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Key message: Comprehensive transcriptome analysis of leaf and root tissues of Nothapodytes nimmoniana unravels several putative pathway genes, transcription factors and CYPs related to camptothecin (CPT) biosynthesis. Additionally, post-transcriptional suppression by artificial microRNA (aMIR) of NnCYP76B6 (geraniol 10-hydroxylase) suggests its role in CPT biosynthesis. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Abstract: Nothapodytes nimmoniana is a rich source of potent anticancer drug camptothecin (CPT) whose biosynthetic pathway is unresolved due to the lack of genomic and transcriptomic information. Present investigation entails deep transcriptome analysis of N. nimmoniana which led to identification of putative pathway genes and regulatory components involved in CPT biosynthesis. Using Illumina HiSeq 2500 sequencing platform a total of 31,172,889 (6.23 Gb) and 31,218,626 (6.24 Gb) raw reads were generated from leaf and root wood, respectively. These were assembled de novo into 138,183 unique contigs. Additionally, 16 cytochrome P450 transcripts related to secondary metabolism were also identified. Further, transcriptome data pool presented 1683 putative transcription factors of which transcripts corresponding to WRKY TFs were the most abundant (14.14%). A total of 2741 transcripts were differentially expressed out of which 478 contigs showed downregulation in root wood and 2263 contigs were up-regulated. Further, comparative analyses of 17 genes involved in CPT biosynthetic pathway were validated by qRT-PCR. On basis of intermediates, two distinct seco-iridoid pathways are involved in the biosynthesis of monoterpene indole alkaloids either through multiple isomers of strictosidinic acid or strictosidine. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Geraniol-10 hydroxylase ( NnCYP76B6) an important enzyme in CPT biosynthesis which specifically shunts geraniol into the secologanin pathway was also cloned from the trancriptome resource. In planta transient expression of NnCYP76B6 showed a significant enhancement in mRNA transcript levels coincident with enhanced CPT accumulation. Further, artificial microRNA (aMIR) mediated downregulation of NnCYP76B6 resulted in reduction of mRNA transcript levels as well as CPT content in comparison to control. These empirical results suggest a plausible regulatory role for NnCYP76B6 in CPT biosynthesis and also establish a valuable repository for deciphering various structural, rate limiting and regulatory genes of CPT biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Rose-scented geranium ( Pelargonium graveolens L' Hért.) is a fragrant and bushy essential oil bearing crop of significant industrial and commercial importance. In an attempt to overcome the constraints imposed by sexual sterility for genetic improvement and restricted environmental adaptability of Pelargonium, the present study was aimed at exploring new niches for its cultivation in northern most sub-tropical part of India (Jammu). The flowering stage was found to be the optimum stage for harvesting in relation to essential oil quantity (0.16-0.18%, Fresh Weight Basis; FWB) and quality (geraniol 18% and citronellol 37%) during different phenophases of plant growth. Dynamics of essential oil turnover correlated well with the glandular trichome ontogeny using scanning electron microscopy (SEM). The present study also entailed induction of somaclonal variations via in vitro organogenesis. Organogenetic callus induction was achieved on B5 (Gamborg's) basal medium supplemented with kinetin/2,4-dichlorophenoxyacetic acid (Kn/2,4-D) and kinetin/indole-3-butyric acid (Kn/IBA). Frequency of callus induction ranged from 54-85%. Field established in vitro regenerated plants/somaclones displayed appreciable variability in fresh leaf biomass (365-2020 g, FWB), essential oil content (0.06%-0.26%) and quality (geraniol and citronellol as major constituents). To evaluate the genetic instability of calliclones, RAPD (Random Amplification of Polymorphic DNA) markers were used to assess the genetic basis of variability. Further, the identity of Pelargonium was confirmed by DNA bar-code analyses using three plant based barcoding loci (ITS, rbcL and psbA/trnH). They may be used as a standard reference system for varietal identification and protection of Intellectual Property Rights (IPR). Overall, the present investigation demonstrates the feasibility of Pelargonium cultivation in sub-tropical niches as a short duration winter crop, with desirable essential oil content and composition for commercial purposes. It also establishes a viable method for induction of variability for genetic amelioration of sexually sterile crop. [ABSTRACT FROM AUTHOR]

Withania somnifera (L.) Dunal synthesizes large array of pharmacologically active secondary metabolites known as withanolides. It has been extensively investigated in terms of chemistry and bioactivity profiling. However, there exists fragmentary information about the dynamics of withanolide biosynthesis at different phenophases in concert with the expression analysis of key pathway genes. In the present study, two morpho-chemovariants of W. somnifera were harvested at five developmental stages, dissected into leaf and root tissues and assayed for three major withanolides viz. withanolide-A (WS-1), withanone (WS-2) and withaferin A (WS-3) content using high performance liquid chromatography. The present investigation also analyzed the expression pattern of five withanolide biosynthetic pathway genes namely squalene synthase, squalene epoxidase, cycloartenol synthase, cytochrome P450 reductase 1, cytochrome P450 reductase 2 to corroborate with the metabolite flux at different developmental stages. The relative transcript profiles of identified genes at various ontogenetic stages illustrated significant variation in leaf and root tissues and were largely concurrent with the alteration in withanolide pool. Comparatively, the concentrations of withanolide A, withanone and withaferin A along with expression levels of all the five genes were appreciably higher in the leaves than in roots. Relative dynamics in terms of quantitative and qualitative profiles of withanolides in leaf and root tissues revealed least correspondence between the pattern of accumulation, possibly indicting towards de novo tissue-specific biosynthesis. [ABSTRACT FROM AUTHOR]

Highlights: [•] We discovered novel cytotypes from Himalayan habitats in Rumex nepalensis. [•] Higher content of anthraquinones was positively related to increasing ploidy levels. [•] Glycone constituents were found to be highest in the dodecaploid type. [•] Elite cytotypes were identified with desirable chemoprofiles. [•] R. nepalensis showed robust ecological plasticity in response to different altitudes. [Copyright &y& Elsevier]