Publications of NIPGR Scientists
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Item miR394 enhances WUSCHEL-induced somatic embryogenesis in Arabidopsis thaliana(John Wiley & Sons, 2023) Lu, Lu; Holt, Anna; Chen, Xinying; Liu, Yang; Knauer, Steffen; Tucker, Elise J.; Sarkar, Ananda K.; Hao, Zhaodong; Roodbarkelari, Farshad; Shi, Jisen; Chen, Jinhui; Laux, ThomasMany plant species can give rise to embryos from somatic cells after a simple hormone treatment, illustrating the remarkable developmental plasticity of differentiated plant cells. However, many species are recalcitrant to somatic embryo formation for unknown reasons, which poses a significant challenge to agriculture, where somatic embryogenesis is an important tool to propagate desired genotypes. The micro RNA394 (miR394) promotes shoot meristem maintenance in Arabidopsis thaliana, but the underlying mechanisms have remained elusive. We analyzed whether miR394 affects indirect somatic embryogenesis and determined the transcriptome of embryogenic callus upon miR394-enhanced somatic embryogenesis. We show that ectopic miR394 expression enhances somatic embryogenesis in the recalcitrant Ler accession when co-expressed with the transcription factor WUSCHEL (WUS) and that miR394 acts in this process through silencing the target LEAF CURLING RESPONSIVENESS (LCR). Furthermore, we show that higher endogenous miR394 levels are required for the elevated embryogenic potential of the Columbia accession compared to Ler, providing a mechanistic explanation for this natural variation. Our transcriptional analysis provides a framework for miR394 function in regulating pluripotency by expanding WUS-mediated direct transcriptional repression.Item Potassium chloroaurate-mediated in vitro synthesis of gold nanoparticles improved root growth by crosstalk with sucrose and nutrient-dependent auxin homeostasis in Arabidopsis thaliana(MDPI AG, 2022) Yadav, Sandeep; Yugandhar, Poli; Alavilli, Hemasundar; Raliya, Ramesh; Singh, Archita; Sahi, Shivendra V.; Sarkar, Ananda K.; Jain, AjayIn a hydroponic system, potassium chloroaurate (KAuCl4 ) triggers the in vitro sucrose (Suc)-dependent formation of gold nanoparticles (AuNPs). AuNPs stimulate the growth of the root system, but their molecular mechanism has not been deciphered. The root system of Arabidopsis (Arabidopsis thaliana) exhibits developmental plasticity in response to the availability of various nutrients, Suc, and auxin. Here, we showed the roles of Suc, phosphorus (P), and nitrogen (N) in facilitating a AuNPs-mediated increase in root growth. Furthermore, the recuperating effects of KAuCl4 on the natural (IAA) auxin-mediated perturbation of the root system were demonstrated. Arabidopsis seedlings harboring the cell division marker CycB1;1::CDB-GUS provided evidence of the restoration efficacy of KAuCl4 on the IAA-mediated inhibitory effect on meristematic cell proliferation of the primary and lateral roots. Arabidopsis harboring synthetic auxin DR5rev::GFP exhibited a reinstating effect of KAuCl4 on IAA-mediated aberration in auxin subcellular localization in the root. KAuCl4 also exerted significant and differential recuperating effects on the IAA-mediated altered expression of the genes involved in auxin signaling and biosynthetic pathways in roots. Our results highlight the crosstalk between KAuCl4 -mediated improved root growth and Suc and nutrient-dependent auxin homeostasis in Arabidopsis.Item Identification and analysis of miRNAs-lncRNAs-mRNAs modules involved in stem-elongation of deepwater rice (Oryza sativa L.)(John Wiley & Sons, 2022) Panda, Alok Kumar; Rawal, Hukam C.; Jain, Priyanka; Mishra, Vishnu; Nishad, Jyoti; Chowrasia, Soni; Sarkar, Ananda K.; Sen, Priyabrata; Naik, Soumendra Kumar; Mondal, Tapan KumarDeepwater is an abiotic stress that limits rice cultivation worldwide due to recurrent floods. The miRNAs and lncRNAs are two non-coding RNAs emerging as major regulators of gene expressions under different abiotic stresses. However, the regulation of these two non-coding RNAs under deepwater stress in rice is still unexplored. In this study, small RNA-seq and RNA-seq from internode and node tissues were analyzed to predict deepwater stress responsive miRNAs and lncRNAs, respectively. Additionally, a competitive endogenous RNA (ceRNA) study revealed about 69 and 25 lncRNAs acting as endogenous target mimics (eTM) with the internode and node miRNAs, respectively. In ceRNA analyses, some of the key miRNAs such as miR1850.1, miR1848 and IN-nov-miR145 were up-regulated while miR159e was down-regulated, and their respective eTM lncRNAs and targets were found to have opposite expressions. Moreover, we have transiently expressed one module (IN-nov-miR145–Cc-TCONS_00011544-Os11g36430.3) in tobacco leaves. The integrated analysis has identified differentially expressed miRNAs, lncRNAs and their target genes, and the complex regulatory network, which might lead to stem elongation under deepwater stress. In this novel attempt to identify and characterize miRNAs and lncRNAs under deepwater stress in rice, we have provided, probably for the first time, a reference platform to study the interactions of these two non-coding RNAs with respective target genes through transient expression analyses.Item A unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating ethylene and the abscisic acid pathway(The Company of Biologists, 2022) Mishra, Vishnu; Singh, Archita; Gandhi, Nidhi; Das, Shabari Sarkar; Yadav, Sandeep; Kumar, Ashutosh; Sarkar, Ananda K.Submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates Galactosyltransferase (GALT9) and their expression is inversely affected at 24 hours of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild type and MIM775 Arabidopsis shoot. A similar recovery phenotype of galt9 mutant indicates the role of miR775-GALT9 module in post-submergence recovery. We predicted Golgi-localized GALT9 to be potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29, and ORE1), ethylene signalling (EIN2 and EIN3) and ABA biosynthesis (NCED3) pathway genes in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role of miR775-GALT9 module in post-submergence recovery through a crosstalk with ethylene and ABA pathway.