Characterization of a CrPME indicates its possible role in determining vindoline accumulation in Catharanthus roseus leaves.

The leaf-specific Catharanthus roseus alkaloid, vindoline, is the major bottleneck precursor in the production of scarce and costly anticancer bisindoles (vincristine and vinblastine). The final steps of its biosynthesis and storage occur in the laticifers. Earlier, we have shown that vindoline content is directly related to laticifer number. Pectin remodeling enzymes, like pectin methylesterase (PME), are known to be involved in laticifer development. A search in the croFGD yielded a leaf-abundant CrPME isoform that co-expressed with a few vindoline biosynthetic genes. Full-length cloning, tissue-specific expression profiling, and in silico analysis of CrPME were carried out. It was found to possess all the specific characteristics of a typical plant PME. Transient silencing (through VIGS) and overexpression of CrPME in C. roseus indicated a direct relationship between its expression and vindoline content. Comparative analysis of transcript abundance and enzyme activity in three familial C. roseus genotypes differing significantly in their vindoline content and laticifer count (CIM-Sushil > Dhawal > Nirmal) also corroborated the positive relationship of CrPME expression with vindoline content. This study highlights the possible role of CrPME, a cell wall remodeling enzyme, in modulating laticifer-associated secondary metabolism.

Protective effect of Achyranthes aspera against compound 48/80, histamine and ovalbumin-induced allergic disorders in murine model.

BACKGROUND: Achyranthes aspera L. (family Amaranthaceae) is a plant species valued in Ayurveda for the treatment of respiratory ailments. Scientific validation of its antiallergic potential was aimed. METHODS AND RESULTS: Three extracts of A. aspera [aqueous (AaAq), hydroalcoholic (AaHA), ethanolic (AaEt)] were evaluated for their potency against C48/80-induced anaphylaxis in mice at 200 mg/kg BW oral dose. The effective dose of the most potent extract was determined through its effect on C48/80-induced anaphylaxis, and was further analyzed through its effect on mast cell degranulation, histamine-induced bronchospasm and ovalbumin (OVA)-induced asthma in a murine model. Among the three extracts, AaAq was found to be most potent at 200 mg/kg BW. AaAq 400 (400 mg/kg BW) was found to be the most effective dose in terms of inhibition of mortality and histamine level. AaAq 400 prevented the peritoneal and mesenteric mast cells from undergoing morphological changes due to degranulation induced by C48/80. Further, AaAq 400 delayed pre-convulsive time in histamine-induced bronchospasm. In the OVA-induced asthma model, AaAq 400 inhibited the level of inflammatory cell count in blood, bronchoalveolar lavage fluid and peritoneal fluid of mice. The Th2 cytokines (IL-4, IL-5, IL-13), TGF-ß and OVA-specific IgE were also reduced as evaluated by ELISA. Also, significant reduction in IL-5 (an eosinophilia indicator) transcript abundance and lung inflammatory score was observed. AaAq was safe up to 4000 mg/kg BW. CONCLUSIONS: Thus AaAq 400 possesses significant antiallergic potential and acts via attenuation of C48/80-induced anaphylaxis and inhibition of mast cell degranulation. It reduces pre-convulsive dyspnea in histamine-induced bronchospasm and Th2 cytokines in asthmatic mice.

Contribution of fossil and biomass-derived secondary organic carbon to winter water-soluble organic aerosols in Delhi, India.

