In this study, transcriptomics and metabolomics were performed to reveal the regulatory mechanism of secondary metabolites during flowering of L. japonica. The results showed that the content of chlorogenic acid (CGA) and luteolin gradually decreased from green bud stage (Sa) to white flower stage (Sc), especially from white flower bud stage (Sb) to Sc. Most of the genes encoding the crucial rate-limiting enzymes, including PAL, C4H, HCT, C3′H, F3′H and FNSII, were down-regulated in three comparisons. Correlation analysis identified some members of the MYB, AP2/ERF, bHLH and NAC transcription factor families that are closely related to CGA and luteolin biosynthesis. Furthermore, differentially expressed genes (DEGs) involved in hormone biosynthesis, signalling pathways and flowering process were analysed in three flower developmental stage.

Mycorrhizal colonization and Streptomyces viridosporus HH1 synergistically up-regulate the polyphenol biosynthesis genes in wheat against stripe rust

BackgroundStripe rust is considered one of the most devastating diseases of wheat all over the world, resulting in a high loss in its production. In this study, time-course changes in expression of the polyphenol biosynthesis pathways genes in wheat against stripe rust were investigated. The defense mechanisms triggered by mycorrhizal colonization and/or spraying with Streptomyces viridosporus HH1 against this disease were also investigated.ResultsResults obtained revealed that C3H, which is considered the key gene in lignin biosynthesis, was the most expressed gene. Furthermore, most of the chlorogenic acid and flavonoid biosynthesis genes were also overexpressed. Volcano plots of the studied genes reveal that the dual treatment led to a high significant overexpression of 10 out of the 13 studied genes. Heatmap of these genes showed that the most frequent expressed gene in response to all applied treatments along the study period was DFR, the key gene in the biosynthesis of anthocyanidins. Gene co-expression network of the studied genes showed that HQT was the most central gene with respect to the other genes, followed by AN2 and DFR, respectively. Accumulation of different flavonoids and phenolic acids were detected in response to the dual treatment, in particular, cinnamic acid, coumarin, and esculetin, which recorded the highest elevation level recording 1000, 488.23, and 329.5% respectively. Furthermore, results from the greenhouse experiment showed that application of the dual treatment led to an 82.8% reduction in the disease severity, compared with the control treatment.ConclusionsWe can conclude that the biosynthesis of lignin, chlorogenic acid, and flavonoids contributed to the synergistic triggering effect of the dual treatment on wheat resistance to stripe rust.

The transcription factor NtERF13a enhances abiotic stress tolerance and phenylpropanoid compounds biosynthesis in tobacco

The AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) transcription factors play multiple roles in modulating the biosynthesis of diverse specialized metabolites in response to various environmental stresses. ERF13 has been shown to participate in plant resistance to biotic stress as well as in repressing the synthesis of fatty acid. However, its full roles in regulating plant metabolism and stress resistance still remains to be further studied. In this study, we identified two NtERF genes from N. tabacum genome that belong to Ⅸa subgroup of ERF family. Over-expression and knock-out of NtERF13a showed that NtERF13a could enhance plant resistance to salt and drought stresses, as well as promoted the biosynthesis of chlorogenic acid (CGA), flavonoids, and lignin in tobacco. Transcriptome analysis between WT and NtERF13a-OE plants revealed 6 differentially expressed genes (DEGs) that encode enzymes catalyzing the key steps of phenylpropanoid pathway. Chromatin immunoprecipitation, Y1H, and Dual-Luc assays further clarified that NtERF13a could directly bind to the fragments containing GCC box or DRE element in the promoters of NtHCT, NtF3'H, and NtANS genes to induce the transcription of these genes. Knock-out of NtHCT, NtF3'H, or NtANS in the NtERF13a-OE background significantly repressed the increase of phenylpropanoid compound contents caused by over-expression of NtERF13a, indicating that the promotion of NtERF13a on the phenylpropanoid compound contents depends on the activity of NtHCT, NtF3'H, and NtANS. Our study demonstrated new roles of NtERF13a in promoting plant resistance to abiotic stresses, and provided a promising target for modulating the biosynthesis of phenylpropanoid compounds in tobacco.

