Integrated analysis of smRNAome, transcriptome, and degradome data to decipher microRNAs regulating costunolide biosynthesis in Saussurea lappa.

Saussurea lappa (S. lappa) has been known to synthesize medicinally important, costunolide. Due to its immense therapeutic importance, understanding of regulatory mechanism associated with its biosynthesis is crucial. The identification of genes and transcription factors (TFs) in S. lappa, created a clear picture of costunolide biosynthesis pathways. Further to understand the regulation of costunolide biosynthesis by miRNAs, an integrated study of transcriptome, miRNAs, and degradome was performed. Identified candidate miRNAs and associated feed-forward loops (FFLs) illustrates their regulatory role in secondary metabolite biosynthesis. Small RNA and degradome sequencing were performed for leaf and root tissues to determine miRNAs-targets pairs. A total of 711 and 525 such targets were obtained for novel and known miRNAs respectively. This data was used to generate costunolide-specific miRNA-TF-gene interactome to perform systematic analyses through graph theoretical approach. Interestingly, miR171c.1 and sla-miR121 were identified as key regulators to connect and co-regulate both mevalonate and sesquiterpenoid pathways to bio-synthesize costunolide. Tissue-specific FFLs were identified to be involved in costunolide biosynthesis which further suggests the evolutionary co-relation of root-specific networks in synthesis of secondary metabolites in addition to leaf-specific networks. This integrative approach allowed us to determine candidate miRNAs and associated tissue-specific motifs involved in the diversification of secondary metabolites. MiRNAs identified in present study can provide alternatives for bioengineering tool to enhance the synthesis of costunolide and other secondary metabolites in S. lappa.

Identification of novel microRNAs and their targets in Chlorophytum borivilianum by small RNA and degradome sequencing.

Plant specific miRNAs (Novel miRNAs) are well known to perform distinctive functions in biological processes. Identification of new miRNAs is necessary to understand their gene regulation. Degradome provides an opportunity to explore the miRNA functions by comparing the miRNA population and their degraded products. In the present study, Small RNA sequencing data was used to identify novel miRNAs. Further, degradome sequencing was carried out to identify miRNAs targets in the plant, Chlorophytum borivilianum. The present study supplemented 40 more novel miRNAs correlating degradome data with smallRNAome. Novel miRNAs, complementary to mRNA partial sequences obtained from degradome sequencing were actually targeting the later. A big pool of miRNA was established by using Oryza sativa, Arabidopsis thaliana, Populus trichocarpa, Ricinus communis, and Vitis vinifera genomic data. Targets were identified for novel miRNAs and total 109 targets were predicted. BLAST2GO analysis elaborate about localization of novel miRNAs' targets and their corresponding KEGG (Kyoto Encyclopedia for Genes and Genomes) pathways. Identified targets were annotated and were found to be involved in significant biological processes like Nitrogen metabolism, Pyruvate metabolism, Citrate cycle (TCA cycle), photosynthesis, and Glycolysis/Gluconeogenesis. The present study provides an overall view of the miRNA regulation in multiple metabolic pathways that are involved in plant growth, pathogen resistance and secondary metabolism of C. borivilianum.

Small RNA profiling for identification of miRNAs involved in regulation of saponins biosynthesis in Chlorophytum borivilianum.

BACKGROUND: MicroRNAs act as molecular regulator of cell signaling, plant growth and development, and regulate various primary and secondary plant metabolic processes. In the present study, deep sequencing of small RNAs was carried out to identify known and novel miRNAs from pharmaceutically important plant, Chlorophytum borivilianum. RESULTS: Total 442 known miRNAs and 5 novel miRNAs were identified from young leaf small RNA library. Experimental validation with stem loop RT-PCR confirmed the in silico identification. Based on transcriptome data of root and leaf of C. borivilianum, Oryza sativa, and Arabidopsis thaliana target gene prediction was done using psRNAtarget and mirRanda. BLAST2GO helped in localization of predicted targets and KEGG (Kyoto Encyclopedia for Genes and Genomes) pathway analysis concluded that miR9662, miR894, miR172, and miR166 might be involved in regulating saponin biosynthetic pathway. The correlation between miRNA and its target gene was further validated by RT-qPCR analysis. CONCLUSION: This study provides first elaborated glimpse of miRNA pool of C. borivilianum, which can help to understand the miRNA dependent regulation of saponin biosynthesis and to design further metabolic engineering experiment to enhance their contents in the plant.