Delhi, among the world's most polluted megacities, is a hotspot of particulate matter emissions, with high contribution from organic aerosol (OA), affecting health and climate in the entire northern India. While the primary organic aerosol (POA) sources can be effectively identified, an incomplete source apportionment of secondary organic aerosol (SOA) causes significant ambiguity in the management of air quality and the assessment of climate change. Present study uses positive matrix factorization analysis on the water-soluble organic aerosol (WSOA) data from the offline-aerosol mass spectrometry (AMS). It revealed POA as the dominant source of WSOA, with biomass-burning OA (31-34 %) and solid fuel combustion OA (∼21 %) being two major contributors. Here we use water-solubility fingerprints to track the SOA precursors, such as oxalates or organic nitrates, instead of identifying them based on their O:C ratio. Non-fossil precursors dominate in more oxidized oxygenated organic carbon (MO-OOC) (∼90 %), a proxy for aged secondary organic carbon (SOC), by coupling offline-AMS with 14C measurements. On the contrary, the oxidation of fossil fuel emissions produces a large quantity of fresh fossil SOC, which accounts for ∼75 % of less oxidized oxygenated organic carbon (LO-OOC). Our study reveals that apart from major POA contributions, large fractions of fossil (10-14 %) and biomass-derived SOA (23-30 %) contribute significantly to the total WSOA load, having impact on climate and air quality of the Delhi megacity. Our study reveals that large-scale unregulated biomass burning was not only found to dominate in POA but was also observed to be a significant contributor to SOA with implications on human health, highlighting the need for effective control strategies.

Temperature-induced lipocalin-mediated membrane integrity: Possible implications for vindoline accumulation in Catharanthus roseus leaves.

Plant lipocalins perform diverse functions. Recently, allene oxide cyclase, a lipocalin family member, has been shown to co-express with vindoline pathway genes in Catharanthus roseus under various biotic/abiotic stresses. This brought focus to another family member, a temperature-induced lipocalin (CrTIL), which was selected for full-length cloning, tissue-specific expression profiling, in silico characterization, and upstream genomic region analysis for cis-regulatory elements. Stress-mediated variations in CrTIL expression were reflected as disturbances in cell membrane integrity, assayed through measurement of electrolyte leakage and lipid peroxidation product, MDA, which implicated the role of CrTIL in maintaining cell membrane integrity. For ascertaining the function of CrTIL in maintaining membrane stability and elucidating the relationship between CrTIL expression and vindoline content, if any, a direct approach was adopted, whereby CrTIL was transiently silenced and overexpressed in C. roseus. CrTIL silencing and overexpression confirmed its role in the maintenance of membrane integrity and indicated an inverse relationship of its expression with vindoline content. GFP fusion-based subcellular localization indicated membrane localization of CrTIL, which was in agreement with its role in maintaining membrane integrity. Altogether, the role of CrTIL in maintaining membrane structure has possible implications for the intracellular sequestration, storage, and viability of vindoline.

Stress responsiveness of vindoline accumulation in Catharanthus roseus leaves is mediated through co-expression of allene oxide cyclase with pathway genes.

Vindoline is an important alkaloid produced in Catharanthus roseus leaves. It is the more important monomer of the scarce and costly anticancer bisindole alkaloids, vincristine, and vinblastine, as unlike catharanthine (the other monomer), its biosynthesis is restricted to the leaves. Here, biotic (bacterial endophyte, phytoplasma, virus) and abiotic (temperature, salinity, SA, MeJa) factors were studied for their effect on vindoline accumulation in C. roseus. Variations in vindoline pathway-related gene expression were reflected in changes in vindoline content. Since allene oxide cyclase (CrAOC) is involved in jasmonate biosynthesis and MeJa modulates many vindoline pathway genes, the correlation between CrAOC expression and vindoline content was studied. It was taken up for full-length cloning, tissue-specific expression profiling, in silico analyses, and upstream genomic region analysis for cis-regulatory elements. Co-expression analysis of CrAOC with vindoline metabolism-related genes under the influence of aforementioned abiotic/biotic factors indicated its stronger direct correlation with the tabersonine-to-vindoline genes (t16h, omt, t3o, t3r, nmt, d4h, dat) as compared to the pre-tabersonine genes (tdc, str, sgd). Its expression was inversely related to that of downstream-acting peroxidase (prx) (except under temperature stress). Direct/positive relationship of CrAOC expression with vindoline content established it as a key gene modulating vindoline accumulation in C. roseus.

Decalepis salicifolia (Bedd. ex Hook. f.) Venter: A steno-endemic and critically endangered medicinal and aromatic plant from Western Ghats, India.