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 Role of abiotic stress responsive miRNAs in Arabidopsis root development(Springer Nature Publishing AG, 2020) Singh, Archita; Gandhi, Nidhi; Mishra, Vishnu; Yadav, Sandeep; Rai, Vandna; Sarkar, Ananda K.Abiotic stresses like drought, extreme temperature, and lack of sufficient water or nutrients adversely affect plant growth and productivity. The physiological responses of higher plants to the environmental stresses are largely influenced by the root system, which can quickly modulate its developmental pattern under changing water, nutrient, and temperature, as an adaptive response. Protein coding genes, phytohormones and microRNAs (miRNAs) are among the key players which imparts crucial intrinsic role in shaping the root development and its environment adaptive growth pattern. Among these factors, miRNAs belong to a class of small non-coding RNAs of 21–24 nucleotides in length, which regulates various aspects of plant growth and development by negatively regulating their target genes through either transcriptional cleavage or translational inhibition. Although many miRNAs have been identified to be differentially regulated under various abiotic stress conditions, only a limited number of them have been characterized, due to the complex nature of its regulation. However, some of the miRNAs, such as miR156, miR165/166, miR169 etc., have recently been shown to be involved in both abiotic stress response and root development, indicating the diverse role of miRNA mediated gene regulation. The field of miRNA mediated gene regulation is dynamically expanding and more miRNAs are being characterized for their function. Current review focuses on miRNAs that are differentially regulated by major abiotic stresses as well as are involved in root development in Arabidopsis thaliana. We highlight their role in regulation of multiple and diverse aspects of developmental and physiological processes in Arabidopsis.Item Tweaking microRNA-mediated gene regulation for crop improvement(Elsevier B.V., 2020) Yadav, Sandeep; Das, Shabari Sarkar; Kumar, Pramod; Mishra, Vishnu; Sarkar, Ananda K.Many beneficial agronomic traits of crops have been lost in the course of domestication and selective introgression processes. One of the best strategies to prevent the exclusion of beneficial traits during the introgression of novel traits is to selectively fine-tune the expression of some candidate genes or microRNAs (miRNAs). Noncoding regulatory miRNAs of 20–24 nucleotide (nt) length have evolved as specific posttranscriptional regulators that negatively regulate the transcript abundance of their target genes via either cleavage of mRNAs or translational inhibition. In this chapter, we summarize the current knowledge on the role of miRNAs and elaborate their use in miRNA-mediated gene regulation for the improvement of agronomic traits in crop plants. Recent CRISPR-Cas9-based genome engineering technology can be applied to selectively tweak the specific miRNA-target regulation, and thus, the function of the candidate genes to improve crop plants by conferring better productivity or resistance to abiotic or biotic stresses.Item Role of chromatin modification and remodeling in stem cell regulation and meristem maintenance in Arabidopsis(Oxford University Press, 2020) Singh, Sharmila; Singh, Alka; Singh, Archita; Mahima; Yadav, Sandeep; Bajaj, Ishita; Kumar, Shailendra; Jain, Ajay; Sarkar, Ananda K.In higher plants, pluripotent stem cells reside in the specialized microenvironment called stem cell niches (SCNs) harbored at the shoot apical meristem (SAM) and root apical meristem (RAM), which give rise to the aerial and underground parts of a plant, respectively. The model plant Arabidopsis thaliana (Arabidopsis) has been extensively studied to decipher the intricate regulatory mechanisms involving some key transcriptions factors and phytohormones that play pivotal roles in stem cell homeostasis, meristem maintenance, and organ formation. However, there is increasing evidence to show the epigenetic regulation of the chromatin architecture, gene expression exerting an influence on an innate balance between the self-renewal of stem cells, and differentiation of the progeny cells to a specific tissue type or organ. Post-translational histone modifications, ATP-dependent chromatin remodeling, and chromatin assembly/ disassembly are some of the key features involved in the modulation of chromatin architecture. Here, we discuss the major epigenetic regulators and illustrate their roles in the regulation of stem cell activity, meristem maintenance, and related organ patterning in Arabidopsis.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.