Browsing by Author "Rout, Biswaranjan"

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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.
Iono-metabolomic guided elucidation of arsenic induced physiological and metabolic dynamics in wheat genotypes
(Elsevier B.V., 2023) Saini, Himanshu; Panthri, Medha; Rout, Biswaranjan; Pandey, Ashutosh; Gupta, Meetu
Despite the growing concerns about arsenic (As) toxicity, information on wheat adaptability in such an aggravating environment is limited. Thus, the present investigation based on an iono-metabolomic approach is aimed to decipher the response of wheat genotypes towards As toxicity. Wheat genotypes procured from natural conditions were characterized as high As-contaminated (Shri ram-303 and HD-2967) and low As-contaminated (Malviya-234 and DBW-17) based on ICP-MS As accumulation analysis. Reduced chlorophyll fluorescence attributes, grain yield and quality traits, and low grain nutrient status were accompanied by remarkable grain As accumulation in high As-contaminated genotypes, thus imposing a higher potential cancer risk and hazard quotient. Contrarily, in low As-contaminated genotypes, the richness of Zn, N, Fe, Mn, Na, K, Mg, and Ca could probably have supported less grain As accumulation, imparting better agronomic and grain quality traits. Additionally, from metabolomic analysis (LC-MS/MS and UHPLC), abundances of alanine, aspartate, glutamate, quercetin, isoliquiritigenin, trans-ferrulic, cinnamic, caffeic, and syringic bestow Malviya-234 as the best edible wheat genotype. Further, the multivariate statistical analysis (HCA, PCA, and PLS-DA) revealed certain other key metabolites (rutin, nobletin, myricetin, catechin, and naringenin) based genotypic discrimination that imparts strength to genotypes for better adaptation in harsh conditions. Out of the 5 metabolic pathways ascertained through topological analysis, the two main pathways vital for plant's metabolic adjustments in an As-induced environment were: 1. The alanine, aspartate and glutamate metabolism pathway, and 2. The flavonoid biosynthesis pathway. This is also evident from network analysis, which stipulates amino acid metabolism as a prominent As regulatory factor closely associated with flavonoids and phenolics. Therefore, the present findings are useful for wheat breeding programs to develop As adaptive genotypes that are beneficial for crop improvement and human health.
Two homeologous MATE transporter genes, NtMATE21 and NtMATE22, are involved in the modulation of plant growth and flavonol transport in Nicotiana tabacum
(Oxford University Press, 2022) Gani, Umar; Nautiyal, Abhishek Kumar; Kundan, Maridul; Rout, Biswaranjan; Pandey, Ashutosh; Misra, Prashant
Multidrug and toxic compound extrusion (MATE) family has been implicated in the transport of a diverse range of molecules, including specialized metabolites. In Nicotiana tabacum, only a limited number of MATE transporters have been functionally characterized, and no MATE transporter has been studied in the context of flavonoid transport in this plant species so far. Here in the present study, we characterize two homeologous MATE genes, NtMATE21 and NtMATE22, and demonstrate their role in flavonol transport and in plant growth and development. The expression of these two genes was reported to be up-regulated in trichomes as compared to the trichome-free leaf. The transcript levels of NtMATE21 and NtMATE22 were found to be higher in flavonol over-producing tobacco transgenic lines as compared to wild type (WT) tobacco. The two transporters were demonstrated to be localized to the plasma membrane. Genetic manipulation of NtMATE21 and NtMATE22 led to altered growth phenotypes and modulated flavonol contents in N. tabacum. The GUS and GFP fusion transgenic lines of promoter regions suggested that NtMATE21 and NtMATE22 exclusively express in the trichome heads in the leaf tissue and petals. Moreover, in transient transactivation assay, NtMYB12, a flavonol-specific MYB transcription factor, was found to transactivate the expression of NtMATE21 and NtMATE22 genes. Together, our results strongly suggest the involvement of NtMATE21 and NtMATE22 in flavonol transport as well as in the regulation of plant growth and development.