Browsing by Author "Gautam, Vibhav"

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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.
Coevolution pattern and functional conservation or divergence of miR167s and their targets across diverse plant species
(Nature Publishing Group, 2015) Barik, Suvakanta; Kumar, Ashutosh; Das, Shabari Sarkar; Yadav, Sandeep; Gautam, Vibhav; Singh, Archita; Singh, Sharmila; Sarkar, Ananda K.
microRNAs (miRNAs), a class of endogenously produced small non-coding RNAs of 20-21 nt length, processed from precursor miRNAs, regulate many developmental processes by negatively regulating the target genes in both animals and plants. The coevolutionary pattern of a miRNA family and their targets underscores its functional conservation or diversification. The miR167 regulates various aspects of plant development in Arabidopsis by targeting ARF6 and ARF8. The evolutionary conservation or divergence of miR167s and their target genes are poorly understood till now. Here we show the evolutionary relationship among 153 MIR167 genes obtained from 33 diverse plant species. We found that out of the 153 of miR167 sequences retrieved from the "miRBase", 27 have been annotated to be processed from the 3' end, and have diverged distinctively from the other miR167s produced from 5' end. Our analysis reveals that gma-miR167h/i and mdm-miR167a are processed from 3' end and have evolved separately, diverged most resulting in novel targets other than their known ones, and thus led to functional diversification, especially in apple and soybean. We also show that mostly conserved miR167 sequences and their target AUXIN RESPONSE FACTORS (ARFs) have gone through parallel evolution leading to functional diversification among diverse plant species.
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.
An efficient LCM-based method for tissue specific expression analysis of genes and miRNAs
(Nature Publishing Group, 2016) Gautam, Vibhav; Singh, Archita; Singh, Sharmila; Sarkar, Ananda K.
Laser Capture Microdissection (LCM) is a powerful tool to isolate and study gene expression pattern of desired and less accessible cells or tissues from a heterogeneous population. Existing LCM-based methods fail to obtain high quality RNA including small RNAs from small microdissected plant tissue and therefore, are not suitable for miRNA expression studies. Here, we describe an efficient and cost-effective method to obtain both high quality RNA and miRNAs from LCM-derived embryonic root apical meristematic tissue, which is difficult to access. We have significantly modified and improved the tissue fixation, processing, sectioning and RNA isolation steps and minimized the use of kits. Isolated RNA was checked for quality with bioanalyzer and used for gene expression studies. We have confirmed the presence of 19-24 nucleotide long mature miRNAs using modified stem-loop RT-PCR. This modified LCM-based method is suitable for tissue specific expression analysis of both genes and small RNAs (miRNAs).
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, Neeti
Seed 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.
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.
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.
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).
Laser assisted microdissection, an efficient technique to understand tissue specific gene expression patterns and functional genomics in plants
(Springer, 2015) Gautam, Vibhav; Sarkar, Ananda K.
Laser assisted microdissection (LAM) is an advanced technology used to perform tissue or cell-specific expression profiling of genes and proteins, owing to its ability to isolate the desired tissue or cell type from a heterogeneous population. Due to the specificity and high efficiency acquired during its pioneering use in medical science, the LAM technique has quickly been adopted for use in many biological researches. Today, it has become a potent tool to address a wide range of questions in diverse field of plant biology. Beginning with comparative transcriptome analysis of different tissues such as reproductive parts, meristems, lateral organs, roots etc., LAM has also been extensively used in plant-pathogen interaction studies, proteomics, and metabolomics. In combination with next generation sequencing and proteomics analysis, LAM has opened up promising opportunities in the area of large scale functional studies in plants. Ever since the advent of this technique, significant improvements have been achieved in term of its instrumentation and method, which has made LAM a more efficient tool applicable in wider research areas. Here, we discuss the advancement of LAM technique with special emphasis on its methodology and highlight its scope in modern research areas of plant biology. Although we put emphasis on use of LAM in transcriptome studies, which is mostly used, we also discuss its recent application and scope in proteome and metabolome studies.
Phylogenetic analysis reveals conservation and diversification of micro RNA166 genes among diverse plant species
(Elsevier B.V., 2014) Barik, Suvakanta; SarkarDas, Shabari; Singh, Archita; Gautam, Vibhav; Kumar, Pramod; Majee, Manoj; Sarkar, Ananda K.
Similar to the majority of the microRNAs, mature miR166s are derived from multiple members of MIR166 genes (precursors) and regulate various aspects of plant development by negatively regulating their target genes (Class III HD-ZIP). The evolutionary conservation or functional diversification of miRNA166 family members remains elusive. Here, we show the phylogenetic relationships among MIR166 precursor and mature sequences from three diverse model plant species. Despite strong conservation, some mature miR166 sequences, such as ppt-miR166m, have undergone sequence variation. Critical sequence variation in ppt-miR166m has led to functional diversification, as it targets non-HD-ZIPIII gene transcript (s). MIR166 precursor sequences have diverged in a lineage specific manner, and both precursors and mature osa-miR166i/j are highly conserved. Interestingly, polycistronic MIR166s were present in Physcomitrella and Oryza but not in Arabidopsis. The nature of cis-regulatory motifs on the upstream promoter sequences of MIR166 genes indicates their possible contribution to the functional variation observed among miR166 species.
