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Item PHYTOCHROME INTERACTING FACTOR1 interactions leading to the completion or prolongation of seed germination(Taylor & Francis Group, 2018) Dirk, Lynnette M. A.; Kumar, Santosh; Majee, Manoj; Downie, A. BruceIn Arabidopsis thaliana, the basic Helix Loop Helix transcription factor, PHYTOCHROME INTERACTING FACTOR1 (PIF1) is known to orchestrate the seed transcriptome such that, ultimately, proteins repressing the completion of germination are produced in darkness. While PIF1-mediated control of abscisic acid (ABA) and gibberellic acid (GA) anabolism/catabolism is indirect, PIF1 action favors ABA while discriminating against GA, firmly establishing ABA’s repressive influence on the completion of germination. The result is tissue that is more sensitive to and producing more ABA; and is less responsive to and deficient in GA. Illumination of the appropriate wavelength activates phytochrome which enters the nucleus, and binds to PIF1, initiating PIF1’s phosphorylation by diverse kinases, subsequent polyubiquitination, and hydrolysis. One mechanism by which phosphorylated PIF1 is eliminated from the cells of the seed upon illumination involves an F-BOX protein, COLD TEMPERATURE GERMINATING10 (CTG10). Discovered in an unbiased screen of activation tagged lines hastening the completion of seed germination at 10°C, one indirect consequence of CTG10 action in reducing PIF1 titer, should be to enhance the transcription of genes whose products work to increase bioactive GA titer, shifting the intracellular milieu from one that is repressive to, toward one conducive to, the completion of seed germination. We have tested this hypothesis using a variety of Arabidopsis lines altered in CTG10 amounts. Here we demonstrate using bimolecular fluorescence complementation that PIF1 interacts with CTG10 and show that, in light exposed seeds, PIF1 is more persistent in ctg10 relative to WT seeds while it is less stable in seeds over-expressing CTG10. These results are congruent with the relative transcript abundance from three genes whose products are involved in bioactive GA accumulation. We put forth a model of how PIF1 interactions in imbibed seeds change during germination and how a permissive light signal influences these changes, leading to the completion of germination of these positively photoblastic propagules.Item Kelch f-box protein positively influences Arabidopsis seed germination by targeting phytochrome-interacting factor1(PNAS, USA, 2018) Majee, Manoj; Kumar, Santosh; Kathare, Praveen Kumar; Wu, Shuiqin; Gingerich, Derek; Nayak, Nihar R.; Salaita, Louai; Dinkins, Randy; Martin, Kathleen; Goodin, Michael; Dirk, Lynnette M. A.; Lloyd, Taylor D.; Zhu, Ling; Chappell, Joseph; Hunt, Arthur G.; Vierstra, Richard; Huq, Enamul; Downie, A. BruceSeeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here we show that the Arabidopsis F-BOX protein COLD TEMPERATURE-GERMINATING (CTG)-10, identified by activation tagging, is a positive regulator of this process. When overexpressed (OE), CTG10 hastens aspects of seed germination. CTG10 is expressed predominantly in the hypocotyl, and the protein is localized to the nucleus. CTG10 interacts with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) and helps regulate its abundance in planta. CTG10-OE accelerates the loss of PIF1 in light, increasing germination efficiency, while PIF1-OE lines fail to complete germination in darkness, which is reversed by concurrent CTG10-OE. Double-mutant (pif1 ctg10) lines demonstrated that PIF1 is epistatic to CTG10. Both CTG10 and PIF1 amounts decline during seed germination in the light but reaccumulate in the dark. PIF1 in turn down-regulates CTG10 transcription, suggesting a feedback loop of CTG10/PIF1 control. The genetic, physiological, and biochemical evidence, when taken together, leads us to propose that PIF1 and CTG10 coexist, and even accumulate, in the nucleus in darkness, but that, following illumination, CTG10 assists in reducing PIF1 amounts, thus promoting the completion of seed germination and subsequent seedling development.Item A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don(Nature Publishing Group, 2016) Kumar, Santosh; Bhatia, SabhyataSimple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5′ flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5′UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both at the level of transcription and translation. Transcript abundance was estimated using Northern analysis and qRT-PCR, whereas the rate of Tdc gene transcription was assessed using in-situ nuclear run-on transcription assay. Translation status of Tdc gene was monitored by quantification of polysome associated Tdc mRNA using qRT-PCR. These observations were validated through transient expression analysis using the fusion construct [CaM35S:(CT)8–21:GUS]. Our study demonstrated that not only does the length of (CT)n -SSRs influences the promoter activity, but the presence of SSRs per se in the 5′-UTR significantly enhances the level of gene expression. We termed this phenomenon as “microsatellite mediated enhancement” (MME) of gene expression. Results presented here will provide leads