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.

Cotton 4-coumarate-CoA ligase 3 enhanced plant resistance to Verticillium dahliae by promoting jasmonic acid signaling-mediated vascular lignification and metabolic flux.

Lignins and their antimicrobial-related polymers cooperatively enhance plant resistance to pathogens. Several isoforms of 4-coumarate-coenzyme A ligases (4CLs) have been identified as indispensable enzymes involved in lignin and flavonoid biosynthetic pathways. However, their roles in plant-pathogen interaction are still poorly understood. This study uncovers the role of Gh4CL3 in cotton resistance to the vascular pathogen Verticillium dahliae. The cotton 4CL3-CRISPR/Cas9 mutant (CR4cl) exhibited high susceptibility to V. dahliae. This susceptibility was most probably due to the reduction in the total lignin content and the biosynthesis of several phenolic metabolites, e.g., rutin, catechin, scopoletin glucoside, and chlorogenic acid, along with jasmonic acid (JA) attenuation. These changes were coupled with a significant reduction in 4CL activity toward p-coumaric acid substrate, and it is likely that recombinant Gh4CL3 could specifically catalyze p-coumaric acid to form p-coumaroyl-coenzyme A. Thus, overexpression of Gh4CL3 (OE4CL) showed increasing 4CL activity that augmented phenolic precursors, cinnamic, p-coumaric, and sinapic acids, channeling into lignin and flavonoid biosyntheses and enhanced resistance to V. dahliae. Besides, Gh4CL3 overexpression activated JA signaling that instantly stimulated lignin deposition and metabolic flux in response to pathogen, which all established an efficient plant defense response system, and inhibited V. dahliae mycelium growth. Our results propose that Gh4CL3 acts as a positive regulator for cotton resistance against V. dahliae by promoting JA signaling-mediated enhanced cell wall rigidity and metabolic flux.

Trait Mapping of Phenolic Acids in an Interspecific (Vaccinium corymbosum var. caesariense × V. darrowii) Diploid Blueberry Population.

Blueberries (Vaccinium sect. Cyanococcus) are a dietary source of phenolic acids, including chlorogenic acid (CGA) and related compounds such as acetylated caffeoylquinic acid (ACQA) and caffeoylarbutin (CA). These compounds are known to be potent antioxidants with potential health benefits. While the chemistry of these compounds has been extensively studied, the genetic analysis has lagged behind. Understanding the genetic basis for traits with potential health implications may be of great use in plant breeding. By characterizing genetic variation related to fruit chemistry, breeders can make more efficient use of plant diversity to develop new cultivars with higher concentrations of these potentially beneficial compounds. Using a large interspecific F1 population, developed from a cross between the temperate V. corymbosum var. ceasariense and the subtropical V. darrowii, with 1025 individuals genotyped using genotype-by-sequencing methods, of which 289 were phenotyped for phenolic acid content, with data collected across 2019 and 2020, we have identified loci associated with phenolic acid content. Loci for the compounds clustered on the proximal arm of Vc02, suggesting that a single gene or several closely associated genes are responsible for the biosynthesis of all four tested compounds. Within this region are multiple gene models similar to hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) and UDP glucose:cinnamate glucosyl transferase (UGCT), genes known to be involved in the CGA biosynthesis pathway. Additional loci on Vc07 and Vc12 were associated with caffeoylarbutin content, suggesting a more complicated biosynthesis of that compound.

Insights into chlorogenic acids' efficient biosynthesis through Carthamus tinctorius cell suspension cultures and their potential mechanism as α-glucosidase inhibitors

