PatWRKY71 transcription factor regulates patchoulol biosynthesis and plant defense response.

Patchoulol, a valuable compound belonging to the sesquiterpenoid family, is the primary component of patchouli oil produced by Pogostemon cablin (P. cablin). It has a variety of pharmacological and biological activities and is widely used in the medical and cosmetic industries. However, despite its significance, there is a lack of research on the transcriptional modulation of patchoulol biosynthesis.Salicylic acid (SA), is a vital plant hormone that serves as a critical signal molecule and plays an essential role in plant growth and defense. However, to date, no studies have explored the modulation of patchoulol biosynthesis by SA. In our study, we discovered that the application of SA can enhance the production of patchoulol. Utilizing transcriptome analysis of SA-treated P. cablin, we identified a crucial downstream transcription factor, PatWRKY71. The transcription level of PatWRKY71 was significantly increased with the use of SA. Furthermore, our research has revealed that PatWRKY71 was capable of binding to the promoter of PatPTS, ultimately leading to an increase in its expression. When PatWRKY71 was silenced by a virus, the expression of both PatWRKY71 and PatPTS was reduced, resulting in the down-regulation of patchoulol production. Through our studies, we discovered that heterologous expression of PatWRKY71 leads to an increase in the sensitivity of Arabidopsis to salt and Cd, as well as an outbreak of reactive oxygen species (ROS). Additionally, we uncovered the regulatory role of PatWRKY71 in both patchoulol biosynthesis and plant defense response. This discovery provided a theoretical basis for the improvement of the content of patchoulol and the resistance of P. cablin through genetic engineering.

Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viruses infecting patchouli (Pogostemon cablin).

Patchouli (Pogostemon cablin), a highly valued medicinal plant, suffers significant economic losses following infection with Broad bean wilt virus 2 (BBWV-2) and Peanut stripe virus (PStV). In this study, a field-based isothermal technique called reverse transcription loop-mediated isothermal amplification (RT-LAMP) was established for an early and specific detection of BBWV-2 and PStV. The oligo primers were designed to target the coat protein genes of PStV and BBWV-2. The reaction conditions, such as temperature and time duration, were optimized to 65 °C for 60 min. The LAMP amplicons positive for PStV and BBWV-2 revealed characteristic ladder-type bands following agarose gel electrophoresis. Further, a colorimetric assay using a metal ion-based indicator (Hydroxy-naphthol blue, HNB) was conducted to visualize the amplified products with the naked eye, thus facilitating accessibility to field practices. The assay developed in this study was found to be virus specific, and was 100 times more sensitive than RT-PCR. Thus, the RT-LAMP assay established in this study is quick, reliable, and cost-effective for the accurate identification of BBWV-2 and PStV. It will facilitate the screening of patchouli planting materials.  Further, it may reduce the risk of virus spread and could be helpful in phytosanitary programs.

Discovery and determination of misuse and chemotypes of Pogostemon cablin by liquid chromatography-quadrupole time-of-flight mass spectrometry and liquid chromatography with diode-array detector.

Traditional Chinese medicine (TCM) has garnered significant scientific interest in healthcare but faces increased regulatory scrutiny due to concerns about uncontrolled usage. This study focuses on characterizing Pogostemon cablin (PC) to mitigate potential misuse and identify chemotype differences. Leveraging untargeted metabolomics, we identified 222 distinctive features effectively differentiating PC from Agastache rugosa (AR), reducing misidentification risks. Pogostone and tilianin emerged as potential markers, leading to a high-performance liquid chromatography-diode array detection (HPLC-DAD) method development for PC and AR discrimination. Evaluation of PC chromatograms revealed notable profile and pogostone level differences among samples, suggesting chemotype associations. Untargeted metabolic profiling identified 78 features with significant differences, highlighting 7,3',4'-tri-O-methyleriodictyol as a potential discriminatory marker between PC chemotypes. The developed HPLC-DAD method quantified pogostone and 7,3',4'-tri-O-methyleriodictyol, effectively discriminating PC chemotypes. This platform differentiates PC and AR and distinguishes chemical types within PC, like pogostone-type and patchoulol-type. Applied to local TCM stores, it ensures PC authenticity. This approach addresses TCM control concerns, enhancing understanding and application of herbal medicine by providing insights into PC chemical composition and discrimination.

