Mining NGS transcriptomes for miRNAs and dissecting their role in regulating growth, development, and secondary metabolites production in different organs of a medicinal herb, Picrorhiza kurroa

Abstract

This study is the first endeavor on mining of miRNAs and analyzing their involvement in development and secondary metabolism of an endangered medicinal herb Picrorhiza kurroa (P. kurroa ). miRNAs are ubiquitous non-coding RNA species that target complementary sequences of mRNA and result in either translational repression or target degradation in eukaryotes. The role of miRNAs has not been investigated in P. kurroa which is a medicinal herb of industrial value due to the presence of secondary metabolites, picroside-I and picroside-II. Computational identification of miRNAs was done in 6 transcriptomes of P. kurroa generated from root, shoot, and stolon organs varying for growth, development, and culture conditions. All available plant miRNA entries were retrieved from miRBase and used as backend datasets to computationally identify conserved miRNAs in transcriptome data sets. Total 18 conserved miRNAs were detected in P. kurroa followed by target prediction and functional annotation which suggested their possible role in controlling various biological processes. Validation of miRNA and expression analysis by qRT-PCR and 5' RACE revealed that miRNA-4995 has a regulatory role in terpenoid biosynthesis ultimately affecting the production of picroside-I. miR-5532 and miR-5368 had negligible expression in field-grown samples as compared to in vitro-cultured samples suggesting their role in regulating P. kurroa growth in culture conditions. The study has thus identified novel functions for existing miRNAs which can be further validated for their potential regulatory role.

Contemporary Understanding of miRNA-Based Regulation of Secondary Metabolites Biosynthesis in Plants

“…Transcription factor SPL9, the target of miRNA156, directly binds to and activates promoter of terpene synthases 21 (TPS21) gene and positively regulates its transcription thereby regulating the synthesis of sesquiterpenoid (Yu et al, 2015). Similarly, miR-4995 was predicted to target mRNA of an enzyme 3-deoxy-7-phosphoheptulonate synthase, which is involved in the picroside biosynthetic pathway in a medicinal herb P. kurroa (Vashisht et al, 2015). In addition, Saifi et al (2015), have mined and validated 11 miRNAs which are involved in steviol glycoside biosynthetic pathway (Table 1) in Stevia and established the relationship pattern with the expression levels of their target mRNAs as well as steviol glycoside contents.…”

New opportunities for the regulation of secondary metabolism in plants: focus on microRNAs

“…Agrobacterium T-DNA oncogenes that perturb the miRNA biosynthesis apparatus and hence affect secondary metabolism Global changes in miRNA levels and corresponding physiological responses appear to be caused by plant (2013) Gossypium ghr-miR8156 a ghr-miR8170 a Potential targets are resistance genes such as mitogenactivated protein kinase genes Terpenoid biosynthesis and isoquinoline alkaloid biosynthesis Picrorhiza kurroa miR4995 a A gene encoding 3-deoxy-7-phosphoheptulonate synthase Potential inhibition of iridoid glycosides biosynthesis Expression of miR4995 was threefold higher in vitrocultured shoots comparing with field-grown shoots Vashisht et al (2015) a High-throughput-sequencing technologies and in silico experiments Biotechnol Lett T-DNA oncogenes. These oncogenes cause a pleiotropic effect on plant growth and development and affect defense reactions and secondary metabolism.…”

“…Apparently, the effect of each miRNA is specific, and its down-regulation or overexpression will not result in global changes in the plant transcriptome. In Taxus mairei, Catharanthus roseus, Papaver somniferum and other economically important plants, several miRNAs have been identified as regulators of secondary metabolism (Hao et al 2012;Pani and Mahapatra 2013;He et al 2014;Boke et al 2015;Vashisht et al 2015).…”

Renaissance in phytomedicines: promising implications of NGS technologies

“…In Picrorhiza kurroa also 18 conserved miRNA sequences were identified as having roles in various metabolic pathways (Vashisht et al 2015). The most imperative discovery was the miRNA sequence complementary to mRNA for the enzyme 3-hydroxy-7-phosphoheptulonate synthase.…”