Decalepis salicifolia (Bedd. ex Hook. f.) Venter is a potential medicinal and highly aromatic plant species confined to the southernmost part of the Western Ghats of India. The plant is well known for its traditional uses among the various tribal communities of south India. The tubers of the plant possess characteristic vanillin-like aroma due to the presence of the compound 2-hydroxy-4-methoxybenzaldehyde. The tubers are used to substitute Hemidesmus indicus in various herbal formulations. The plants in the wild are continuously uprooted for their roots, leading to the irreversible destruction of the whole plant. The resulting tremendous loss of populations in the wild led to the species being declared as critically endangered by IUCN. Our group is working on the various aspects of this species including population status, distribution mapping, prospection, and conservation management. In the present review, we have brought out the available information till date on D. salicifolia, including taxonomy, ethno-medicinal uses, phytochemistry, pharmacology, population status, and conservation efforts along with research gap and lacunae to provide direction for further research into this less explored medicinal and aromatic plant.

Adventitious root cultures of Decalepis salicifolia for the production of 2-hydroxy-4-methoxybenzaldehyde, a vanillin isomer flavor metabolite.

Decalepis salicifolia (Bedd. ex. Hook.f.) Venter is a potential natural source of the vanillin isomer, 2-hydroxy-4-methoxybenzaldehyde (2H4MB), an aromatic compound. However, the utilization of the plant is hindered especially due to its critically endangered status and the root-specific accumulation of the compound. The use of in vitro culture techniques offers a sustainable means for the production of valuable metabolites. In this study, an efficient system was established for the production of 2H4MB in the adventitious root cultures of D. salicifolia. Leaf explants of in vitro grown plants produced on an average 4.33 ± 2.07 number of roots with root initiation frequency of 95.69 ± 3.74% in woody plant medium supplemented with 0.5 mg/L α-naphthalene acetic acid (NAA) and 1.0 mg/L kinetin (Kn). The adventitious root biomass accumulation of 10.61 ± 0.89 g fresh weight (FW) was obtained in woody plant liquid media containing 0.5 mg/L NAA and 0.3 mg/L indole-3-butyric acid (IBA) in 60 days of inoculation. Field-grown plants of the same age produced 0.30 ± 0.02 g FW, which was 35-fold lower than the adventitious root culture. The total production of 2H4MB in the same growth period was 4.9-fold higher in adventitious root culture (139.54 µg) as compared to field-grown plants (28.62 µg). Furthermore, sucrose concentration of 2% was favorable for biomass accumulation, whereas 5% was favorable for 2H4MB production. On the other hand, media pH 5.0 was suitable for biomass production and pH 7.0 was best suited for accumulation of 2H4MB. The adventitious roots also showed stable production of biomass and 2H4MB over 2 years. The established adventitious root culture system is suitable for further large-scale production of 2H4MB for flavor and fragrance industrial applications. KEY POINTS: • Biomass accumulation was higher in adventitious root cultures than in field-grown plants. • Manipulation of sucrose concentration and media pH led to increased 2H4MB production. • Adventitious roots showed stable biomass and 2H4MB production over 2 years.

lncRNADetector: a bioinformatics pipeline for long non-coding RNA identification and MAPslnc: a repository of medicinal and aromatic plant lncRNAs.

Long non-coding RNAs (lncRNAs) are an emerging class of non-coding RNAs and potent regulatory elements in the living cells. High throughput RNA sequencing analyses have generated a tremendous amount of transcript sequence data. A large proportion of these transcript sequences does not code for proteins and are known as non-coding RNAs. Among them, lncRNAs are a unique class of transcripts longer than 200 nucleotides with diverse biological functions and regulatory mechanisms. Recent emerging studies and next-generation sequencing technologies show a substantial amount of lncRNAs within the plant genome, which are yet to be identified. The computational identification of lncRNAs from these transcripts is a challenging task due to the involvement of a series of filtering steps. We have developed lncRNADetector, a bioinformatics pipeline for the identification of novel lncRNAs, especially from medicinal and aromatic plant (MAP) species. The lncRNADetector has been utilized to analyse and identify more than 88,459 lncRNAs from 21 species of MAPs. To provide a knowledge resource for the plant research community towards elucidating the diversity of biological roles of lncRNAs, the information generated about MAP lncRNAs (post-filtering steps) through lncRNADetector has been stored and organized in MAPslnc database (MAPslnc, https://lncrnapipe.cimap.res.in). The lncRNADetector web server and MAPslnc database have been developed in order to facilitate researchers for accurate identification of lncRNAs from the next-generation sequencing data of different organisms for downstream studies. To the best of our knowledge no such MAPslnc database is available till date.

