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Item Whole mount in situ localization of miRNAs and target mRNA transcripts in plants(Springer Nature Publishing AG, 2019) Gautam, Vibhav; Singh, Archita; Verma, Swati; Singh, Sharmila; Chatterjee, Sourav; Sarkar, Ananda K.The functional characterization of miRNAs often involves understanding of their spatiotemporal expression, which mostly relies on reporter-based or in situ hybridization studies. The available in situ localization methods follow separate protocols for pre-hybridization, hybridization, post-hybridization, and detection steps for both miRNA and mRNA transcripts in plants. In this study, we present a single method which can be used for whole mount in situ localization of both miRNAs and mRNAs in different plant tissues. Our modified method provides enhanced sensitivity for the localization of miRNA and their target transcripts. Consequently, a less laborious, time-saving, economic and efficient method has been proposed by the modification of pre-hybridization, hybridization, post-hybridization and detection steps.Item Improved laser capture microdissection (LCM)‑based method for isolation of RNA, including miRNA and expression analysis in woody apple bud meristem(Springer Nature, 2019) Verma, Swati; Gautam, Vibhav; Sarkar, Ananda K.Isolation of high-quality RNA, including miRNA, from microscopic woody apple bud meristem using laser capture microdissection-based method. It is often challenging to study the expression of microRNAs (miRNAs) or genes in less accessible inner tissues of tree species rich in polyphenols or polysaccharides. Here, we report a laser capture microdissection (LCM)-based method for efficient and cost-effective isolation and expression analysis of miRNAs and genes in the meristem tissue of woody apple bud. The tissue fixation, processing, infiltration, and sectioning steps were optimized for LCM-based excision and subsequent RNA isolation. Further, we have confirmed that RNA isolated from LCM-derived apple bud meristem contained miRNAs and was of good quantity and quality, sufficient for downstream expression analysis.Item Improved method of RNA isolation from laser capture microdissection (LCM)-derived plant tissues(Springer Nature, 2019) Gautam, Vibhav; Singh, Archita; Singh, Sharmila; Verma, Swati; Sarkar, Ananda K.Laser capture microdissection (LCM) is a tool to isolate desired and/or less accessible cells or tissues from a heterogeneous population. In the current method, we describe an efficient and cost-effective method to obtain both high-quality mRNA and miRNAs in sufficient quantity from LCM-derived plant tissues. The quality of the isolated RNA can be assessed using Bioanalyzer. Using modified stem-loop RT-PCR, we confirmed the presence of 21–24 nucleotide (nt) long mature miRNAs. This modified LCM-based method has been found to be suitable for the tissue-specific expression analysis of both genes and small RNAs (miRNAs).Item Identification and co-evolution pattern of stem cell regulator miR394s and their targets among diverse plant species(BioMed Central Ltd, 2019) Kumar, Ashutosh; Gautam, Vibhav; Kumar, Pramod; Mukherjee, Shalini; Verma, Swati; Sarkar, Ananda K.Background Micro RNAs (miRNAs), a class of small non-coding RNAs, have been implicated in various aspects of plant development. miR394 is required for shoot apical meristem organization, stem cell maintenance and abiotic stress responses in Arabidopsis, where it functions by negatively regulating the transcript level of target LEAF CURLING RESPONSIVENESS (LCR), which is an F-box protein-coding gene. The evolutionary conservation of stem cell regulatory miR394-LCR module among plants remains elusive. Results Our study has identified 79 miR394 and 43 target sequences across 40 plant species using various homology based search tools and databases, and analysed their co-evolution pattern. We customised an annotation workflow which computationally validates 20 novel miR394s from 14 plant species. Independent phylogenetic trees were reconstructed with precursor MIR394s, mature miR394s, and their target sequences along with complementary miR394 binding sites. The phylogeny revealed that mature sequences of miR394s as well as their targets belonging to the F-box protein encoding gene families, were highly conserved. Though, miR394–3p were complementary to miR394s/miR394–5p, they clustered separately. Conclusion The existence and separate clustering of miR394–3p and miR394s/miR394–5p indicate their independent regulation. The phylogeny also suggests that miR394s had evolved at the beginning of gymnosperm-angiosperm divergence. Despite strong conservation, some level of sequence variation in miR394s and the complementary binding sites of their targets suggests possible functional diversification of miR394-LCR mediated stem cell regulation in plants.Item Plant small RNAs: advancement in the understanding of biogenesis and role in plant development(Springer Nature, 2018) Singh, Archita; Gautam, Vibhav; Singh, Sharmila; Das, Shabari Sarkar; Verma, Swati; Mishra, Vishnu; Mukherjee, Shalini; Sarkar, Ananda K.Main conclusion: Present review addresses the advances made in the understanding of biogenesis of plant small RNAs and their role in plant development. We discuss the elaborate role of microRNAs (miRNAs) and trans-acting small interfering RNAs (ta-siRNAs) in various aspects of plant growth and development and highlight relevance of small RNA mobility. Small non-coding RNAs regulate various aspects of plant development. Small RNAs (sRNAs) of 21–24 nucleotide length are derived from double-stranded RNAs through the combined activity of several biogenesis and processing components. These sRNAs function by negatively regulating the expression of target genes. miRNAs and ta-siRNAs constitute two important classes of endogenous small RNAs in plants, which play important roles in plant growth and developmental processes like embryogenesis, organ formation and patterning, shoot and root growth, and reproductive development. Biogenesis of miRNAs is a multistep process which includes transcription, processing and modifcation, and their loading onto RNA-induced silencing complex (RISC). RISC-loaded miRNAs carry out post-transcriptional silencing of their target(s). Recent studies identifed orthologues of diferent biogenesis components of novel and conserved small RNAs from diferent model plants. Although many small RNAs have been identifed from diverse plant species, only a handful of them have been functionally characterized. In this review, we discuss the advances made in understanding the biogenesis, functional conservation/divergence in miRNA-mediated gene regulation, and the developmental role of small RNAs in diferent plant species.Item Role of miRNAs in root development of model plant Arabidopsis thaliana(Springer, 2017) Gautam, Vibhav; Singh, Archita; Verma, Swati; Kumar, Ashutosh; Kumar, Pramod; Mahima; Singh, Sharmila; Mishra, Vishnu; Sarkar, Ananda K.The molecular regulation of root development is relatively well studied in model plant Arabidopsis as compared to other plants. Besides phytohormones, transcription factors and environmental factors, other important regulators which have recently been shown to play crucial roles in controlling root development are the non-coding RNAs. Small non-coding RNAs of 21–24 nt length (miRNAs and ta-siRNAs) regulate various aspects of plant development by negatively regulating their target genes through transcript cleavage or translational inhibition. In recent past the microRNA-mediated regulation of root development has drawn significant interest in the area of plant research. Several reports have highlighted the role of many miRNAs and ta-siRNAs in root growth, vascular patterning, lateral root (LR) formation and elongation, and adventitious root development, Phytohormones like auxin, cytokinin and environmental factors like light, abiotic and biotic stresses, and nutrient availability influence many miRNA-mediated regulation of root growth and branching. In current review, we summarize the recent advances made in understanding the miRNA-mediated regulation of root development in the model plant Arabidopsis thaliana. The molecular crosstalk between different miRNAs, ta-siRNAs, and concerned target genes that regulate root growth and branching have been addressed.