Comparative temporal metabolomics studies to investigate interspecies variation in three Ocimum species.

Ocimum is one of the most revered medicinally useful plants which have various species. Each of the species is distinct in terms of metabolite composition as well as the medicinal property. Some basil types are used more often as an aromatic and flavoring ingredient. It would be informative to know relatedness among the species which though belong to the same genera while exclusively different in terms of metabolic composition and the operating pathways. In the present investigation the similar effort has been made in order to differentiate three commonly occurring Ocimum species having the high medicinal value, these are Ocimum sanctum, O. gratissimum and O. kilimandscharicum. The parameters for the comparative analysis of these three Ocimum species comprised of temporal changes in number leaf trichomes, essential oil composition, phenylpropanoid pathway genes expression and the activity of important enzymes. O. gratissimum was found to be richest in phenylpropanoid accumulation as well as their gene expression when compared to O. sanctum while O. kilimandscharicum was found to be accumulating terpenoid. In order to get an overview of this qualitative and quantitative regulation of terpenes and phenylpropenes, the expression pattern of some important transcription factors involved in secondary metabolism were also studied.

Ocimum metabolomics in response to abiotic stresses: Cold, flood, drought and salinity.

Ocimum tenuiflorum is a widely used medicinal plant since ancient times and still continues to be irreplaceable due to its properties. The plant has been explored chemically and pharmacologically, however, the molecular studies have been started lately. In an attempt to get a comprehensive overview of the abiotic stress response in O. tenuiflorum, de novo transcriptome sequencing of plant leaves under the cold, drought, flood and salinity stresses was carried out. A comparative differential gene expression (DGE) study was carried out between the common transcripts in each stress with respect to the control. KEGG pathway analysis and gene ontology (GO) enrichment studies exhibited several modifications in metabolic pathways as the result of four abiotic stresses. Besides this, a comparative metabolite profiling of stress and control samples was performed. Among the cold, drought, flood and salinity stresses, the plant was most susceptible to the cold stress. Severe treatments of all these abiotic stresses also decreased eugenol which is the main secondary metabolite present in the O. tenuiflorum plant. This investigation presents a comprehensive analysis of the abiotic stress effects in O. tenuiflorum. Current study provides an insight to the status of pathway genes' expression that help synthesizing economically valuable phenylpropanoids and terpenoids related to the adaptation of the plant. This study identified several putative abiotic stress tolerant genes which can be utilized to either breed stress tolerant O. tenuiflorum through pyramiding or generating transgenic plants.

RNAi of Sterol Methyl Transferase1 Reveals its Direct Role in Diverting Intermediates Towards Withanolide/Phytosterol Biosynthesis in Withania somnifera.

The medicinal properties of Ashwagandha (Withania somnifera) are accredited to a group of compounds called withanolides. 24-Methylene cholesterol is the intermediate for sterol biosynthesis and a proposed precursor of withanolide biogenesis. However, conversion of 24-methylene cholesterol to withaferin A and other withanolides has not yet been biochemically dissected. Hence, in an effort to fill this gap, an important gene, encoding S-adenosyl l-methionine-dependent sterol-C24-methyltransferase type 1 (SMT1), involved in the first committed step of sterol biosynthesis, from W. somnifera was targeted in the present study. Though SMT1 has been characterized in model plants such as Nicotiana tabacum and Arabidopsis thaliana, its functional role in phytosterol and withanolide biosynthesis was demonstrated for the first time in W. somnifera. Since SMT1 acts at many steps preceding the withanolide precursor, the impact of this gene in channeling of metabolites for withanolide biosynthesis and its regulatory nature was illustrated by suppressing the gene in W. somnifera via the RNA interference (RNAi) approach. Interestingly, down-regulation of SMT1 in W. somnifera led to reduced levels of campesterol, sitosterol and stigmasterol, with an increase of cholesterol content in the transgenic RNAi lines. In contrast, SMT1 overexpression in transgenic N. tabacum enhanced the level of all phytosterols except cholesterol, which was not affected. The results established that SMT1 plays a crucial role in W. somnifera withanolide biosynthesis predominantly through the campesterol and stigmasterol routes.

Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal.

The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

Molecular cloning, characterization and expression analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Centella asiatica L.

3-Hydroxy-3-methylglutaryl-CoA reductases (HMGR) plays an important role in catalyzing the first committed step of isoprenoid biosynthesis in the mevelonic (MVA) pathway (catalyzes the conversion of HMG-CoA to MVA) in plants. The present manuscript reports the full length cDNA cloning of HMGR (CaHMGR, GenBank accession number: KJ939450.2) and its characterization from Centella asiatica. Sequence analysis indicated that the cDNA was of 1965 bp, which had an open reading frame of 1617 bp and encoded a protein containing 539 amino-acids with a mol wt of 57.9 kDa. A BLASTp search against non-redundant (nr) protein sequence showed that C. asiatica HMGR (CaHMGR) has 65-81% identity with HMGRs from different plant species and multi-alignment comparison analysis showed the presence of two motif each corresponding to HMG-CoA-binding and NADP(H)-binding. The Conserved Domain Database analysis predicted that CaHMGR belongs to Class I hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase. Three-dimensional modeling confirmed the novelty of CaHMGR with a spatial structure similar to Homo sapiens (PDB id: 1IDQ8_A). Tissue Expression analysis indicates that CaHMGR is ubiquitous albeit differentially expressed among different tissues analysed, Strong expression was recorded in the nodes and leaves and low in the roots. The present investigation confirmed that nodes are vital to terpenoid synthesis in C. asiatica. Thus, the cloning of full length CDS, characterization and structure-function analysis of HMGR gene in Centella facilitate to understand the HMGR's functions and regulatory mechanisms involved in mevalonate pathway in C. asiatica at genetic level.

