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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 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 Balanced activity of microRNA166/165 and its target transcripts from the class III homeodomain leucine-zipper family regulates root growth in Arabidopsis thaliana(Springer, 2014) Singh, Archita; Singh, Sharmila; Panigrahi, Kishore C. S.; Reski, Ralf; Sarkar, Ananda K.Overexpression of miR166/165 down-regulates target HD - ZIP IIIs and promotes root growth by enhancing cell division and meristematic activity, whereas overexpression of HD - ZIP IIIs inhibits root growth in Arabidopsis thaliana. Post-embryonic growth of higher plants is maintained by active meristems harbouring undifferentiated cells. Shoot and root apical meristems (SAM and RAM) utilize both similar and distinct signalling mechanisms for their maintenance in Arabidopsis thaliana. An important regulatory role in this context has the interaction of microRNAs with their target mRNAs, mostly encoding transcription factors. One class of microRNA166/165 (miR166/165) has been implicated in the maintenance of SAM and vascular patterning. Here, we show that miR166/165 plays an important role in root growth also by negatively regulating its target transcripts, HD-ZIP IIIs, in the RAM. While overexpression of miR166 promotes RAM activity, overexpression of its targets reduces RAM activity. These results reveal a conserved role of miR166/165 in the maintenance of SAM and RAM activity in A. thaliana.