Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

Abstract

BackgroundTaxol® (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control.ResultsSix separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line.ConclusionsThis study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil

“…Lectins, callose synthase, and thaumatin-like proteins that presumably do not have any role in secondary metabolite accumulation were also identified among the MeJA upregulated transcripts (Supplemental Table S4). These genes function as plant defense components and were also found to be induced by MeJA in other plant species (Cheong and Choi, 2003;Lenka et al, 2012;Ee et al, 2013). The data also suggest MeJA responsiveness of the MEP and MEV pathways that provide 5C building blocks for the biosynthesis of the diverse terpene metabolites (Table II).…”

“…The plant endogenous signaling molecule MeJA, in addition to regulating the normal plant developmental processes, induces defense responses during herbivore and pathogen attacks by accumulating diverse secondary metabolites (Cheong and Choi, 2003). Because MeJA has been shown to be a powerful inducer of secondary metabolite production in diverse plants (Aerts et al, 1994;Suzuki et al, 2005;Kim et al, 2006;De Geyter et al, 2012;Lenka et al, 2012), we studied MeJA-elicited transcriptional changes with the goal to identify the candidate genes involved in the biosynthesis of secondary metabolites in sweet basil. SSH is a powerful genomics tool for identification of differentially expressed genes for nonmodel organisms such as sweet basil, in which genomic information is currently scarce (Ohlrogge and Benning, 2000).…”

“…The SSH library was also enriched with the transcripts that presumably have no direct role in cellular metabolism. Some of them were related to the plant defense responses and are known to be induced by MeJA in other plant species as well (Supplemental Table S4; Cheong and Choi, 2003;Lenka et al, 2012;Ee et al, 2013).…”

“…When plants experience various biotic and abiotic stresses, biosynthesis of the secondary metabolites is triggered that helps plants adapt to the challenging environment (Gershenzon and Dudareva, 2007;Hartmann, 2007;Reichling, 2010). Consequently, transcript and metabolite profiling of stress/elicitor-treated plants or cell cultures represents a powerful approach to determine gene function in the biosynthesis of the secondary metabolites (Aerts et al, 1994;Goossens et al, 2003;Suzuki et al, 2005;Zhao et al, 2005;Naoumkina et al, 2008;De Geyter et al, 2012;Lenka et al, 2012;Yu et al, 2012;Ee et al, 2013;Mishra et al, 2013;Sun et al, 2013).…”

Regulation of Specialized Metabolism by WRKY Transcription Factors

“…The phytohormone jasmonate is a key elicitor of specialized metabolism (Aerts et al, 1994;Singh et al, 1998;Qi et al, 2011;Lenka et al, 2012), primarily through the induction of key TFs, such as MYC2 and ethylene response factors, that regulate the biosynthetic genes (van der Fits and Memelink, 2000;Shoji et al, 2010;Shoji and Hashimoto, 2011;Zhang et al, 2011;Patra et al, 2013;Schweizer et al, 2013;Sears et al, 2014). Study of the grape (Vitis vinifera) WRKY family found that 80% of WRKY genes are jasmonate responsive .…”