Unravelling the genome of Holy basil: an "incomparable" "elixir of life" of traditional Indian medicine.

BACKGROUND: Ocimum sanctum L. (O. tenuiflorum) family-Lamiaceae is an important component of Indian tradition of medicine as well as culture around the world, and hence is known as "Holy basil" in India. This plant is mentioned in the ancient texts of Ayurveda as an "elixir of life" (life saving) herb and worshipped for over 3000 years due to its healing properties. Although used in various ailments, validation of molecules for differential activities is yet to be fully analyzed, as about 80 % of the patents on this plant are on extracts or the plant parts, and mainly focussed on essential oil components. With a view to understand the full metabolic potential of this plant whole nuclear and chloroplast genomes were sequenced for the first time combining the sequence data from 4 libraries and three NGS platforms. RESULTS: The saturated draft assembly of the genome was about 386 Mb, along with the plastid genome of 142,245 bp, turning out to be the smallest in Lamiaceae. In addition to SSR markers, 136 proteins were identified as homologous to five important plant genomes. Pathway analysis indicated an abundance of phenylpropanoids in O. sanctum. Phylogenetic analysis for chloroplast proteome placed Salvia miltiorrhiza as the nearest neighbor. Comparison of the chemical compounds and genes availability in O. sanctum and S. miltiorrhiza indicated the potential for the discovery of new active molecules. CONCLUSION: The genome sequence and annotation of O. sanctum provides new insights into the function of genes and the medicinal nature of the metabolites synthesized in this plant. This information is highly beneficial for mining biosynthetic pathways for important metabolites in related species.

Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance.

Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (ß-amyrin synthase) and ß-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides formation in W. somnifera leaves.

De novo sequencing and comparative analysis of holy and sweet basil transcriptomes.

BACKGROUND: Ocimum L. of family Lamiaceae is a well known genus for its ethnobotanical, medicinal and aromatic properties, which are attributed to innumerable phenylpropanoid and terpenoid compounds produced by the plant. To enrich genomic resources for understanding various pathways, de novo transcriptome sequencing of two important species, O. sanctum and O. basilicum, was carried out by Illumina paired-end sequencing. RESULTS: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively. Out of the total transcripts, 59648 (86.30%) and 105470 (81.10%) from O. sanctum and O. basilicum, and respectively were annotated by uniprot blastx against Arabidopsis, rice and lamiaceae. KEGG analysis identified 501 and 952 transcripts from O. sanctum and O. basilicum, respectively, related to secondary metabolism with higher percentage of transcripts for biosynthesis of terpenoids in O. sanctum and phenylpropanoids in O. basilicum. Higher digital gene expression in O. basilicum was validated through qPCR and correlated to higher essential oil content and chromosome number (O. sanctum, 2n = 16; and O. basilicum, 2n = 48). Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism. Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes. CONCLUSION: This is the first report of a comparative transcriptome analysis of Ocimum species and can be utilized to characterize genes related to secondary metabolism, their regulation, and breeding special chemotypes with unique essential oil composition in Ocimum.

Enhancement of artemisinin content through four cycles of recurrent selection with relation to heritability, correlation and molecular marker in Artemisia annua L.

Due to the high demand and low yield of the anti-malarial drug artemisinin in natural populations of Artemisia annua (Quinghao), an attempt has been made to enhance the artemisinin content through 4 cycles of recurrent selection (C(0)-C(3)) using selected genotypic and phenotypic traits. Based on their phenotypic and genotypic characteristics, the top 5% plants of each cycle were selected, and their seedlings were planted in poly-cross block to produce seeds for the subsequent generation. A significant increase in the artemisinin content (0.15% in C (0) to 1.16% in C (3), i.e., about 40% genetic gain over the generation) was observed. This enhancement was directly correlated with the plant height and branching intensity in all four cycles. Similarly, the PCV (phenotypic coefficient of variation) and GCV (genotypic coefficient of variation) have been observed to have a higher value for artemisinin content. The DNA marker (MAP 12) with relation to artemisinin was also identified for high yielding genotypes in all four cycles of selection. Over the four cycles of recurrent selection, the plant developed an oval appearance (Variety: CIM-Arogya) and a high artemisinin content (1.16%).

SCAR markers for correct identification of Phyllanthus amarus, P. fraternus, P. debilis and P. urinaria used in scientific investigations and dry leaf bulk herb trade.

The trade in Phyllanthus material as bulk herb is rampant and mainly involves herbaceous species such as Phyllanthus amarus, P. fraternus, P . debilis and P. urinaria. These species are very important in herbal medicines and have varied activities. In India these species grow sympatrically and there are chances of deliberate or ignorant adulteration of crude drugs, lowering the efficiency of the medication for its intended purpose. Secondly, incorrect identification may also lead to erroneous reports on activities/molecules. To overcome this problem in crude drug (dry leaf powder) and compliment morphological identification in live plant, we have developed SCAR markers for all four species. In each species, we selected one fragment as being monomorphic between accessions but differing in size between species. These species-specific fragments were selected, cloned and sequenced. Based on the sequences, primer pairs were designed and amplification conditions standardized. SCAR markers were isolated from population DNA amplification profiles and validated by sequencing. The species-specific SCAR primers could retrieve the same size and sequence of fragments as in the RAPD profile. These fragments are 1150 bp, 317 bp, 980 bp and 550 bp in size for P. amarus, P. fraternus, P. debilis and P. urinaria, respectively. Additional fragments in P. debilis and P. urinaria indicate different alleles. The retrieval of same size and sequence of species-specific unique SCAR markers from the respective accessions (mixed DNA sample of same accessions) indicates the usefulness to study natural hybridization between the species in addition to adulteration.