Phytohormonal crosstalk modulates the expression of miR166/165s, target Class III HD-ZIPs, and KANADI genes during root growth in Arabidopsis thaliana
(Nature Publishing Group, 2017) Singh, Archita; Roy, Shradha; Singh, Sharmila; Das, Shabari Sarkar; Gautam, Vibhav; Yadav, Sandeep; Kumar, Ashutosh; Singh, Alka; Samantha, Sukanya; Sarkar, Ananda K.
Both phytohormones and non-coding microRNAs (miRNAs) play important role in root development in Arabidopsis thaliana. Mature miR166/165 s, which are derived from precursor transcripts of concerned genes, regulate developmental processes, including leaf and root patterning, by targeting Class III HOMEODOMAIN LEUCINE-ZIPPER (HD-ZIP III) transcription factors (TFs). However, their regulation through hormones remained poorly understood. Here, we show that several phytohormones dynamically regulate the spatio-temporal expression pattern of miR166/165 and target HD-ZIP IIIs in developing roots. Hormone signaling pathway mutants show differential expression pattern of miR166/165, providing further genetic evidence for multilayered regulation of these genes through phytohormones. We further show that a crosstalk of at least six different phytohormones regulate the miR166/165, their target HD-ZIP IIIs, and KANADI (KANs). Our results suggest that HD-ZIP IIIs mediated root development is modulated both transcriptionally through phytohormones and KANs, and post-transcriptionally by miR166/165 that in turn are also regulated by the phytohormonal crosstalk.
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.
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.
Root transcriptome of two contrasting indica rice cultivars uncovers regulators of root development and physiological responses
(Nature Publishing Group, 2016) Singh, Alka; Kumar, Pramod; Gautam, Vibhav; Rengasamy, Balakrishnan; Adhikari, Bijan; Udayakumar, Makarla; Sarkar, Ananda K.
The huge variation in root system architecture (RSA) among different rice (Oryza sativa) cultivars is conferred by their genetic makeup and different growth or climatic conditions. Unlike model plant Arabidopsis, the molecular basis of such variation in RSA is very poorly understood in rice. Cultivars with stable variation are valuable resources for identification of genes involved in RSA and related physiological traits. We have screened for RSA and identified two such indica rice cultivars, IR-64 (OsAS83) and IET-16348 (OsAS84), with stable contrasting RSA. OsAS84 produces robust RSA with more crown roots, lateral roots and root hairs than OsAS83. Using comparative root transcriptome analysis of these cultivars, we identified genes related to root development and different physiological responses like abiotic stress responses, hormone signaling, and nutrient acquisition or transport. The two cultivars differ in their response to salinity/dehydration stresses, phosphate/nitrogen deficiency, and different phytohormones. Differential expression of genes involved in salinity or dehydration response, nitrogen (N) transport, phosphate (Pi) starvation signaling, hormone signaling and root development underlies more resistance of OsAS84 towards abiotic stresses, Pi or N deficiency and its robust RSA. Thus our study uncovers gene-network involved in root development and abiotic stress responses in rice.
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.
Sirtinol, a Sir2 protein inhibitor, affects stem cell maintenance and root development in Arabidopsis thaliana by modulating auxin-cytokinin signaling components
(Nature Publishing Group, 2017) Singh, Sharmila; Singh, Alka; Yadav, Sandeep; Gautam, Vibhav; Singh, Archita; Sarkar, Ananda K.
In Arabidopsis thaliana, besides several key transcription factors and chromatin modifiers, phytohormones auxin and cytokinin play pivotal role in shoot and root meristem maintenance, and lateral root (LR) development. Sirtinol, a chemical inhibitor of Sir2 proteins, is known to promote some auxin induced phenotypes in Arabidopsis. However, its effect on plant stem cell maintenance or organ formation remained unaddressed. Here we show that sirtinol affects meristem maintenance by altering the expression of key stem cell regulators, cell division and differentiation by modulating both auxin and cytokinin signaling in Arabidopsis thaliana. The expression of shoot stem cell niche related genes WUSCHEL (WUS) and CLAVATA3 (CLV3) was upregulated, whereas SHOOT MERISTEMLESS (STM) was downregulated in sirtinol treated seedlings. The expression level and domain of key root stem cell regulators PLETHORA (PLTs) and WUS-Related Homeobox 5 (WOX5) were altered in sirtinol treated roots. Sirtinol affects LR development by disturbing proper auxin transport and maxima formation, similar to 2,4-dichlorophenoxyacetic acid (2,4-D). Sirtinol also affects LR formation by altering cytokinin biosynthesis and signaling genes in roots. Therefore, sirtinol affects shoot and root growth, meristem maintenance and LR development by altering the expression of cytokinin-auxin signaling components, and regulators of stem cells, meristems, and LRs.
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.