for engineering plants with enhanced amounts of medicinally important alkaloids.Item Identification of novel miRNAs from drought tolerant rice variety Nagina 22(Nature Publishing Group, 2016) Mutum, Roseeta Devi; Kumar, Santosh; Balyan, Sonia; Kansal, Shivani; Mathur, Saloni; Raghuvanshi, SaurabhMicroRNAs regulate a spectrum of developmental and biochemical processes in plants and animals. Thus, knowledge of the entire miRNome is essential to understand the complete regulatory schema of any organism. The current study attempts to unravel yet undiscovered miRNA genes in rice. Analysis of small RNA libraries from various tissues of drought-tolerant 'aus' rice variety Nagina 22 (N22) identified 71 novel miRNAs. These were validated based on precursor hairpin structure, small RNA mapping pattern, 'star' sequence, conservation and identification of targets based on degradome data. While some novel miRNAs were conserved in other monocots and dicots, most appear to be lineage-specific. They were segregated into two different classes based on the closeness to the classical miRNA definition. Interestingly, evidence of a miRNA-like cleavage was found even for miRNAs that lie beyond the classical definition. Several novel miRNAs displayed tissue-enriched and/or drought responsive expression. Generation and analysis of the degradome data from N22 along with publicly available degradome identified several high confidence targets implicated in regulation of fundamental processes such as flowering and stress response. Thus, discovery of these novel miRNAs considerably expands the dimension of the miRNA-mediated regulation in rice.Item Isolation of Catharanthus roseus (L.) G. don nuclei and measurement of rate of Tryptophan decarboxylase gene transcription using nuclear run-on transcription assay(PLOS, 2015) Kumar, Santosh; Bhatia, SabhyataBACKGROUND: An accurate assessment of transcription 'rate' is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription 'rate'. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a 'molecular hub' to understand gene expression profiles. RESULTS: The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli. CONCLUSIONS: Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear 'run-on' transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run-on transcript could be further subjected to RNA-Seq for global nuclear run-on assay (GNRO-Seq) for genome wide in-situ measurement of transcription rate of plant genes.Item Insights into the small RNA-mediated networks in response to abiotic stress in plants(Springer, 2015) Balyan, Sonia C.; Mutum, Roseeta D.; Kansal, Shivani; Kumar, Santosh; Mathur, Saloni; Raghuvanshi, SaurabhUnder natural conditions, plants are constantly exposed to various environmental stresses such as drought, extreme temperature, salt, UV, mechanical, or nutrient starvation. To cope with these adverse conditions, plants have evolved cascade of molecular networks to perceive and transduce the stress signals, resulting into the reprogramming of gene expression. The stress-regulated reprogramming of gene expression at post-transcriptional regulation has been emphasized with the discovery of small regulatory RNAs. Plant small RNAs represent non-coding RNAs in the size range of 20–24 nucleotides and categorized into hairpin RNAs (hpRNAs) and siRNAs. The first category includes miRNAs, lmiRNAs, and nat-miRNAs while the siRNA group includes hc-siRNA, secondary siRNAs and nat-siRNAs. Studies have shown that small RNAs, especially miRNAs, are dynamically regulated by a variety of abiotic stress conditions. Such sRNAs target a variety of downstream targets including regulatory proteins as well as metabolic enzymes and thus play pivotal role in the regulation of plant abiotic stress response. Stress appears to regulate miRNA biogenesis as well as its activity. Several miRNA gene:target pairs respond to multiple stress conditions and are conserved in various plant species indicating that miRNAs may define pivotal regulatory nodes involved in the regulation of the plant stress response. On the other hand, miRNAs also show variety-/cultivar-specific stress response indicating that they themselves are under a very dynamic regulation. The world of small RNAs is gradually unfolding and much remains to be explored, nevertheless, it has been conclusively demonstrated that small RNAs define a new dimension in the molecular regulatory network regulating the plant stress response.Item Large scale in-silico identification and characterization of simple sequence repeats (SSRs) from de novo assembled transcriptome of Catharanthus roseus (L.) G. Don(Springer, 2014) Kumar, Santosh; Shah, Niraj; Garg, Vanika; Bhatia, SabhyataTranscriptomic data of C. roseus offering ample sequence resources for providing better insights into gene diversity: large resource of genic SSR markers to accelerate genomic studies and breeding in Catharanthus . Next-generation sequencing is an efficient system for generating high-throughput complete transcripts/genes and developing molecular markers. We present here the transcriptome sequencing of a 26-day-old Catharanthus roseus seedling tissue using Illumina GAIIX platform that resulted in a total of 