Botany - Research Publications

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    DNA Barcoding, Phylogeny and Phylogeography of Green Sea Weed Ulva from Indian Subcontinent
    (Central University of Punjab, 2019) Rani, Pooja; Bast, Felix
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    DNA barcoding and phylogeny based comparative evaluation of anti-cancer properties of Caulerpa (J V Lamouroux) spp. from Indian coasts
    (Central University of Punjab, 2019) Mehra, Richa; Bast, Felix and Singh, Sandeep
    A total of 15 Caulerpa samples were collected from Indian coasts and identified based on morphological and molecular data inferred from ITS, 18S, tufA and rbcL. Seven different species viz. C. scalpelliformis, C. racemosa, C. sertularioides, C. verticillata, C. taxifolia, and C. corynephora; and their geographical isolates were identified. Barcode data for these species was generated using aforementioned molecular markers and used for phylogenetic assessment. Phylogenetic trees using Bayesian inference (BI) and Maximum Likelihood (ML) function were generated for each molecular marker. tufA was found to be most suitable marker for the genus Caulerpa, resolving the species into 17 different lineages, with 15 corresponding to already known sections and 2 new lineages. Besides, a database named DbIndAlgae of Indian algae was generated and all the morphological as well as molecular data generated in this study is uploaded on the database. In addition, the phycochemical analysis revealed the presence of alkaloids, terpenoids, steroids, tannins, saponins, flavonoids, and phenols in different Caulerpa species. The selective cytotoxicity of methanolic extracts of Caulerpa (CMEs) was evaluated on MDA-MB-231, T47-D and H1299 cells, and the results revealed significant cytotoxicity of all species. C. racemosa KNY-254 and C. taxifolia TEN-158 were found to be most potent on MDA-MB-231 cells with IC50 value of 0.226 ± 0.004 and 0.246 ± 0.009 µg/µL. The mitochondrial membrane perturbation was revealed by JC-1 and apoptotic cell death was confirmed by Annexin V/FITC staining. CMEs also induced ROS in MDA-MB-231 cells as depicted by DHE, and increased activity of SOD, decreased activity of gluthatione reductase. The CMEs also exhibit anti-invasion activity and inhibited up to 71% migration across the artificially scratched wound in MDA-MB-231, w.r.t. untreated control cells. Moreover, chemical probing of C. racemosa KNY-254 by LC-MS analysis revealed six previously reported and six unreported molecules. The molecular docking analysis revealed weak to moderate interactions with all of the protein targets viz. Bcl2, AMPK, mTOR, BID, PERK, IGF-1R, PI3K, PTP1B and Akt2, known to play important role in cancer cell signaling. Additionally, a moderately positive correlation amongst the phylogeny and anti-cancer activity was observed suggesting that phylogeny might provide cues for anti-cancer activity, subject to further validations.
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    Transcriptomic investigations of gene networks in response to arsenic accumulation in Brassica juncea (L.) Czern & Coss
    (Central University of Punjab, 2019) Thakur, Sapna; Bhardwaj, Pankaj
    Arsenic (As), a widespread toxic metalloid is class I carcinogen known to cause adverse health effects in human. In the present study, As accumulation potential and differential gene expression in B. juncea is investigated. The amount of arsenic accumulated varied in the range of 15.99 to 1138.70 mg/Kg on dry weight basis in five cultivars. A decrease in chlorophyll content and increase in membrane damage and enzymatic activities of antioxidants was observed with increase in As concentration in the B. juncea cultivars. Using maximum As accumulating cultivar (RLM514), a total of 10,870 significantly differentially expressed transcripts in response to As treatment were identified. Further, the pathway analysis revealed a large scale reprogramming of genes involving carbon metabolism (2.5%), plant hormone signaling (1.4%), and glutathione metabolism (0.6%). Moreover, a comparative account of Cd toxicity revealed a total of 11,294 transcripts to be significantly differentially expressed. The genes related to response to chemical, oxidative stress, transport, and secondary metabolism were upregulated whereas multicellular organismal development, developmental process, photosynthesis were downregulated by Cd treatment. Furthermore, 616 membrane transport proteins were found to be significantly differentially expressed. Cd-related transporters such as metal transporter (Nramp1), metal tolerance protein (MTPC2, MTP11), cadmiumtransporting ATPase, and plant cadmium resistance protein (PCR2, PCR6) were upregulated while cadmium/zinc- transporting ATPase (HMA2, HMA3, HMA4), highaffinity calcium antiporter (CAX1), and iron transport protein (IRT1) were downregulated by Cd treatment. Pathway analysis revealed signaling cascades including plant hormones signaling, MAPK signaling and Ca signaling was modulated suggesting their role in Cd-stress tolerance. The regulation overview using MapMan also revealed gene expression related to plant hormones, calcium regulation and MAP kinases were altered under Cd-stress.