Carthamus tinctorius is commonly used as food and medicine because it contains various bioactive components, particularly chlorogenic acids (CGAs). However, few research works focused on the development and application of C. tinctorius-derived CGAs. In this study, the technology system for efficient CGAs biosynthesis through C. tinctorius cells was successfully developed, and the feasibility of C. tinctorius cells-derived CGAs as α-glucosidase inhibitors was evaluated. At first, the inflorescence explant was used to induce C. tinctorius embryogenic callus (induction rate: 95%), in which a total of 10 CGAs, including rare caffeoyl and feruloyl-substituted derivatives, were identified and quantified by liquid chromatography-mass spectrometry. Secondly, an excellent C. tinctorius cell suspension culture was established. To further promote CGAs biosynthesis, parameters of elicitation combined with precursor feeding were optimized through Plackett-Burman’s Design and Central Composite Design. Under the optimal condition [cinnamic acid: 283.05 μM, methyl jasmonate: 80.98 μM and yeast extract: 666.66 mg·L−1], CGAs content could reach up to 103.234 mg·g−1 (DW), which was 2.55 times that of the cells in the initial condition. The CGAs production first time reached the highest level among plant-cultured cells in the available literature. At last, the inhibition activity test results indicated good inhibitory activities of C. tinctorius cells-derived CGAs against α-glucosidase (IC50: 0.15–1.38 g·L−1), especially di- and tri-acyl CGAs. Moreover, molecular docking simulation showed that these CGAs could interact with the active site pocket of α-glucosidase to thereby achieve the inhibitory activity. Therefore, these results provided a solid foundation for the large-scale production of CGAs and revealed the application prospects of CGAs in functional food and pharmaceutical industries as α-glucosidase inhibitor.

Transcriptome Analysis Identifies Genes Associated with Chlorogenic Acid Biosynthesis during Apple Fruit Development

As a predominant phenolic compound in apple fruits, chlorogenic acid (CGA) benefits human health due to its various antioxidant properties. However, little has been known regarding the molecular mechanism underlying the CGA accumulation in apple fruits. In this study, we measured the CGA content and relative enzymes’ activities during fruit development in two different flesh-colored cultivars ‘Huashuo’ and ‘Red Love’. The CGA content in both cultivars decreased sharply from 30 days after full bloom (DAFB) to 60 DAFB. Notably, the CGA content in fruit flesh was relatively higher than that in the peel. Further, the activities of C3H and HCT enzymes downstream of the CGA biosynthesis showed the similar changing trend as CGA content. Based on the transcriptome data of ‘Huashuo’ fruit at 30 DAFB and 60 DAFB, 23 differentially expressed CGA synthesis-related genes were screened. Gene expression analyses further showed that MdHCT1/2/4/5/6 and MdC3H1/2/3 were positively correlated to the variation of CGA content in two cultivars. These findings establish a theoretical foundation for further mechanism study on CGA biosynthesis and provide guidance for nutrient improvement in apple breeding programs.

Effects of Preharvest Aminoethoxyvinylglycine (AVG) Treatment on Fruit Ripening, Core Browning and Related Gene Expression in ‘Huangguan’ Pear (Pyrus bretschneideri Rehd.)

‘Huangguan’ pear (Pyrus bretschneideri Rehd. cv. Huangguan) is a widely planted cultivar in China. However, it is susceptible to core browning after harvest. In this study, aminoethoxyvinylglycine (AVG) was applied at 200 mg L−1 one and two weeks prior to harvest, and its effects on fruit quality, ripening and core browning were investigated during fruit storage at ambient temperature (25 ± 1 °C). The results showed that there was higher firmness, soluble solids content (SSC) and titratable acid (TA) content, but a lower ethylene production rate and core browning index in AVG-treated fruit than in control (water). Compared with the control fruit, AVG treatment decreased the malondialdehyde (MDA) content and polyphenol oxidase (PPO) activity, delayed the peak of chlorogenic acid (CGA) content in the core tissue, and significantly inhibited the expression of genes such as ACC synthase (PbACS2, PbACS3a, PbACS5a and PbASC5b), ACC oxidase (PbACO1 and PbACO2), ethylene receptors (PbETR2 and PbERS1), ethylene response factor (PbERF1), phenylalanine ammonia lyase (PbPAL1), cinnamate 4-hydroxylase (PbC4H4), 4-hydroxycinnamoyl- CoA ligase (Pb4CL2), hydroxycinnamoyl- CoA shikimate hydroxycinnamoyl transferase (PbHCT1 and PbHCT3), and polyphenol oxidase (PbPPO1 and PbPPO5), as well as phospholipase D (PbPLD) and lipoxygenase (PbLOX1 and PbLOX5). Thus, these results suggested that the reduction in core browning by preharvest application of AVG might be due to an inhibitory effect on the expression of genes associated with ethylene biosynthesis and signaling pathways, CGA biosynthesis, PPO and cell membrane degradation in ‘Huangguan’ pear.

