Publications of NIPGR Scientists
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Item MEDIATOR SUBUNIT17 is required for transcriptional optimization of root system architecture in Arabidopsis(Oxford University Press, 2023) Agrawal, Rekha; Singh, Amrita; Giri, Jitender; Magyar, Zoltan; Thakur, Jitendra K.Sucrose and auxin are well-known determinants of root system architecture (RSA). However, the factors that connect the signaling pathways evoked by these two critical factors during root development are poorly understood. In this study, we report the role of MEDIATOR SUBUNIT17 (MED17) in RSA and its involvement in the transcriptional integration of sugar and auxin signaling pathways in Arabidopsis (Arabidopsis thaliana). Sucrose regulates root meristem activation through the TARGET OF RAPAMYCIN-E2 PROMOTER BINDING FACTOR A TOR-E2FA pathway, and auxin regulates lateral root (LR) development through AUXIN RESPONSE FACTOR-LATERAL ORGAN BOUNDARIES DOMAIN ARF-LBDs. Both sucrose and auxin play a vital role during primary and LR development. However, there is no clarity on how sucrose is involved in the ARF-dependent regulation of auxin-responsive genes. This study establishes MED17 as a nodal point to connect sucrose and auxin signaling. Transcription of MED17 was induced by sucrose in an E2FA/B-dependent manner. Moreover, E2FA/B interacted with MED17, which can aid in the recruitment of the Mediator complex on the target promoters. Interestingly, E2FA/B and MED17 also occupied the promoter of ARF7, but not ARF19, leading to ARF7 expression, which then activates auxin signaling and thus initiates LR development. MED17 also activated cell division in the root meristem by occupying the promoters of cell-cycle genes, thus regulating their transcription. Thus, MED17 plays an important role in relaying the transcriptional signal from sucrose to auxin-responsive and cell-cycle genes to regulate primary and lateral root development, highlighting the role of the Mediator as the transcriptional processor for optimal root system architecture in Arabidopsis.Item Mediator subunit OsMED14_1 plays an important role in rice development(John Wiley & Sons, 2020) Malik, Naveen; Ranjan, Rajeev; Parida, Swarup K.; Agarwal, Pinky; Tyagi, Akhilesh K.Mediator, a multi‐subunit coactivator complex, regulates transcription in eukaryotes and is involved in diverse processes in Arabidopsis through its different subunits. Here, we have explored developmental aspects of one of the rice Mediator subunit gene OsMED14_1. We analyzed its expression pattern through RNA in‐situ hybridization and pOsMED14_1:GUS transgenics which showed its expression in roots, leaves, anthers and seeds prominently at younger stages, indicating possible involvement of this subunit in multiple aspects of rice development. To understand developmental roles of OsMED14_1 in rice, we generated and studied RNAi based knockdown rice plants which showed multiple effects including less height, narrower leaves and culms with reduced vasculature, lesser lateral root branching, defective microspore development, reduced panicle branching and seed set, and smaller seeds. Histological analyses showed that slender organs were caused by reduction in both cell number and cell size in OsMED14_1 knockdown plants. Flow cytometric analyses and expression analyses of cell‐cycle related genes revealed that defective cell‐cycle progression led to these defects. Expression analyses of auxin related genes and IAA immuno‐localization study indicated altered auxin level in these knockdown plants. Reduction of lateral root branching in knockdown plants was corrected by exogenous IAA supplement. OsMED14_1 physically interacts with transcription factors YABBY5, TDR and MADS29, possibly regulating auxin homeostasis and ultimately leading to lateral organ/leaf, microspore and seed development.Item The Mediator subunit OsMED15a is a transcriptional co-regulator of seed size/weight-modulating genes in rice(Elsevier B.V., 2019) Dwivedi, Nidhi; Maji, Sourobh; Waseem, Mohd; Thakur, Pallabi; Kumar, Vinay; Parida, Swarup K.; Thakur, Jitendra K.Although several transcription factors (TFs) that regulate seed size/weight in plants are known, the molecular landscape regulating this important trait is unclear. Here, we report that a Mediator subunit, OsMED15a, links rice grain size/weight-regulating TFs to their target genes. Expression analysis and high-resolution quantitative trait loci (QTL) mapping suggested that OsMED15a is involved in rice seed development. OsMED15a has an N-terminal, three-helical KIX domain. Two of these helices, α1 and α3, and three amino acids, 76LRC78, within OsMED15a helix α3 were important for its interaction with several proteins, including interactions with the transactivation domains of two NAC-type TFs, OsNAC024 and OsNAC025. Moreover, OsMED15a, OsNAC024, and OsNAC025 all exhibited increased expression during seed development, and we identified several grain size/weight-associated SNPs in these genes in 509 low- and high-grain-weight rice genotypes. RNAi-mediated repression of OsMED15a expression down-regulated the expression of the grain size/weight regulating genes GW2, GW5 and DR11 and reduced grain length, weight, and yield. Of note, both OsNAC024 and OsNAC025 bound to the promoters of these three genes. We conclude that the transactivation domains of OsNAC024 and OsNAC025 target the KIX domain of OsMED15a in the regulation of grain size/weight-associated genes such as GW2, GW5, and D11. We propose that the integrated molecular-genetics approach used here could help identify networks of functional alleles of other regulator and co-regulator genes and thereby inform efforts for marker-assisted introgression of useful alleles in rice crop improvement.Item