Publications of NIPGR Scientists

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Author: search.filters.author.Pandey, AshutoshSubject: search.filters.subject.Transcription factor

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    CRISPR/dCas9-KRAB mediated transcriptional suppression of NtbHLH47 enhances tolerance to iron stress and modulates iron content in tobacco
    (Elsevier B.V., 2025) Alok, Anshu; Chauhan, Hanny; Rout, Biswaranjan; Pandey, Ashutosh; Singh, Kashmir
    Iron homeostasis is a multifaceted regulatory process that needs to be studied to elucidate iron distribution, uptake, and storage in plants. NtbHLH47, a homologue to AtbHLH47, is a negative regulator of iron. The current study deploys CRISPR interference-dCas9-KRAB (Krüppel-associated box) in the transcriptional suppression of NtbHLH47 and its effect on iron uptake by plants. The pHSN6I01 harbouring dCas9-KRAB and gRNA targeting NtbHHLH47 was constructed. Four gRNAs were designed, G1, G2, G3, and G4, located at + 19, + 111, + 232, and + 335 bp upstream from the ATG start codon in the promoter region of NtbHLH47. The NtbHLH47 was repressed in the developed transgenic lines of tobacco and the qRT-PCR analysis showed that target sites G1 and G2 suppressed NtbHLH47 effectively. The transgenic pHSN6I01 +G1 plants were tolerant to the elevated levels of iron, copper, zinc, and magnesium. The root Ferric chelate reductase activity of pHSN6I01 +G1 lines was reduced against wild type. The Perl staining showed high iron content in the roots of the pHSN6I01 +G1 plants. ICP-MS analysis showed increased Fe content in the roots of pHSN6I01 +G1 line suggesting that NtbHLH47 modulates it. The expression of NtbHLH38, NtbHLH100, NtbHLH101, and NtFIT was found to be upregulated in the pHSN6I01 +G1 line. This is the first report of using CRISPRi based on dCas9-KRAB in tobacco and its application in the functional validation of a gene. Using this, NtbHLH47 was transcriptionally suppressed and the generated lines expressed increased levels of iron in the roots of N. tabacum and gave insight in the iron homeostasis.
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    Deciphering the complexity of terpenoid biosynthesis and its multi-level regulatory mechanism in plants
    (Springer Nature Publishing AG, 2024) Singh, Samar; Chhatwal, Himani; Pandey, Ashutosh
    Terpenoids are one of the essential plant metabolites which are known to play vital roles in plants' primary growth and development including protection from biotic and abiotic stresses. They have huge structural diversity and are known to provide various health benefits, flavors, fragrances, essential oils, cosmetics, pigments, insecticides, etc. The biosynthesis of terpenoids by mevalonic acid (MVA) and methylerythritol pathway (MEP) occurs inside the cytoplasm and the chloroplast, respectively. Here, we provide a comprehensive overview of synthesis, metabolic, and regulatory pathways of terpenoid, their different classes and ecological roles. We also discuss in detail the key transcription factors (TFs) like WRKY, AP2/ERF, bHLH, MYB, NAC, and bZIP which reprogram and modulate the terpenoid pathway in plants. It also includes the post-transcriptional and post-translational modifications of these pathways. The post-transcriptional regulation by microRNA plays a critical role in the synthesis and regulation of the terpenoid. MAP kinases also regulate the stability of biosynthetic enzymes and transcription factors to regulate terpenoid biosynthesis. High-throughput sequencing technology and functional genomics have further strengthened our understanding of this pathway and associated regulatory genes which control it. As a future perspective, modifying these transcription factors via various strategies holds promise for improvement of agricultural crop plants in terms of nutritional enrichment, stress responsiveness, and resistance.
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    The R2R3-MYB gene family in Cicer arietinum: genome-wide identification and expression analysis leads to functional characterization of proanthocyanidin biosynthesis regulators in the seed coat
    (Springer Nature Publishing AG, 2022) Rajput, Ruchika; Tyagi, Shivi; Naik, Jogindra; Pucker, Boas; Stracke, Ralf; Pandey, Ashutosh
    Chickpea (Cicer arietinum) is among the eight oldest crops and has two main types, i.e., desi and kabuli, whose most obvious difference is the color of their seeds. We show that this color difference is due to differences in proanthocyanidin content of seed coats. Using a targeted approach, we performed in silico analysis, metabolite profiling, molecular, genetic, and biochemical studies to decipher the transcriptional regulatory network involved in proanthocyanidin biosynthesis in the seed coat of C. arietinum. Based on the annotated C. arietinum reference genome sequence, we identified 119 typical CaMYB encoding genes, grouped in 32 distinct clades. Two CaR2R3-MYB transcription factors, named CaPAR1 and CaPAR2, clustering with known proanthocyanidin regulators (PARs) were identified and further analyzed. The expression of CaPAR genes correlated well with the expression of the key structural proanthocyanidin biosynthesis genes CaANR and CaLAR and with proanthocyanidin levels. Protein–protein interaction studies suggest the in vivo interaction of CaPAR1 and CaPAR2 with the bHLH-type transcription factor CaTT8. Co-transfection analyses using Arabidopsis thaliana protoplasts showed that the CaPAR proteins form a MBW complex with CaTT8 and CaTTG1, able to activate the promoters of CaANR and CaLAR in planta. Finally, transgenic expression of CaPARs in the proanthocyanidin-deficient A. thaliana mutant tt2-1 leads to complementation of the transparent testa phenotype. Taken together, our results reveal main components of the proanthocyanidin regulatory network in C. arietinum and suggest that CaPARs are relevant targets of genetic engineering toward improved agronomic traits.