3.37 Gb of nucleotide sequence data comprising 29,964,104 reads which were de novo assembled into 26,581 unigenes. Based on similarity searches 58 % of the unigenes were annotated of which 13,580 unique transcripts were assigned 5016 gene ontology terms. Further, 7,687 of the unigenes were found to have Cluster of Orthologous Group classifications, and 4,006 were assigned to 289 Kyoto Encyclopedia of Genes and Genome pathways. Also, 5,221 (19.64 %) of transcripts were distributed to 81 known transcription factor (TF) families. In-silico analysis of the transcriptome resulted in identification of 11,004 SSRs in 26.62 % transcripts from which 2,520 SSR markers were designed which exhibited a non-random pattern of distribution. The most abundant was the trinucleotide repeats (AAG/CTT) followed by the dinucleotide repeats (AG/CT). Location specific analysis of SSRs revealed that SSRs were preferentially associated with the 5'-UTRs with a predicted role in regulation of gene expression. A PCR validation of a set of 48 primers revealed 97.9 % successful amplification, and 76.6 % of them showed polymorphism across different Catharanthus species as well as accessions of C. roseus. In summary, this study will provide an insight into understanding the seedling development and resources for novel gene discovery and SSR development for utilization in marker-assisted selective breeding in C. roseus.Item Ectopic overexpression of vacuolar and apoplastic Catharanthus roseus peroxidases confers differential tolerance to salt and dehydration stress in transgenic tobacco(Springer Science, 2012) Kumar, Santosh; Jaggi, Monika; Sinha, Alok KrishnaCrPrx and CrPrx1 are class III peroxidases previously cloned and characterized from Catharanthus roseus. CrPrx is known to be apoplastic in nature, while CrPrx1 is targeted to vacuoles. In order to study their role in planta, these two peroxidases were expressed in Nicotiana tabacum. The transformed plants exhibited increased peroxidase activity. Increased oxidative stress tolerance was also observed in transgenics when treated with H(2)O(2) under strong light conditions. However, differential tolerance to salt and dehydration stress was observed during germination of T1 transgenic seeds. Under these stresses, the seed germination of CrPrx-transformed plants and wild-type plants was clearly suppressed, whereas CrPrx1 transgenic lines showed improved germination. CrPrx-transformed lines exhibited better cold tolerance than CrPrx1-transformed lines. These results indicate that vacuolar peroxidase plays an important role in salt and dehydration stress over cell wall-targeted peroxidase, while cell wall-targeted peroxidase renders cold stress tolerance.Item Cloning and characterization of two new Class III peroxidase genes from Catharanthus roseus(Elsevier, 2011) Kumar, Santosh; Jaggi, Monika; Taneja, Jyoti; Sinha, Alok KrishnaWe report here the cloning and characterization of two new class III peroxidase genes, CrPrx3 and CrPrx4 from Catharanthus roseus. The full length cDNA of CrPrx3 is 1233 bp long encoding 330 amino acid residues. The CrPrx4 cDNA contains an ORF of 1055 bp, and encodes for 318 amino acids. Hypothetical 3-D structure model of CrPrx3 and CrPrx4 detected the presence of two Ca(2+) ion binding sites at proximal and distal end, and a heme group coordinate at central position. CrPrx3 and CrPrx4 are present in single copy number in C. roseus. Confocal scanning microscopy and phylogenetic analysis showed that CrPrx3 and CrPrx4 are apoplastic in nature. Phylogenetic analysis revealed that both genes have diverse expression patterns in a variety of plant tissues. Quantitative real-time PCR confirmed their maximum expression in stem tissues followed by flower tissue. The expression levels of CrPrx3 and CrPrx4 were down-regulated under salt and dehydration stress, respectively.Item Overexpression of an apoplastic peroxidase gene CrPrx in transgenic hairy root lines of Catharanthus roseus(Springer Science, 2011) Jaggi, Monika; Kumar, Santosh; Sinha, Alok KrishnaPeroxidases are a family of isoenzymes found in all higher plants and are known to be involved in a broad range of physiological processes. However, very little information is available concerning their role in Catharanthus roseus. The present study describes the impact of both overexpression and suppression of a peroxidase gene, CrPrx in C. roseus transgenic hairy root lines. Real-time PCR analysis in 35S-CrPrx and CrPrx-RNAi transgenic lines indicated differential transcript profile for peroxidases as well as for genes and regulators involved in MIA (monoterpenoid indole alkaloid) pathway of C. roseus. Comparative analysis revealed that MIA pathway genes showing elevated levels of expression in 35S-CrPrx transgenic lines showed a significant reduction in their transcript level in CrPrx-RNAi transgenic lines. Metabolite analysis detected higher levels of ajmalicine and serpentine accumulation in overexpressed lines. It was observed that all overexpressed transgenic lines produced more amount of H(2)O(2). These results indicate a role of CrPrx gene in the regulation of MIA pathway genes and regulators, thus affecting the production of specific alkaloids.