KIX domain of AtMed15a, a Mediator subunit of Arabidopsis, is required for its interaction with different proteins(Taylor & Francis Group, 2018) Kumar, Vinay; Waseem, Mohd; Dwivedi, Nidhi; Maji, Sourobh; Kumar, Angad; Thakur, Jitendra K.Med15 is an important subunit of Mediator tail module and is characterized by a KIX domain present in the amino terminal. In yeast and metazoans, Med15 KIX domain has been found to interact with various transcription factors, regulating several processes including carbohydrate metabolism, lipogenesis, stress response and multidrug resistance. Mechanism of Med15 functioning in Arabidopsis is largely unknown. In this study, interactome of Arabidopsis Med15, AtMed15a, was characterized. We found 45 proteins that interact with AtMed15a KIX domain, including 11 transcription factors, 3 single strand nucleic acid-binding proteins and 1 splicing factor. The third helix of the KIX domain was found to be involved in most of the interactions. Mapping of the regions participating in the interactions revealed that the activation domain of a transcription factor, UKTF1 interacted with AtMed15a KIX domain. Thus, our results suggest that in Arabidopsis, activation domain of transcription factors target KIX domain of AtMed15a for their transcriptional responses.Item Emerging functions of multi-protein complex Mediator with special emphasis on plants(Taylor & Francis Group, 2017) Malik, Naveen; Agarwal, Pinky; Tyagi, Akhilesh K.Mediator is a multi-subunit protein complex which is involved in transcriptional regulation in yeast and other eukaryotes. As a co-activator, it connects information from transcriptional activators/repressors to transcriptional machinery including RNA polymerase II and general transcription factors. It is not only involved in transcription initiation but also has important roles to play in transcription elongation and termination. Functional attributes of different Mediator subunits have been largely defined in yeast and mammalian systems earlier, while such studies in plants have gained momentum recently. Mediator regulates various processes related to plant development and is also involved in biotic and abiotic stress response. Thus, plant Mediator, like yeast and mammalian Mediator complex, is indispensable for plant growth and survival. Interaction of its multiple subunits with other regulatory proteins and their ectopic expression or knockdown in model plant like Arabidopsis and certain crop plants are paving the way to biochemical analysis and unravel molecular mechanisms of action of Mediator in plants.Item Sequence and expression analyses of KIX domain proteins suggest their importance in seed development and determination of seed size in rice, and genome stability in Arabidopsis(Springer, 2013) Thakur, Jitendra K.; Agarwal, Pinky; Parida, Swarup K.; Bajaj, Deepak; Pasrija, RichaThe KIX domain, which mediates protein-protein interactions, was first discovered as a motif in the large multidomain transcriptional activator histone acetyltransferase p300/CBP. Later, the domain was also found in Mediator subunit MED15, where it interacts with many transcription factors. In both proteins, the KIX domain is a target of activation domains of diverse transcription activators. It was found to be an essential component of several specific gene-activation pathways in fungi and metazoans. Not much is known about KIX domain proteins in plants. This study aims to characterize all the KIX domain proteins encoded by the genomes of Arabidopsis and rice. All identified KIX domain proteins are presented, together with their chromosomal locations, phylogenetic analysis, expression and SNP analyses. KIX domains were found not only in p300/CBP- and MED15-like plant proteins, but also in F-box proteins in rice and DNA helicase in Arabidopsis, suggesting roles of KIX domains in ubiquitin-mediated proteasomal degradation and genome stability. Expression analysis revealed overlapping expression of OsKIX_3, OsKIX_5 and OsKIX_7 in different stages of rice seeds development. Moreover, an association analysis of 136 in silico mined SNP loci in 23 different rice genotypes with grain-length information identified three non-synonymous SNP loci in these three rice genes showing strong association with long- and short-grain differentiation. Interestingly, these SNPs were located within KIX domain encoding sequences. Overall, this study lays a foundation for functional analysis of KIX domain proteins in plants.Item Analysis of differential expression of mediator subunit genes in Arabidopsis(Landes Bioscience, 2012) Pasrija, Richa; Thakur, Jitendra K.Mediator is a conserved eukaryotic multiprotein complex required by RNA polymerase II for transcription of its target genes. Till date, there is no report explaining the signals that affect the overall concentration of individual Med subunits. In this report, we have analyzed the effect of different phytohormones and stresses on the transcript level of Med genes in Arabidopsis. Hormones like auxin and JA, and cold stress did not show significant effect. ABA moderately increased the transcript abundance of more than 70% of AtMed genes analyzed in this study. However, there was noticeable change in the transcript level of several AtMed genes in response to BR. Stresses like high light, dark and salt also caused significant change in the transcript abundance of many AtMed genes. These data reveal that different environmental cues can affect stoichiometric concentration of Med subunits by affecting the transcription of their respective genes. This may, in turn, affect the overall arrangement of functional Mediator complex. This also suggests that some subunits may have some specific functions to play in response different signals.