Biochar amendment reduced the risk associated with metal uptake and improved metabolite content in medicinal herbs.

Contaminations of heavy metals such as lead (Pb) and cadmium (Cd) in medicinal plants (MPs) not only restrict their safe consumption due to health hazards but also lower their productivity. Biochar amendments in the soil are supposed to immobilize the toxic metals, improve the soil quality and agricultural productivity. However, the impact of biochar on growth attributes, metal accumulation, pharmacologically active compounds of MPs, and health risk is less explored. An experiment was performed on three medicinal plants (Bacopa monnieri (L.), Andrographis paniculata (Burmf.) Nees, and Withaniasomnifera (L.)) grown in a greenhouse in soil co-contaminated with Pb and Cd (at two concentrations) without and with biochar amendments (2 and 4% application rates). The fractionation of Pb and Cd, plant growth parameters, stress enzymes, photosynthetic capacity, pharmacologically active compounds, nutrient content, uptake and translocation of metals, antioxidant activities, and metabolite content were examined in the three MPs. The accumulation of Pb and Cd varied from 3.25-228 mg kg1 and 1.29-20.2 mg kg-1 , respectively, in the three MPs, while it was reduced to 0.08-18 mg kg-1 and 0.03-6.05 mg kg-1 upon biochar treatments. Plants grown in Pb and Cd co-contaminated soil had reduced plant biomass (5-50% depending on the species) compared to control and a deleterious effect on photosynthetic attributes and protein content. However, biochar amendments significantly improved plant biomass (21-175%), as well as photosynthesis attributes, chlorophyll, and protein contents. Biochar amendments in Pb and Cd co-contaminated soil significantly reduced the health hazard quotient (HQ) estimated for the consumption of these medicinal herbs grown on metal-rich soil. An enhancement in secondary metabolite content and antioxidant properties was also observed upon biochar treatments. These multiple beneficial effects of biochar supplementation in Pb and Cd co-contaminated soil suggested that a biochar amendment is a sustainable approach for the safe cultivation of MPs. This article is protected by copyright. All rights reserved.

Arsenic-induced differential expression of oxidative stress and secondary metabolite content in two genotypes of Andrographis paniculata.

The present study explores the differential responses of two genotypes (APwC: wild collection and APMS: mass selection line) of A. paniculata against the three application rates of arsenic (42, 126, and 200 mg kg-1). The oxidative enzymes, As accumulation in different tissues, plant growth, and content of pharmacologically important ent-labdane-related diterpenes (ent-LRDs) of the two genotypes were evaluated in the study. Results demonstrated that As uptake significantly reduced plant biomass in APwC and APMS by 5-41.5% and 9-33% in a dose-response manner, respectively. The APMS exhibited lower bioconcentration and translocation factors, higher As tolerance index, and higher content of ent-LRDs as compared to APWC. As treatment induced a decrease in the sum of four metabolite content of APMS (1.43 times) and an increase in that of APWC (1.12 times) as compared to control. Likewise, variance in the production of 5,7,2',3'-tetramethoxyflavanone, and stress enzymes was also observed between APwC and APMS. The increase in the expression of ApCPS2 suggested its involvement in channeling of metabolic flux towards the biosynthesis of ent-LRDs under As stress.

Author Correction: Character-based DNA barcoding for authentication and conservation of IUCN Red listed threatened species of genus Decalepis (Apocynaceae).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Population Genetics Coupled Chemical Profiling for Conservation Implications of Decalepis salicifolia (Bedd. ex Hook.f.) Venter, an Endemic and Critically Endangered Species of Western Ghats, India.