Global dissection of R2R3-MYB in Pogostemon cablin uncovers a species-specific R2R3-MYB clade.

MYB family is one of the largest transcription factor families in plants and plays a crucial role in regulating plant biochemical and physiological processes. However, R2R3-MYBs in patchouli have not been systematically investigated. Here, based on the gene annotation of patchouli genome sequence, 484 R2R3-MYB transcripts were detected. Further in-depth analysis of the gene structure and expression of R2R3-MYBs supported the tetraploid hybrid origin of patchouli. When combined with R2R3-MYBs from Arabidopsis, a phylogenetic tree of patchouli R2R3-MYBs was constructed and divided into 31 clades. Interestingly, a patchouli-specific R2R3-MYB clade was found and confirmed by homologous from other Lamiaceae species. The syntenic analysis demonstrated that tandem duplication contributed to its evolution. This study systematically analysed the R2R3-MYB family in patchouli, providing information on its gene characterization, functional prediction, and species evolution.

The Flow Rate of the Condenser Cooling Water in the Distillation Process Increases the Quality and Quantity of Patchouli Oil.

Indonesia is an important essential oil-exporting country globally, where 40 types of essential oils have been traded on the international market and are products of Indonesia. However, the quality and quantity of patchouli oil produced in Indonesia are still low. Most essential oil processing units use simple or traditional technology and generally have limited production capacity. This study aimed to obtain the optimum water flow rate in a condenser system for patchouli oil production in Maluku, Indonesia. Patchouli oil extraction from fresh patchouli leaves and twigs was carried out by increasing the condenser water discharge rate. Patchouli oil extraction with a condenser cooling water discharge treatment of 1.74 L/min and drying time for 5 days produced the highest patchouli oil yield of 1.4%. The greater the condenser water discharge rate, the better the yield and accumulation of patchouli oil recovery obtained. In addition, based on the results of the analysis of the composition of patchouli oil compounds with GCMS, it can be seen that 13 compounds can be detected in patchouli oil. The three main components of patchouli oil in all condenser cooling water treatments were alpha-guaiene, delta-guaiene, and patchouli alcohol. Considering the results of all parameters mentioned above, the treatment of the condenser cooling water discharge of 1.74 L/min and drying time for 5 days increases the quality and quantity of patchouli oil.

Effectiveness of repellent from patchouli (Pogestemon cablin) varieties of Southeast Sulawesi against Aedes aegypti.

BACKGROUND OBJECTIVES: The incidence of Dengue Hemorrhagic Fever (DHF) continues to increase over time in the world, including Indonesia. One of the prevention efforts against dengue virus transmission is to avoid vector mosquito bites by the use of repellants. Using repellents can reduce exposure to mosquito bites that may cause infection with the dengue virus. This study aimed to determine the effectiveness of repellent lotion composed of patchouli batik extract (Pogostemon cablin) from Southeast Sulawesi varieties against Aedes aegypti mosquitoes. METHODS: The research subjects were Aedes aegypti adult mosquitoes. The research consisted of three stages. The first stage was a phytochemical test (qualitative method), the second stage was the analysis of patchouli essential oil (GC-MS method) and the third stage was a test of the effectiveness of lotions made from patchouli extract in lotion preparations against Aedes aegypti. RESULTS: The results of the effectiveness test of patchouli leaf repellent (Pogestemon cablin) lotion preparations were as follows: for a concentration of 2.5%, protective power 81.5%; concentration 5%, protection power 83.67%; concentration 7.5%, protection power 88.64 %; the concentration of 10%, protection power 90.44%, and the concentration of 12.5% had protection power 90.89%. Probit analysis and linear regression showed the value of ProbitLc 50 was 6.631. INTERPRETATION CONCLUSION: The results of the effectiveness test of Pogestemon cablin repellent lotion preparations with the most effective concentrations were 10% and 12.5%. The results of ANOVA test indicated there was no difference in the average value of the protection power in patchouli leaves.

Pogostemon cablin (Blanco) Benth granule revealed a positive effect on improving intestinal barrier function and fecal microbiota in mice with irinotecan-induced intestinal mucositis.

