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Item Single cell type specific RNA isolation and gene expression analysis in rice using laser capture microdissection (LCM)-based method(Springer Nature Publishing AG, 2021) Gautam, Vibhav; Chatterjee, Sourav; Sarkar, Ananda K.The success of single cell type-specific gene expression or functional study largely depends on the efficient isolation of high-quality RNA from them. Laser capture microdissection (LCM) is an efficient technique that allows accessing and dissecting out a specific individual cell or cell type from a microscopic heterogeneous tissue in a minimally disruptive way. Here, we describe an efficient and inexpensive LCM-based method for the extraction of RNAs with high yield and integrity from laser-microdissected mesophyll and bundle sheath cells of rice leaf. The integrity of isolated RNA is assessed with bioanalyzer analysis, and the presence of mRNA of a specific gene is validated through RT-PCR. This RNA could further be used for uncovering single cell type-specific gene expression signature using next-generation transcriptome sequence or through regular RT-PCR.Item Conserved LBL1-ta-siRNA and miR165/166-RLD1/2 modules regulate root development in maize(The Company of Biologists, 2021) Gautam, Vibhav; Singh, Archita; Yadav, Sandeep; Singh, Sharmila; Kumar, Pramod; Das, Shabari Sarkar; Sarkar, Ananda K.Root system architecture and anatomy of monocotyledonous maize is significantly different from dicotyledonous model Arabidopsis. The molecular role of non-coding RNA (ncRNA) is poorly understood in maize root development. Here we address the role of LEAFBLADELESS1 (LBL1), a component of maize trans-acting short-interfering RNA (ta-siRNA), in maize root development. We report that the root growth, anatomical patterning, number of lateral roots (LRs) and monocot-specific crown roots (CRs) and seminal roots (SRs) are significantly affected in lbl1-rgd1 mutant, which is defective in production of ta-siRNA, including tasiR-ARF that targets AUXIN RESPONSE FACTOR3 (ARF3) in maize. Altered accumulation and distribution of auxin, due to differential expression of auxin biosynthesis and transporter genes, created an imbalance in auxin signaling. Altered expression of microRNA165/166 (miR165/166) and its targets ROLLED1/2 (RLD1/2) contributed to the changes in lbl1-rgd1 root growth and vascular patterning, as was evident by altered root phenotype of Rld1-O semi-dominant mutant. Thus, LBL1/ta-siRNA module regulates root development, possibly by affecting auxin distribution and signaling, in crosstalk with miR165/166-RLD1/2 module. We further showed that ZmLBL1 and its Arabidopsis homolog AtSGS3 proteins are functionally conserved.Item Auxin signaling modulates LATERAL ROOT PRIMORDIUM1 (LRP1) expression during lateral root development in Arabidopsis(John Wiley & Sons, 2020) Singh, Sharmila; Yadav, Sandeep; Singh, Alka; Mahima; Singh, Archita; Gautam, Vibhav; Sarkar, Ananda K.Auxin signaling mediated by various Auxin/Indole-3-Acetic Acid (Aux/IAAs) and AUXIN RESPONSE FACTORs (ARFs) regulate lateral root (LR) development by controlling the expression of downstream genes. LATERAL ROOT PRIMORDIUM1 (LRP1), a member of SHORT INTERNODES/STYLISH (SHI/STY) family, was identified as an auxin inducible gene. Precise developmental role and molecular regulation of LRP1 in root development remains to be understood. Here we show that LRP1 is expressed in all stages of LR development, besides primary root. The expression of LRP1 is regulated by histone deacetylation in an auxin dependent manner. Our genetic interaction studies showed that LRP1 acts downstream of auxin responsive Aux/IAAs-ARFs modules during LR development. We showed that auxin mediated induction of LRP1 is lost in slr-1 and arf7arf19 mutants roots. NPA treatment studies showed that LRP1 acts after LR founder cell specification and asymmetric division. Overexpression of LRP1 (LRP1 OE) showed increased number of LRPs at stage I, IV and V, resulting in reduced emerged LR density, which suggests that it is involved in LRP development. Interestingly, LRP1 induced expression of YUC4, which is involved in auxin biosynthesis, contributes to increased accumulation of endogenous auxin in LRP1 OE roots. LRP1 interacts with SHI, STY1, SRS3 and SRS7 proteins of SHI/STY family indicating their possible redundant role during root development. Our results suggest that auxin and histone deacetylation affect LRP1 expression, and it acts downstream of LR forming auxin response modules to negatively regulate LRP development by modulating auxin homeostasis in Arabidopsis thaliana.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 Expression dynamics of miRNAs and their targets in seed germination conditions reveals miRNA-ta-siRNA crosstalk as regulator of seed germination(Nature Publishing Group, 2018) Das, Shabari Sarkar; Yadav, Sandeep; Singh, Archita; Gautam, Vibhav; Sarkar, Ananda K.; Nandi, Asis K.; Karmakar, Prakash; Majee, Manoj; Sanan-Mishra, NeetiSeed germination paves the way for the dormant embryo to establish itself as a new plant marking the first critical step in postembryonic plant growth and development. Germination starts with the uptake of water (imbibition), followed by induction of transcription, translation, energy metabolism, and cell division processes. Although small RNAs have been implicated in many developmental processes, their role during seed germination stages and conditions remained elusive. Here we show that seed germination conditions, like imbibition and temperature, dynamically regulate the expression of many developmentally important miRNAs and their targets. We have identified 58 miRNAs belonging to 30 different families at different seed germination conditions. Amongst these, 15 miRNAs and their targets were significantly differentially expressed in Arabidopsis seeds in dry and 12 h, 24 h and 48 h of imbibition. Interestingly, differential expression of miR390, which targets trans-acting siRNA locus (TAS3) derived transcripts, resulted in alteration of tasiR-ARF mediated regulation of expression of target AUXIN RESPONSE FACTORs (ARF2/3/4). Our results suggest that the dynamic expression of several miRNAs, their targets, and a crosstalk between miRNA and ta-siRNA pathways contribute to the regulation of seed germination in Arabidopsis thaliana.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.