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.
Sorghum is a self-pollinated crop with multiple economic uses as cereal, forage, and biofuel feedstock. Hybrid breeding is a cornerstone for sorghum improvement strategies that currently relies on cytoplasmic male sterile lines. To engineer genic male sterility, it is imperative to examine the genetic components regulating anther/pollen development in sorghum. To this end, we have performed transcriptomic analysis from three temporal stages of developing anthers that correspond to meiotic, microspore and mature pollen stages. A total of 5286 genes were differentially regulated among the three anther stages with 890 of them exhibiting anther-preferential expression. Differentially expressed genes could be clubbed into seven distinct developmental trajectories using K-means clustering. Pathway mapping revealed that genes involved in cell cycle, DNA repair, regulation of transcription, brassinosteroid and auxin biosynthesis/signalling exhibit peak expression in meiotic anthers, while those regulating abiotic stress, carbohydrate metabolism, and transport were enriched in microspore stage. Conversely, genes associated with protein degradation, post-translational modifications, cell wall biosynthesis/modifications, abscisic acid, ethylene, cytokinin and jasmonic acid biosynthesis/ signalling were highly expressed in mature pollen stage. High concurrence in transcriptional dynamics and cis-regulatory elements of differentially expressed genes in rice and sorghum confirmed conserved developmental pathways regulating anther development across species. Comprehensive literature survey in conjunction with orthology analysis and anther-preferential accumulation enabled shortlisting of 21 prospective candidates for in-depth characterization and engineering male fertility in sorghum. Sorghum, grown widely for food, feed, and forage, is a gluten-free substitute for staple grains, and a promising feedstock for biofuels 1. It exhibits huge phenotypic and morphological diversity in key agronomic traits including photoperiod sensitivity, biomass, grain yield, disease resistance, abiotic stress tolerance etc., with considerable scope for genetic enhancement of cultivated sorghum. However, sorghum is a self-pollinated crop with 75 to 95% rate of self-pollination observed under natural conditions with outcrossing rates varying with panicle type, distance between plants, wind direction, etc 2,3. Therefore, overcoming reproductive constraints is a major challenge to utilize sorghum diversity for breeding programs. Plant breeders have been using cytoplasmic (CMS) and nuclear male sterility (NMS) systems to take advantage of hybrid vigour in many self-pollinated crop species 4. While CMS, caused by specific nuclear and mitochondrial genetic interactions, is maternally inherited; NMS results due to defects in nuclear genes usually inherited as a recessive trait 5. Studies in rice suggest that hybrids produced through NMS give higher yields and germplasm utilization efficiency with better genetic stability under diverse environmental condit...
Transcriptional regulation of picrosides biosynthesis, the iridoid glycosides of an endangered medicinal herb, Picrorhiza kurroa, is completely unknown. P. kurroa plants obtained from natural habitat accumulate higher picrosides than in-vitro cultured plants, which necessitates identification of transcription factors (TFs) regulating their differential biosynthesis. The current study investigates complete spectrum of different TF classes in P. kurroa transcriptomes and discerns their association with picrosides biosynthesis. Transcriptomes of differential picroside-I content shoots and picroside-II content roots were mined for seven classes of TFs implicated in secondary metabolism regulation in plants. Key TFs were identified through in silico transcript abundance and qPCR analysis was performed to confirm transcript levels of TFs under study in differential content tissues and genotypes. Promoter regions of key picrosides biosynthetic pathway genes were explored to hypothesize which TFs can possibly regulate target genes. A total of 131, 137, 107, 82 and 101 transcripts encoding different TFs families were identified in PKS-25, PKS-15, PKSS, PKR-25 and PKSR transcriptomes, respectively. ERF-18, bHLH-104, NAC-25, 32, 94 and SUF-4 showed elevated expression in roots (up to 37 folds) and shoots (up to 195 folds) of plants obtained from natural habitat, indicating their role as activators of picrosides biosynthesis whereas, elevated expression of WRKY-17, 40, 71 and MYB-4 in low picrosides content conditions suggested their down-regulatory role. In silico analysis of key picrosides biosynthetic pathway gene promoter regions revealed binding domains for ERF-18, NAC-25, WRKY-40 and MYB-4. Identification of candidate TFs contributing towards picrosides biosynthesis is a pre-requisite for designing appropriate metabolic engineering strategies aimed at enhancing picrosides content in vitro and in vivo.
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