Pogostemon cablin (Blanco) Benth (PCB), a medicinal and edible homologous Chinese herb, has a protective effect on the structure and function of intestine. In this study, we aimed to investigate the effect of PCB granule (PCBG) on the improvement of irinotecan-induced intestinal mucositis and the regulation of intestinal microorganisms in mice. Our results demonstrated that PCBG supplementation significantly improved diarrhea symptoms caused by irinotecan, as evidenced by inhibiting weight loss, reversing intestinal atrophy, protecting against splenomegaly and balancing oxidative stress. Furthermore, compared with the model group, PCBG restored the intestinal morphology and improved intestinal barrier dysfunction by promoting the expression of tight junction proteins and mucin. Moreover, high-throughput sequencing analysis revealed that PCBG improved the flora disorder caused by irinotecan and regulated microbial community structure, such as decreasing the relative abundance of Bacteroides as well as increasing the relative abundance of Lactobacillus. Meanwhile, the disordered microbial functions in intestinal mucositis mice were recovered more closely to the controls by PCBG. Finally, we found that a robust correlation between the specific microbiota and intestinal mucositis-related index. In summary, these findings revealed the beneficial effects of PCBG on the intestinal barrier and gut microbiota of irinotecan-induced intestinal mucositis, which may be one of the potential strategies to reduce the clinical side effects of irinotecan.

PcENO3 interacts with patchoulol synthase to positively affect the enzymatic activity and patchoulol biosynthesis in Pogostemon cablin.

Patchouli alcohol, a significant bioactive component of the herbal plant Pogostemon cablin, has considerable medicinal and commercial potential. Several genes and transcription factors involved in the biosynthesis pathway of patchouli alcohol have been identified. However, so far, regulatory factors directly interacting with patchouli synthase (PTS) have not been reported. This study was conducted to analyze the interaction between PcENO3 and PcPTS to explore the molecular regulation effect of PcENO3 on patchouli alcohol biosynthesis. PcENO3, a homologous protein of Arabidopsis ENO3 belonging to the enolase family, was identified and characterized. Subcellular localization experiments in Arabidopsis protoplast cells indicated that the PcENO3 protein was localized in both the cytoplasm and nucleus. The physical interaction between PcENO3 and PcPTS was confirmed through yeast two-hybrid (Y2H), GST pull-down, and bimolecular fluorescence complementation assays. Furthermore, the Y2H assay demonstrated that PcENO3 could also interact with JAZ proteins in the JA pathway. Enzymatic assays showed that the interaction with PcENO3 increased the catalytic activity of patchoulol synthase. Additionally, suppression of PcENO3 expression with VIGS (virus-induced gene silencing) decreased patchouli alcohol content compared to the control. These findings suggest that PcENO3 interacts with patchoulol synthase and modulates patchoulol biosynthesis by enhancing the enzymatic activity of PcPTS.

Whole-Genome Resequencing Reveals the Diversity of Patchouli Germplasm.

As an important medicinal and aromatic plant, patchouli is distributed throughout most of Asia. However, current research on patchouli's genetic diversity is limited and lacks genome-wide studies. Here, we have collected seven representative patchouli accessions from different localities and performed whole-genome resequencing on them. In total, 402,650 single nucleotide polymorphisms (SNPs) and 153,233 insertions/deletions (INDELs) were detected. Based on these abundant genetic variants, patchouli accessions were primarily classified into the Chinese group and the Southeast Asian group. However, the accession SP (Shipai) collected from China formed a distinct subgroup within the Southeast Asian group. As SP has been used as a genuine herb in traditional Chinese medicine, its unique molecular markers have been subsequently screened and verified. For 26,144 specific SNPs and 16,289 specific INDELs in SP, 10 of them were validated using Polymerase Chain Reaction (PCR) following three different approaches. Further, we analyzed the effects of total genetic variants on genes involved in the sesquiterpene synthesis pathway, which produce the primary phytochemical compounds found in patchouli. Eight genes were ultimately investigated and a gene encoding nerolidol synthetase (PatNES) was chosen and confirmed through biochemical assay. In accession YN, genetic variants in PatNES led to a loss of synthetase activity. Our results provide valuable information for understanding the diversity of patchouli germplasm resources.

A Comprehensive Review on Pharmacological Activities of Pachypodol: A Bioactive Compound of an Aromatic Medicinal Plant Pogostemon Cablin Benth.