Information on the genetic diversity and population structure is essential for developing conservational management programs, especially for threatened species. Decalepis salicifolia (Bedd. ex Hook.f.) Venter is a steno-endemic and critically endangered species of the south Western Ghats of India. The present study used ISSR markers as well as essential oil profiling to reveal the extent and distribution of genetic as well as the chemical diversity of all the twelve known populations of D. salicifolia. A total of 84 amplicons generated using 17 ISSR primers represented an overall 72.34% polymorphism. The highest percentage of polymorphic loci was recorded in the population of Theemalai (40.48%) and lowest in Kokanmalai (4.76%) with an average of 20.04% across all the studied populations. At the species level, the Nei's genetic diversity observed was 0.255 ± 0.186, while Shannon's information index observed was 0.385 ± 0.260. The genetic similarity-based unweighted pair-group method with arithmetic average dendrogram grouped the populations according to their geographic locations, which was corroborated by principal component analysis and Bayesian clustering. Distribution of genetic variance through analysis of molecular variance indicated that 38% variance resides within the population, and 62% variance resides among the populations (P < 0.001). Gas chromatography analyses of root volatiles showed significant variation in the percent content of 2-hydroxy-4-methoxybenzaldehyde. The Mantel test analyses showed a positive correlation between the genetic versus geographic distances. Based on the results, both ex situ and in situ conservation strategies are suggested to maximally preserve the genetic resources of this endangered species.

Characterization of a class III peroxidase from Artemisia annua: relevance to artemisinin metabolism and beyond.

KEY MESSAGE: A class III peroxidase from Artemisia annua has been shown to indicate the possibility of cellular localization-based role diversity, which may have implications in artemisinin catabolism as well as lignification. Artemisia annua derives its importance from the antimalarial artemisinin. The -O-O- linkage in artemisinin makes peroxidases relevant to its metabolism. Earlier, we identified three peroxidase-coding genes from A. annua, whereby Aa547 showed higher expression in the low-artemisinin plant stage whereas Aa528 and Aa540 showed higher expression in the artemisinin-rich plant stage. Here we carried out tertiary structure homology modelling of the peroxidases for docking studies. Maximum binding affinity for artemisinin was shown by Aa547. Further, Aa547 showed greater binding affinity for post-artemisinin metabolite, deoxyartemisinin, as compared to pre-artemisinin metabolites (dihydroartemisinic hydroperoxide, artemisinic acid, dihydroartemisinic acid). It also showed significant binding affinity for the monolignol, coniferyl alcohol. Moreover, Aa547 expression was related inversely to artemisinin content and directly to total lignin content as indicated by its transient silencing and overexpression in A. annua. Artemisinin reduction assay also indicated inverse relationship between Aa547 expression and artemisinin content. Subcellular localization using GFP fusion suggested that Aa547 is peroxisomal. Nevertheless, dual localization (intracellular/extracellular) of Aa547 could not be ruled out due to its effect on both, artemisinin and lignin. Taken together, this indicates possibility of localization-based role diversity for Aa547, which may have implications in artemisinin catabolism as well as lignification in A. annua.

Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM) Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs.

Senna alexandrina (Fabaceae) is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes (rbcL and matK) and intergenic spacers (psbA-trnH and ITS) were developed for S. alexandrina along with the allied species. The ability and performance of the ITS1 region to discriminate among the Senna species resulted in the present proposal of the ITS1 tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM) curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM) assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of Senna species. The melting profiles of S. alexandrina and S. italica subsp. micrantha were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs)]. HSA01 (S. alexandrina crude drug sample from Bangalore) and HSA06 (S. alexandrina crude drug sample from Tuticorin, Tamil Nadu, India) were found to be highly contaminated with S. italica subsp. micrantha. Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate Senna herbal products in the market for quality control in the drug supply chain. CIMAP Communication Number: CIMAP/PUB/2017/31.