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    Insights into the Molecular Mechanism of Arsenic Phytoremediation
    (Springer New York LLC, 2019) Thakur S.; Choudhary S.; Majeed A.; Singh A.; Bhardwaj P.
    Arsenic (As) is a widespread carcinogenic pollutant. Phytoremediation is the most suited technology for alleviating the As contamination of soil. In this review, we have discussed the uptake mechanism and the associated transporters for different As species. Glutathione, phytochelatins, metallothionins, and secondary metabolites play important role in As detoxification and enhancing tolerance. The roles of MAPK signaling and calcium signaling are highlighted in the perception of As stress along with phytohormones signaling in stress tolerance. Furthermore, transcription factors involved in regulation of gene expression under As stress are discussed. High-throughput sequencing has reduced the time duration and enhanced the knowledge regarding understanding the molecular mechanism of phytoremediation. The role of CRISPR/Cas9 and synthetic genes in context to phytoremediation is discussed. We have provided a holistic understanding of the present knowledge about phytoremediation in the context of mechanisms of the As uptake and tolerance. A complete understanding of the phytoremediation process is essential for As-risk mitigation and will help in augmenting its efficiency and true potential.
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    Comparative analysis of metabolites in contrasting chickpea cultivars
    (Springer, 2019) Ghosh A.; Dadhich A.; Bhardwaj P.; Babu J.N.; Kumar V.
    Chickpea (Cicer arietinum L.) is a good source of nutrients for animals and human consumption. In the present study, we analyzed the anthocyanin and total phenolic contents in two contrasting (desi and kabuli) chickpea cultivars. The quantification of anthocyanins showed higher amount in desi as compared to kabuli chickpea. The total phenolic contents was estimated in desi and kabuli chickpea using two different solvents (50% Acetone and 70% Methanol extracts) for coverage of all potential phenolic compounds. In continuation, desi chickpea culitvars (himchana and ICC4958) were found to be significantly higher total phenolic contents (in both solvent extracts) as compared to kabuli cultivars (JGK-03 and L-552). Higher phenolic contents was found to be directly correlated to higher anthocyanin contents in desi as compared to kabuli chickpea. The volatile organic compounds were also analyzed using gas chromatography mass spectroscopy technique in both cultivars. The significant compositional differences in volatile organic composition (polar and non-polar) of desi and kabuli cultivars were also found to be noticed using two different solvent extractions (methanol and chloroform). The comparative analysis of volatile organic acids in methanolic and chloroform extracts of desi cultivars (himchana and ICC4958), kabuli cultivars (JGK-03 and L-552) and between desi and kabuli cultivars was also carried out for in-depth understanding of the differential patterns of low molecular weight metabolites. Six metabolites were found to be common in all four selected cultivars in chloroform extracted samples, while four were found to be common in all four selected cultivars in methanolic extracted samples. The remaining detected metabolites are uncommon among different cultivars and represented as cultivar specific signatory metabolites. In conclusion, the present investigation revealed higher anthocyanin and phenolic contents in desi cultivars as compared to kabuli cultivars and differential accumulation of volatile organic compounds in chickpea cultivars. The metabolite alterations among desi and chickpea cultivars could be the potential attribute for diversity, resilience and commercial usuages.
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    Marine macroalga Caulerpa: role of its metabolites in modulating cancer signaling
    (Springer, 2019) Mehra, R; Bhushan, S; Bast, Felix; Singh, S.
    Cancer, the leading causes of death worldwide, causes multiple metabolic and physiological alterations, leading to an unregulated proliferation of cells. The existing anticancer therapies are usually nonspecific with side effects and or are extremely expensive, thus hunt for better therapeutics is still on, specially efforts are made to look for naturally occurring molecules. Sea harbors several organisms which are unexplored for their biological potentials. Green macroalga genus, Caulerpa, is one such invaluable repository of bioactive metabolites like alkaloids, terpenoids, flavonoids, steroids and tannins with reported bioactivities against many diseases including cancer. Anti-cancerous metabolites of Caulerpa like caulerpenyne (Cyn), caulerpin, caulersin, and racemosin C, possess unique structural moieties and are known to exhibit distinct effects on cancer cells. Theses metabolites are reported to affect microtubule dynamics, unfolded protein response, mitochondrial health, cell cycle progression, metabolic and stress pathways by their cross-talk with signalling proteins like AMPK, GRP78, GADD153, Bid, Bax, AIF, Bcl2, P21, cyclin D, cyclin E, caspase 9, and PTP1B. Targeting of multiple cancer hallmarks by Caulerpa metabolites, with concomitant modulations of multiple signalling cascades, displays its multifactorial approach against cancer. Evaluation of anti-cancer properties of this genus is particularly important as Caulerpa species are widely edible and utilized in several delicacies in the coastal countries. This is the first review article providing a consolidated information about the role of Caulerpa in cancer with major contributing metabolites and plausible modulations in cancer signaling and prospects. © 2019, Springer Nature B.V.