As is well known, plant products have been increasingly utilized in the pharmaceutical industry in recent years. By combining conventional techniques and modern methodology, the future of phytomedicines appears promising. Pogostemon Cablin (patchouli) is an important herb used frequently in the fragrance industries and has various therapeutic benefits. Traditional medicine has long used the essential oil of patchouli (P. cablin) as a flavoring agent recognized by the FDA. This is a gold mine for battling pathogens in China and India. In recent years, this plant has seen a significant surge in use, and approximately 90% of the world's patchouli oil is produced by Indonesia. In traditional therapies, it is used for the treatment of colds, fever, vomiting, headaches, and stomachaches. Patchouli oil is used in curing many diseases and in aromatherapy to treat depression and stress, soothe nerves, regulate appetite, and enhance sexual attraction. More than 140 substances, including alcohols, terpenoids, flavonoids, organic acids, phytosterols, lignins, aldehydes, alkaloids, and glycosides, have been identified in P. cablin. Pachypodol (C18H16O7) is an important bioactive compound found in P. cablin. Pachypodol (C18H16O7) and many other biologically essential chemicals have been separated from the leaves of P. cablin and many other medicinally significant plants using repeated column chromatography on silica gel. Pachypodol's bioactive potential has been shown by a variety of assays and methodologies. It has been found to have a number of biological activities, including anti-inflammatory, antioxidant, anti-mutagenic, antimicrobial, antidepressant, anticancer, antiemetic, antiviral, and cytotoxic ones. The current study, which is based on the currently available scientific literature, intends to close the knowledge gap regarding the pharmacological effects of patchouli essential oil and pachypodol, a key bioactive molecule found in this plant.

Residual Change of Four Pesticides in the Processing of Pogostemon cablin and Associated Factors.

Before use as medicines, most traditional Chinese medicine (TCM) plants are processed and decocted. During processing, there may be some changes in pesticide residues in TCM. In recent years, reports have studied the changes of pesticides during the processes of boiling, drying and peeling of TCM materials but have rarely involved special processing methods for TCM, such as ethanol extraction and volatile oil extraction. The changes of carbendazim, carbofuran, pyridaben and tebuconazole residues in common processing methods for P. cablin products were systemically assessed in this study. After each processing step, the pesticides were quantitated by UPLC-MS/MS. The results showed amount decreases in various pesticides to different extents after each processing procedure. Processing factor (PF) values for the four pesticides after decoction, 75% ethanol extraction and volatile oil extraction were 0.02~0.75, 0.40~0.98 and 0~0.02, respectively, which indicated that residual pesticide concentrations may depend on the processing technique. A risk assessment according to the hazard quotient with PF values showed that residual pesticide amounts in P. cablin were substantially lower than levels potentially posing a health risk. Overall, these findings provide insights into the safety assessment of P. cablin.

Fabrication and Evaluation of Polyvinyl Alcohol/Corn Starch/Patchouli Oil Hydrogel Films Loaded with Silver Nanoparticles Biosynthesized in Pogostemon cablin Benth Leaves' Extract.

Research on the manufacture of hydrogel films from polyvinyl alcohol, corn starch, patchouli oil, and silver nanoparticles, (PVA/CS/PO/AgNPs, respectively) was completed. The silver nanoparticles used in this study resulted from green synthesis using local patchouli plants (Pogostemon cablin Benth). Aqueous patchouli leaf extract (APLE) and methanol patchouli leaf extract (MPLE) are used in the synthesis of phytochemicals (green synthesis), which are then blended in the production of PVA/CS/PO/AgNPs hydrogel films, which are then cross linked with glutaraldehyde. The results demonstrated that the hydrogel film was flexible, easy to fold, and free of holes and air bubbles. The presence of hydrogen bonds between the functional groups of PVA, CS, and PO was revealed using FTIR spectroscopy. SEM analysis revealed that the hydrogel film was slightly agglomerated and did not exhibit cracking or pinholes. The analysis of pH, spreadability, gel fraction, and swelling index showed that the resulting PVA/CS/PO/AgNP hydrogel films met expected standards except for the organoleptic properties of the resulting colors, which tended to be slightly darker in color. The formula with silver nanoparticles synthesized in methanolic of patchouli leaf extract (AgMENPs) had the highest thermal stability compared to hydrogel films with silver nanoparticles synthesized in aqueous of patchouli leaf extract (AgAENPs). The hydrogel films can be safely used up to 200 °C. The antibacterial studies revealed that the films inhibited the growth of both Staphylococcus aureus and Staphylococcus epidermis, as determined by the disc diffusion method, with the best antibacterial activity being against Staphylococcus aureus. In conclusion, the hydrogel film F1, loaded with silver nanoparticles biosynthesized in aqueous of patchouli leave extract (AgAENPs) and light fraction of patchouli oil (LFoPO) performed the best activity against both Staphylococcus aureus and Staphylococcus epidermis.

[Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae].

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Traditional Patchouli essential oil modulates the host's immune responses and gut microbiota and exhibits potent anti-cancer effects in ApcMin /+ mice.

Patchouli Essential Oil (PEO) has been used as a scent for various healing purposes since the ancient Egyptian period. The primary source of the oil is Pogostemon cablin (PC), a medicinal plant for treating gastrointestinal symptoms. However, the pharmacological function has not been addressed. Here, we report the cancer prevention and gut microbiota (GM) modulating property of PEO and its derivatives patchouli alcohol (PA) and pogostone (PO) in the ApcMin /+ colorectal cancer mice model. We found that PEO, PA, and PO significantly reduced the tumor burden. At the same time, it strengthened the epithelial barrier, evidenced by substantially increasing the number of the goblet and Paneth cells and upregulation of tight junction and adhesion molecules. In addition, PEO, PA, and PO shifted M1 to M2 macrophage phenotypes and remodeled the inflammatory milieu of ApcMin /+ mice. We also found suppression of CD4+CD25+ and stimulation CD4+ CD8+ cells in the spleen, blood, mesenteric lymph nodes (MLNs), and Peyer's patches (PPs) of the treated mice. The composition of the gut microbiome of the drug-treated mice was distinct from the control mice. The drugs stimulated the short-chain fatty acids (SCFAs)-producers and the key SCFA-sensing receptors (GPR41, GPR43, and GPR109a). The activation of SCFAs/GPSs also triggered the alterations of PPAR-γ, PYY, and HSDCs signaling mediators in the treated mice. Our work showed that PEO and its derivatives exert potent anti-cancer effects by modulating gut microbiota and improving the intestinal microenvironment of the ApcMmin /+ mice.

Complete genome sequence of patchouli chlorosis-associated cytorhabdovirus, a new cytorhabdovirus infecting patchouli plants in Brazil.

A cytorhabdovirus, tentatively named "patchouli chlorosis-associated cytorhabdovirus" (PCaCV), was identified in a patchouli plant, using high-throughput sequencing, and its genome sequence was confirmed by Sanger sequencing. The PCaCV genome consists of 12,913 nucleotides and contains six open reading frames in the order 3'-N-P'-P-P3-M-(G)-L-5'. The glycoprotein gene was found to contain stop codons in the coding frame; hence, this gene is considered defective. PCaCV is most closely related to tomato yellow mottle-associated virus, sharing 61.1% nucleotide sequence identity in the complete genome and 73.9% amino acid sequence identity in the L protein. These data suggest that PCaCV should be considered a new member of the genus Cytorhabdovirus, and the binomial species name "Cytorhabdovirus patchoulii" is proposed.

Lignans and Phenylethanoid Glycosides from the Aerial Parts of Pogostemon cablin.

Two new lignans (1-2) and a new octaketide (12), together with twenty-nine known compounds (3-11, 13-32) were isolated and identified from the aerial part of Pogostemon cablin. Their chemical structures were revealed mainly through NMR and MS data. The absolute configuration of 1 and 2 was deduced by comparing its experimental CD with the calculated ECD spectra. The inhibitory activities of the isolated compounds on LPS-stimulated nitric oxide (NO) production in RAW 264.7 cells were investigated. At a concentration of 25 µM, compounds 1 and 11 showed approximately equal NO inhibitory effects to that of aminoguanidine.

Characterization of Bioactive Compounds from Patchouli Extracted via Supercritical Carbon Dioxide (SC-CO2) Extraction.