SMRT sequencing and characterization of putative hydroxycinnamoyl-CoA: Quinate/shikimate acid hydroxycinnamoyl transferase (HQT/HCT) genes responsible for biosynthesis of structurally diverse chlorogenic acids in Bidens pilosa

Bidens pilosa L. is an underutilised plant that serves as an ingredient for traditional herbal medicine and as food. This plant has received considerable attention because of its chemistry, which comprises of chemicals from various classes, with those from the phenylpropanoid pathway (e.g., chlorogenic acids) existing in abundance. B. pilosa L. produces chemically diverse chlorogenic acids (CGAs), characterised mainly by isomers thereof. The enzymes involved in the biosynthesis of chlorogenic acids have been decoded and characterised in many plants. However, genes that play a role in the biosynthesis of chlorogenic acids in B. pilosa L. are not yet described and, as such, the aim of the current study was to identify different isoforms of the Hydroxycinnamoyl-CoA: quinate/shikimate acid hydroxycinnamoyl transferase (HQT/HCT) genes that play a role in the diversification of chlorogenic acids in Bidens pilosa L. Herein, a robust gene sequencing technology through Single Molecule Real Time (SMRT) approach was applied to establish the gene sequences encoding transferases responsible for CGA production in B. pilosa L. Sequence homology of the established genes was evaluated by means of multiple sequence alignment against already published orthologous genes from closely related plants. Thereafter, phylogeny trees were constructed to graphically display the taxonomical relationship with other chlorogenic acid producing plants. To further demonstrate the functionality of these genes in plants, chlorogenic acid profiling was carried out using Liquid chromatography quadrupole time of flight mass spectrometer (LC-QTOF-MS). B. pilosa L. plants produced isomeric forms of mono, di and tri-acylated CGAs. A total of three HQT genes and one HCT gene have been identified in this study and have been shown through homology to be related with other related genes from other CGAs producing plants. Future work should involve expression of the identified genes in other non-chlorogenic acid producing plants to enhance their nutraceutical value.

Transcriptome profiling of two Moringa species and insights into their antihyperglycemic activity

BackgroundMoringa concanensis Nimmo (MC), a plant that resembles Moringa oleifera Lam. (MO), has less scientific information but has traditionally been used as a medicinal plant. Moringa species have long been known for their medicinal qualities, which include antioxidant, anti-inflammatory, anticancer, and antihyperglycemic effects. We investigated the antidiabetic potential of MC and MO species in this study by using transcriptome profiling, metabolite analysis, and in vitro assay studies.ResultsOur transcriptome analysis revealed the expression of enzymes involved in the biosynthesis of quercetin, chlorogenic acid, and benzylamine, all of which have previously been shown to have antidiabetic activity. We compared the expression patterns of five different tissues from MC and MO and it was found that the key enzymes involved in the biosynthesis of these compounds were highly expressed in leaf tissue. The expression estimated by MC transcriptome data in different tissues was verified using RT-qPCR analysis. The amount of these compounds was further quantified in the crude leaf extract of both species and found that MC had a higher abundance of quercetin and chlorogenic acid than MO. The crude leaf extract from both MC and MO were further tested in vitro, and the results demonstrated strong inhibitory activity for α-glucosidase and DPP-IV enzymes. Our findings suggest that compounds in leaf tissue, such as quercetin, benzylamine, and chlorogenic acid, could play a significant role in this antidiabetic activity. In addition, when comparing MO plants, we found that MC had a slightly higher effect in expression, abundance, and inhibitory activity.ConclusionsThis study presents the first report of MC transcriptome data, as well as a comparison of its anti-diabetic activity to MO. Our analysis discussed the significance of leaf tissue in antidiabetic activity compared to other tissues of both species. Overall, this study not only provides transcriptome resources for Moringa species, but also sheds light on antidiabetic potential of both species.