ISSR-Derived Species-Specific SCAR Marker for Rapid and Accurate Authentication of Ocimum tenuiflorum L.

Ocimum tenuiflorum has been widely used in traditional medicine and has high medicinal value. High volume trade of this potential medicinal plant species led to unscrupulous adulteration of both crude drugs as well as formulations. Morphology-based authentication is difficult in cases of incomplete or damaged samples and in dried herbal materials. In such cases, PCR-based molecular methods may aid in accurate identification. The present study aimed at developing species-specific DNA marker(s) for the authentication of O. tenuiflorum. A species-specific amplicon (279 bp) generated through an inter-simple sequence repeat marker (UBC 835) in all individuals of O. tenuiflorum was cloned, sequenced, and a primer pair was developed (designated as CIM-OT-835F/CIM-OT-835R). The newly developed sequence characterized amplified region marker was validated through PCR amplification in all available seven species of Ocimum, and its specificity for O. tenuiflorum was confirmed with the consistent generation of an amplicon of 177 bp. The developed marker can be used for accurate and rapid identification of the species for certification purposes and will be useful in quality control of medicinal preparations containing this important medicinal species.

Character-based DNA barcoding for authentication and conservation of IUCN Red listed threatened species of genus Decalepis (Apocynaceae).

The steno-endemic species of genus Decalepis are highly threatened by destructive wild harvesting. The medicinally important fleshy tuberous roots of Decalepis hamiltonii are traded as substitute, to meet the international market demand of Hemidesmus indicus. In addition, the tuberous roots of all three species of Decalepis possess similar exudates and texture, which challenges the ability of conventional techniques alone to perform accurate species authentication. This study was undertaken to generate DNA barcodes that could be utilized in monitoring and curtailing the illegal trade of these endangered species. The DNA barcode reference library was developed in BOLD database platform for candidate barcodes rbcL, matK, psbA-trnH, ITS and ITS2. The average intra-specific variations (0-0.27%) were less than the distance to nearest neighbour (0.4-11.67%) with matK and ITS. Anchoring the coding region rbcL in multigene tiered approach, the combination rbcL + matK + ITS yielded 100% species resolution, using the least number of loci combinations either with PAUP or BLOG methods to support a character-based approach. Species-specific SNP position (230 bp) in the matK region that is characteristic of D. hamiltonii could be used to design specific assays, enhancing its applicability for direct use in CITES enforcement for distinguishing it from H. indicus.

Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia.

DNA barcoding is used as a universal tool for delimiting species boundaries in taxonomically challenging groups, with different plastid and nuclear regions (rbcL, matK, ITS and psbA-trnH) being recommended as primary DNA barcodes for plants. We evaluated the feasibility of using these regions in the species-rich genus Terminalia, which exhibits various overlapping morphotypes with pantropical distribution, owing to its complex taxonomy. Terminalia bellerica and T. chebula are ingredients of the famous Ayurvedic Rasayana formulation Triphala, used for detoxification and rejuvenation. High demand for extracted phytochemicals as well as the high trade value of several species renders mandatory the need for the correct identification of traded plant material. Three different analytical methods with single and multilocus barcoding regions were tested to develop a DNA barcode reference library from 222 individuals representing 41 Terminalia species. All the single barcodes tested had a lower discriminatory power than the multilocus regions, and the combination of matK+ITS had the highest resolution rate (94.44%). The average intra-specific variations (0.0188±0.0019) were less than the distance to the nearest neighbour (0.106±0.009) with matK and ITS. Distance-based Neighbour Joining analysis outperformed the character-based Maximum Parsimony method in the identification of traded species such as T. arjuna, T. chebula and T. tomentosa, which are prone to adulteration. rbcL was shown to be a highly conservative region with only 3.45% variability between all of the sequences. The recommended barcode combination, rbcL+matK, failed to perform in the genus Terminalia. Considering the complexity of resolution observed with single regions, the present study proposes the combination of matK+ITS as the most successful barcode in Terminalia.

Feasibility of nuclear ribosomal region ITS1 over ITS2 in barcoding taxonomically challenging genera of subtribe Cassiinae (Fabaceae).