Patchouli extracts and oils extracted from Pogostemon cablin are essential raw material for the perfume and cosmetics industries, in addition to being used as a natural additive for food flavoring. Steam distillation is a standard method used for plant extraction. However, this method causes thermal degradation of some essential components of the oil. In this study, patchouli was extracted with supercritical carbon dioxide (SC-CO2) under different conditions of pressure (10-30 MPa) and temperature (40-80 °C). The chemical components of the crude extracted oil and the functional group were characterized using gas chromatography-mass spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FT-IR). The extraction with supercritical carbon dioxide was shown to provide a higher yield (12.41%) at a pressure of 20 MPa and a temperature of 80 °C. Patchouli alcohol, Azulene, δ-Guaiene, and Seychellene are the main bioactive compounds that GC-MS results have identified. FTIR spectra showed alcohol, aldehyde, and aromatic ring bond stretching peaks. Extraction of patchouli with supercritical carbon dioxide provided a higher yield and a better quality of the crude patchouli oil.

[Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter].

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

Characterization of a Cytosolic Acyl-Activating Enzyme Catalyzing the Formation of 4-Methylvaleryl-CoA for Pogostone Biosynthesis in Pogostemon Cablin.

Pogostone, a compound with various pharmaceutical activities, is a major constituent of the essential oil preparation called Pogostemonis Herba, which is obtained from the plant Pogostemon cablin. The biosynthesis of pogostone has not been elucidated, but 4-methylvaleryl-CoA (4MVCoA) is a likely precursor. We analyzed the distribution of pogostone in P. cablin using gas chromatography-mass spectrometry (GC-MS) and found that pogostone accumulates at high levels in the main stems and leaves of young plants. A search for the acyl-activating enzyme (AAE) that catalyzes the formation of 4MVCoA from 4-methylvaleric acid was launched, using an RNAseq-based approach to identify 31 unigenes encoding putative AAEs including the PcAAE2, the transcript profile of which shows a strong positive correlation with the distribution pattern of pogostone. The protein encoded by PcAAE2 was biochemically characterized in vitro and shown to catalyze the formation of 4MVCoA from 4-methylvaleric acid. Phylogenetic analysis showed that PcAAE2 is closely related to other AAE proteins in P. cablin and other species that are localized to the peroxisomes. However, PcAAE2 lacks a peroxisome targeting sequence 1 (PTS1) and is localized in the cytosol.

Nematicidal and Molecular Docking Investigation of Essential Oils from Pogostemon cablin Ecotypes against Meloidogyne incognita.

Root-knot nematode, Meloidogyne incognita is one of the most destructive nematodes worldwide. Essential oils (EOs) are being extensively utilized as eco-benign bionematicides, although the precise mechanism of action remains unclear. Pogostemon cablin Benth. is well-known as "Patchouli". It is native to South East Asia and known for ethno-pharmacological properties. In this study, chemical composition and potential nematicidal effect of EOs hydrodistilled from the leaves of P. cablin grown at three different locations in India were comprehensively investigated to correlate their mechanism of action for target specific binding affinities toward nematode proteins. Aromatic volatile Pogostemon essential oils (PEO) from Northern India (PEO-NI), Southern India (PEO-SI) and North Eastern India (PEO-NEI) were analyzed by Gas Chromatography-Mass Spectrometry (GC/MS) to characterize forty volatile compounds. Maximum thirty-three components were identified in PEO-NEI. Sesquiterpenes were predominant with higher content of α-guaiene (2.3-24.4 %), patchoulol (6.1-32.7 %) and α-bulnesene (5.9-27.1 %). Patchoulol was the major component in PEO-SI (32.7±1.2 %) and PEO-NEI (29.2±1.1 %), while α-guaiene in PEO-NI (24.4±1.2 %). In vitro nematicidal assay revealed significant nematicidal action (LC50 44.6-87.0 µg mL-1 ) against juveniles of M. incognita within 24 h exposure. Mortality increases with increasing time to 48 h (LC50 33.6-71.6 µg mL-1 ) and 72 h (LC50 27.7-61.2 µg mL-1 ). Molecular modelling and in silico studies revealed multi-modal inhibitive action of α-bulnesene (-22 to -13 kJ mol-1 ) and α-guaiene (-22 to -12 kJ mol-1 ) against three target proteins namely, acetyl cholinesterase (AChE), odorant response gene-1 (ODR1), odorant response gene-3 (ODR3). Most preferable binding mechanism was observed against AChE due to pi-alkyl, pi-sigma, and hydrophobic interactions. Structure nematicidal activity relationship suggested the presence of hydroxy group for nematicidal activity is nonessential, rather highly depends on synergistic composition of sesquiterpene hydrocarbons.