Identification and expression analysis of chlorogenic acid biosynthesis key gene PpHCT in peach

Shikimic acid/quinic acid hydroxy cinnamyl transferase (HCT) is one of the key enzymes in the phenylpropanoid pathway. However, the role of the HCT gene in chlorogenic acid (CGA) biosynthesis in peach fruit remains unclear. For this, we identified the accumulation pattern of CGA in four peach cultivars, cloned and characterized 11 PpHCT gene members, and further analyzed the expression patterns of these PpHCT genes during fruit development. The contents of CGAs in the four peach cultivars all exhibited a trend of increasing and then decreasing during the fruit growth and development. Moreover, the contents of CGAs in the peel and flesh were tissue-specific. Gene structure analysis indicated that the PpHCT genes were highly conserved, containing two exons and one intron. The protein structure analysis demonstrated that the PpHCT proteins contained two conserved motifs (HXXXD, DFGWG) and a transferase domain (PF02458), which belonged to the BAHD acyltransferase family. The cis-acting element analysis suggested that the promoters of PpHCT genes contained many light-related, hormone-related, stress-related, tissue-specific, and circadian-related elements, and they could participate in a variety of biological processes. Phylogenetic analysis showed that the HCT proteins of peach were closely related to the HCT proteins of plum and had a close evolutionary relationship. The qRT-PCR analysis indicated that the expression levels of PpHCT1 and PpHCT2 showed an opposite trend to the accumulation of CGA, whereas the expression levels of PpHCT4, PpHCT5, PpHCT7, PpHCT8, and PpHCT11 demonstrated the same trend as CGA accumulation. It was worth noting that only PpHCT4 and PpHCT5 were highly expressed in the two high-CGA cultivars but showed low levels of expression in the two low-CGA cultivars. Therefore, it was hypothesized that these two genes might be key genes to the synthesis of CGA in peach fruit. Those findings provide a theoretical basis for further study on the biological functions of the HCT gene and help to reveal the molecular mechanism of CGA.

BAHD acyltransferase induced by histone deacetylase inhibitor catalyzes 3-O-hydroxycinnamoylquinic acid formation in bamboo cells.

Suspension-cultured cells of a bamboo species (Bambusa multiplex; Bm) produce 3-O-feruloylquinic acid (3-FQA) and 3-O-p-coumaroylquinic acid (3-pCQA) by treatment with the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA). Acyltransferases catalyzing the formation of 5-O-hydroxycinnamoylquinic acid esters by transesterification from hydroxycinnamoyl-CoAs to the C-5 hydroxy group of quinic acid (hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase, HQT) have been identified in the biosynthesis of chlorogenic acids and monolignols; however, an HQT that catalyzes the acylation of the C-3 hydroxy group of quinic acid has not been identified previously. In the present study, we purified a native HQT from SBHA-treated Bm cells. The purified enzyme preferentially accepted feruloyl-/p-coumaroyl-CoAs as acyl-donors and quinic acid as the acyl-acceptor, and the enzyme specifically formed 3-FQA and 3-pCQA but not 5-O-hydroxycinnamoylquinic acid esters or esters with shikimic acid. A cDNA (BmHQT1) encoding this HQT was isolated. Although BmHQT1 is a phylogenetically unique member of the BAHD acyltransferase superfamily that does not cluster with other HQTs, functional characterization of the recombinant enzyme verified that BmHQT1 catalyzes the regiospecific formation of 3-O-hydroxycinnamoylquinic acid esters. Transcript levels of BmHQT1 markedly increased in Bm cells cultured in the presence of SBHA. Moreover, elevated acetylation levels of histone H3 were observed in the coding region of BmHQT1 in the presence of SBHA, indicating that the induced accumulation of 3-FQA/3-pCQA by SBHA is caused by transcriptional activation of BmHQT1 by the action of SBHA as a histone deacetylase inhibitor. The results demonstrate the utility of HDAC inhibitors for discovery of cryptic secondary metabolites and unknown biosynthetic enzymes.