Ashutosh K. Shukla

DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market

The past couple of decades have witnessed global resurgence of herbal-based health care. As a result, the trade of raw drugs has surged globally. Accurate and fast scientific identification of the plant(s) is the key to success for the herbal drug industry. The conventional approach is to engage an expert taxonomist, who uses a mix of traditional and modern techniques for precise plant identification. However, for bulk identification at industrial scale, the process is protracted and time-consuming. DNA barcoding, on the other hand, offers an alternative and feasible taxonomic tool box for rapid and robust species identification. For the success of DNA barcode, the barcode loci must have sufficient information to differentiate unambiguously between closely related plant species and discover new cryptic species. For herbal plant identification, matK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA and 18S-rRNA have been used as successful DNA barcodes. Emerging advances in DNA barcoding coupled with next-generation sequencing and high-resolution melting curve analysis have paved the way for successful species-level resolution recovered from finished herbal products. Further, development of multilocus strategy and its application has provided new vistas to the DNA barcode-based plant identification for herbal drug industry. For successful and acceptable identification of herbal ingredients and a holistic quality control of the drug, DNA barcoding needs to work harmoniously with other components of the systems biology approach. We suggest that for effectively resolving authentication challenges associated with the herbal market, DNA barcoding must be used in conjunction with metabolomics along with need-based transcriptomics and proteomics.

Differentially Expressed Genes during Contrasting Growth Stages of Artemisia annua for Artemisinin Content

Artemisia annua is the source of antimalarial phytomolecule, artemisinin. It is mainly produced and stored in the glandular secretory trichomes present in the leaves of the plant. Since, the artemisinin biosynthesis steps are yet to be worked out, in this investigation a microarray chip was strategized for the first time to shortlist the differentially expressing genes at a stage of plant producing highest artemisinin compared to the stage with no artemisinin. As the target of this study was to analyze differential gene expression associated with contrasting artemisinin content in planta and a genotype having zero/negligible artemisinin content was unavailable, it was decided to compare different stages of the same genotype with contrasting artemisinin content (seedling - negligible artemisinin, mature leaf - high artemisinin). The SCAR-marked artemisinin-rich (∼1.2%) Indian variety ‘CIM-Arogya’ was used in the present study to determine optimal plant stage and leaf ontogenic level for artemisinin content. A representative EST dataset from leaf trichome at the stage of maximal artemisinin biosynthesis was established. The high utility small scale custom microarray chip of A. annua containing all the significant artemisinin biosynthesis-related genes, the established EST dataset, gene sequences isolated in-house and strategically selected candidates from the A. annua Unigene database (NCBI) was employed to compare the gene expression profiles of two stages. The expression data was validated through semiquantitative and quantitative RT-PCR followed by putative annotations through bioinformatics-based approaches. Many candidates having probable role in artemisinin metabolism were identified and described with scope for further functional characterization.

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.

Precursor feeding studies and molecular characterization of geraniol synthase establish the limiting role of geraniol in monoterpene indole alkaloid biosynthesis in Catharanthus roseus leaves

The monoterpene indole alkaloids (MIAs) are generally derived from strictosidine, which is formed by condensation of the terpene moiety secologanin and the indole moiety tryptamine. There are conflicting reports on the limitation of either terpene or indole moiety in the production of MIAs in Catharanthus roseus cell cultures. Formation of geraniol by geraniol synthase (GES) is the first step in secologanin biosynthesis. In this study, feeding of C. roseus leaves with geraniol, but not tryptophan (precursor for tryptamine), increased the accumulation of the MIAs catharanthine and vindoline, indicating the limitation of geraniol in MIA biosynthesis. This was further validated by molecular and in planta characterization of C. roseus GES (CrGES). CrGES transcripts exhibited leaf and shoot specific expression and were induced by methyl jasmonate. Virus-induced gene silencing (VIGS) of CrGES significantly reduced the MIA content, which was restored to near-WT levels upon geraniol feeding. Moreover, over-expression of CrGES in C. roseus leaves increased MIA content. Further, CrGES exhibited correlation with MIA levels in leaves of different C. roseus cultivars and has significantly lower expression relative to other pathway genes. These results demonstrated that the transcriptional regulation of CrGES and thus, the in planta geraniol availability plays crucial role in MIA biosynthesis.

Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

Abstract High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.

Evaluation of Somaclonal Variation for Genetic Improvement of Patchouli (Pogostemon patchouli), an Exclusively Vegetatively Propagated Aromatic Plant

Patchouli (Pogostemon patchouli) is an important, exclusively vegetatively propagated aromatic plant, whose essential oil is widely used in perfumery and cosmetic products. Forty SC1 generation (first generation following in vitro phase) somaclones selected randomly from about 400 somaclones developed from the variety Johore, were multiplied through stem cuttings and evaluated in SC2 and SC3 generations to study the extent of somaclonal variation generated for plant height, herb yield, essential oil content, essential oil yield, and seven constituents of the essential oil. Significant or highly significant somaclonal variation was observed for plant height, herb yield, essential oil content, essential oil yield, and contents of patchouli alcohol, α-guaiene, α,δ-patchoulene, and α-bulnesene in the essential oil. The number of somaclones significantly superior to the parental variety for plant height, herb yield, essential oil content, and patchouli alcohol content in the essential oil ranged from 8–16 and the maximum superiority over the parental variety for these traits ranged from 21–79%. Broad-sense heritability estimates of plant height, herb yield, and essential oil content were 0.60–0.70 while those of essential oil yield and patchouli alcohol content were 0.44 and 0.47, respectively. Heritability estimates of other studied essential oil constituents were generally low (0.12–0.38). A high positive correlation was observed between essential oil yield and herb yield suggesting that selection for herb yield would be effective in improving essential oil yield. Patchouli alcohol content in the essential oil was negatively correlated with all the studied traits. Somaclonal variation, heritabilities of traits, and inter-trait correlations are reported for the first time in patchouli.

Proteomics indicates modulation of tubulin polymerization by L-menthol inhibiting human epithelial colorectal adenocarcinoma cell proliferation†

Menthol is a naturally occurring cyclic monoterpene used in oral hygiene products, confectionary, pharmaceuticals, cosmetics, pesticides, and as a flavoring agent. In the present study, we analyzed the differentially expressing proteome in L‐menthol‐treated Caco‐2 cell line as it was found to inhibit cell proliferation. Interestingly, free tubulin proteins were observed to be limited after menthol treatment. Semiquantitative RT‐PCR with α‐tubulin primers showed no change in the level of RNA expression in menthol‐treated cell line. However, tubulin polymerization assay with menthol indicated a trend similar to taxol in promoting microtubule assembly. Further, physical counting of apoptotic nuclei and active caspase‐3 assays confirmed onset of apoptosis though the rate was slower as compared with that of taxol treatment. This study is the first report of a monoterpene L‐menthol modulating tubulin polymerization and apoptosis to inhibit cancer cell proliferation.

Natural infection of periwinkle (Catharanthus roseus) with Cucumber mosaic virus, subgroup IB

This paper reports the characterisation of a naturally occurringCucumber mosaic virus (CMV) infection, causing mosaic, leaf distortion and stunting of periwinkle (Catharanthus roseus). The virus isolate showed wide host range, biophysical properties similar to CMV, isometric particles of 28 nm, capsid protein of 26 kDa, serological reaction with CMV-S and non-persistent transmission by aphids. The CP gene of the virus was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) with CP-specific primers, cloned and sequenced (657 bp). Sequence analysis of the PCR product with other CMV isolates revealed the closest identity withRauvolfia serpentina isolate of CMV (98%) and the phylogram revealed that CMV naturally infecting periwinkle belongs to subgroup IB.

Medicinal and Aromatic Plants

Many of the industrially and commercially used pharmaceuticals are products of secondary metabolism in microbial or plant systems. Out of the 350,000 plant species known so far, about 35,000 (some estimate up to 70,000) are used worldwide for medicinal purposes and less than about 0.5% of these have been chemically investigated. About 100 plant species are involved in 25% of all drugs prescribed in advanced countries (Comer and Debus 1996). The annual market value of herbal drugs used worldwide was estimated to be around US

A transcriptomic approach for exploring the molecular basis for dosha-balancing property-based classification of plants in Ayurveda

45 billion in the late 1990s and the value varies in different reports (US