PREMISE OF THE STUDY: The internal transcribed spacer (ITS) region is situated between 18S and 26S in a polycistronic rRNA precursor transcript. It had been proved to be the most commonly sequenced region across plant species to resolve phylogenetic relationships ranging from shallow to deep taxonomic levels. Despite several taxonomical revisions in Cassiinae, a stable phylogeny remains elusive at the molecular level, particularly concerning the delineation of species in the genera Cassia, Senna and Chamaecrista. This study addresses the comparative potential of ITS datasets (ITS1, ITS2 and concatenated) in resolving the underlying morphological disparity in the highly complex genera, to assess their discriminatory power as potential barcode candidates in Cassiinae. METHODOLOGY: A combination of experimental data and an in-silico approach based on threshold genetic distances, sequence similarity based and hierarchical tree-based methods was performed to decipher the discriminating power of ITS datasets on 18 different species of Cassiinae complex. Lab-generated sequences were compared against those available in the GenBank using BLAST and were aligned through MUSCLE 3.8.31 and analysed in PAUP 4.0 and BEAST1.8 using parsimony ratchet, maximum likelihood and Bayesian inference (BI) methods of gene and species tree reconciliation with bootstrapping. DNA barcoding gap was realized based on the Kimura two-parameter distance model (K2P) in TaxonDNA and MEGA. PRINCIPAL FINDINGS: Based on the K2P distance, significant divergences between the inter- and intra-specific genetic distances were observed, while the presence of a DNA barcoding gap was obvious. The ITS1 region efficiently identified 81.63% and 90% of species using TaxonDNA and BI methods, respectively. The PWG-distance method based on simple pairwise matching indicated the significance of ITS1 whereby highest number of variable (210) and informative sites (206) were obtained. The BI tree-based methods outperformed the similarity-based methods producing well-resolved phylogenetic trees with many nodes well supported by bootstrap analyses. CONCLUSION: The reticulated phylogenetic hypothesis using the ITS1 region mainly supported the relationship between the species of Cassiinae established by traditional morphological methods. The ITS1 region showed a higher discrimination power and desirable characteristics as compared to ITS2 and ITS1 + 2, thereby concluding to be the locus of choice. Considering the complexity of the group and the underlying biological ambiguities, the results presented here are encouraging for developing DNA barcoding as a useful tool for resolving taxonomical challenges in corroboration with morphological framework.

Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis.

Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229-403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant.

Repurposing L-Menthol for Systems Medicine and Cancer Therapeutics? L-Menthol Induces Apoptosis through Caspase 10 and by Suppressing HSP90.

The objective of the present study was to repurpose L-menthol, which is frequently used in oral health and topical formulations, for cancer therapeutics. In this article, we argue that monoterpenes such as L-menthol might offer veritable potentials in systems medicine, for example, as cheaper anti-cancer compounds. Other monoterpenes such as limonene, perillyl alcohol, and geraniol have been shown to induce apoptosis in various cancer cell lines, but their mechanisms of action are yet to be completely elucidated. Earlier, we showed that L-menthol modulates tubulin polymerization and apoptosis to inhibit cancer cell proliferation. In the present report, we used an apoptosis-related gene microarray in conjunction with proteomics analyses, as well as in silico interpretations, to study gene expression modulation in human adenocarcinoma Caco-2 cell line in response to L-menthol treatment. The microarray analysis identified caspase 10 as the important initiator caspase, instead of caspase 8. The proteomics analyses showed downregulation of HSP90 protein (also corroborated by its low transcript abundance), which in turn indicated inhibition of AKT-mediated survival pathway, release of pro-apoptotic factor BAD from BAD and BCLxL complex, besides regulation of other factors related to apoptosis. Based on the combined microarray, proteomics, and in silico data, a signaling pathway for L-menthol-induced apoptosis is being presented for the first time here. These data and literature analysis have significant implications for "repurposing" L-menthol beyond oral medicine, and in understanding the mode of action of plant-derived monoterpenes towards development of cheaper anticancer drugs in future.