An MYB transcription factor regulating specialized metabolisms in <i>Ophiorrhiza pumila</i>

Abstract

Camptothecin is a plant-derived alkaloid and important precursor of clinically used anti-tumor drugs, but little is known about regulatory mechanism of camptothecin production in plants. We show here that a MYB transcription factor, OpMYB1, isolated from Ophiorrhiza pumila is a regulator of camptothecin biosynthesis. OpMYB1 has an EAR-like motif and exhibits a transcriptional repression activity in an in vivo assay using Arabidopsis thaliana leaves. Overexpression of OpMYB1 in hairy roots of O. pumila resulted in reduced production of camptothecin and reduced expression of OpTDC encoding triptophane decarboxylase catalyzing the earliest step in camptothecin biosynthesis. From the deep transcriptome analysis, GO enrichment in secondary (specialized) metabolisms, especially in phenylpropanoid pathway was observed in the hairy roots over-expressing OpMYB1. Furthuremore, gene suppression by OpMYB1 was revealed in biosynthetic pathways of seco-iridoids, monoterpene indole alkaloids, anthraquinone and chlorogenic acid. These results suggested that OpMYB1 is a negative regulator to fine-tune the general specialized metabolisms in O. pumila.

Function of AP2/ERF Transcription Factors Involved in the Regulation of Specialized Metabolism in Ophiorrhiza pumila Revealed by Transcriptomics and Metabolomics

“…Therefore, using comparative expression analysis, we identified AP2/ERFs that are specially expressed in HR when compared with CSC. From the data derived by PCR-select cDNA subtraction (Rohani et al, 2016), six out of 353 HR-specific fragments were annotated as ERF-like proteins. Full-length cDNA cloning revealed that all these six fragments constitute a part of 903-bp cDNA sequence (designated as OpERF1 ).…”

“…Removal of adapter sequences, low-quality reads, and reads shorter than 50 bp yielded a total of over 84 million high-quality paired end clean reads (Table 2). Clean reads from individual libraries were aligned with previously described and annotated O. pumila de novo transcriptome assembly (Rohani et al, 2016) using Bowtie 2.0 (Langmead et al, 2009), and transcript abundance in FPKM was estimated using the RSEM program (Li and Dewey, 2011). More than 88% of the clean reads from each library could be aligned to a reference de novo assembly.…”

“…Raw read pre-processing, alignment of clean reads to a reference O. pumila de novo assembly (Rohani et al, 2016), transcript abundant estimation and expression level in fragment per kilobase exon per million mapped fragment (FPKM) were performed as described by Rai et al (2016a). Raw read sequences of six RNAi HR lines were deposited in NCBI's Gene Expression Omnibus (Accession number GSE89674; Edgar et al, 2002).…”

“…Using deep transcriptome analysis and untargeted metabolic profiling of O. pumila HR, the candidate genes in the specialized metabolism were profiled in comparison with those in the cell suspension culture (CSC) that doesn't produce CPT (Yamazaki et al, 2013). Recently, Rohani et al (2016) reported the characterization of a suppressor, Op MYB1, involved in the regulation of specialized metabolism in O. pumila . Their study demonstrated that overexpression of OpMYB1 in HR negatively affected the transcript level of TDC and resulted in reduced accumulation of CPT.…”

Integrated omics analysis of specialized metabolism in medicinal plants

“…Transcriptome and metabolome analysis for engineered O. pumila cultured cell lines producing (hairy roots) or not producing (cell suspension culture) camptothecin-type alkaloids led to the identification of candidate genes involved in the biosynthesis of alkaloids and anthraquinones . Further, transcription factors, namely OpMYB1 and OpERF2, of O. pumila with functions in regulating the biosynthesis of secondary metabolites were identified using RNA-seq-based transcriptome profiling and metabolome analysis (Rohani et al, 2016;Udomsom et al, 2016). Comprehensive transcriptome and metabolome analysis, and their integration, led to the elucidation of morphine biosynthetic pathways and functional characterization of most of the genes coding for associated biosynthetic enzymes (Hagel and Facchini, 2013;Higashi and Saito, 2013).…”