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    An overview of designing and selection of sgRNAs for precise genome editing by the CRISPR-Cas9 system in plants
    (Springer, 2019) Uniyal, A.P; Mansotra, K; Yadav, S.K; Kumar, Vinay
    A large number of computational tools have been documented in recent years for identification of target-specific valid single-guide (sg) RNAs (18–20 nucleotide long sequence) that is an important component for the efficient utilization of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats—CRISPR-associated Protein) system. Despite optimization of Cas9, other major concerns are on-target efficiency and off-target activity that depend upon the sequence(s) of target-specific sgRNA(s). However, a very little attention has been paid for identification of the best-hit sgRNA for precise targeting as well as minimizing the off-target effects. The aim of this present work is to offer comparative insight into existing CRISPR software tools with their unique features (including targeted genome) and utilities. These available web tools were found to be designed based upon only a few limited mathematical models. Among all these available web tools, three (Benchling, Desktop and CRISPR-P) have been curated as exclusively available for plant genome-editing purpose. These three software tools have been comprehensively described and analyzed with single same target enquiry from two randomly selected genes (IDM2 and IDM3 from Arabidopsis thaliana). Interestingly, all these selected tools generated different results (sgRNAs) even for the same query. In fact, the sequence of sgRNA is considered an important parameter to determine the efficiency and specificity of sgRNAs for precise genome editing. Thus, there is an urgent requirement to pay attention for a validated sgRNA-designing tool for precise DNA editing in plants. In conclusion, this work will encourage building up a consensus for developing a universal valid sgRNA designing for different organisms including plants. © 2019, King Abdulaziz City for Science and Technology.
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    The CRISPR–Cas9, genome editing approach: a promising tool for drafting defense strategy against begomoviruses including cotton leaf curl viruses
    (Springer, 2019) Uniyal, A.P; Yadav, S.K; Kumar, Vinay
    The CRISPR–Cas9 is emerging genome editing tool and very easy and straightforward in operation that has been tested and explored for introduction of new traits in plant systems. Recently, a number of reports have documented utilization of this technology for providing tolerance against viral diseases mediated by begomoviruses. Begomoviruses infect dicot and are transmitted by white flies and cause devastating losses to yield of important agricultural crops including tomato, cassava and cotton. An overview of genomic structure of begomoviruses has been presented to understand the potential strategy for designing of effective sgRNAs to combat the viral replication for generating resistance against infection. This review provides the introduction, recent developments, and applications of the CRISPR–Cas9 system in plants and proposes a holistic methodology for generating cotton plant an example having resistance against begomoviruses. The genome editing using CRISPR–Cas9 system against complex of begomoviruses collectively termed as cotton leaf curl virus, which a major contributor to reduction of the cotton yield especially in Northern India and Pakistan is also discussed thoroughly. In conclusion, this potential strategy could be a sustainable approach for development of tolerant crops against diseases mediated by DNA viruses. © 2019, Society for Plant Biochemistry and Biotechnology.
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    Natural products as multidrug resistance modulators in cancer
    (Elsevier, 2019) Kumar, Amit; Jaitak, Vikas
    Cancer is a prominent cause of death globally. Currently, many drugs that are in clinical practice are having a high prevalence of side effect and multidrug resistance. Risk of tumors acquiring resistance to chemotherapy (multidrug resistance) remains a significant hurdle to the successful treatment of various types of cancer. Membrane-embedded drug transporters, generally overexpressed in cancer, are the leading cause among multiple mechanisms of multidrug resistance (MDR). P-glycoprotein (P-gp) also MDR1/ABCB1, multidrug resistance associated protein 1 (MRP1/ABCC1), MRP2 and breast cancer resistance protein (BCRP/ABCG2) are considered to be a prime factor for induction of MDR. To date, several chemical substances have been tested in a number of clinical trials for their MDR modulatory activity which are not having devoid of any side effects that necessitates to find newer and safer way to tackle the current problem of multidrug resistance in cancer. The present study systematically discusses the various classes of natural products i.e flavonoids, alkaloids, terpenoids, coumarins (from plants, marine, and microorganisms) as potential MDR modulators and/or as a source of promising lead compounds. Recently a bisbenzyl isoquinoline alkaloid namely tetrandrine, isolated from Chinese herb Stephania tetrandra (Han-Fang-Chi) is in clinical trials for its MDR reversal activity. © 2019 Elsevier Masson SAS
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    Molecular basis of transitivity in plant RNA silencing
    (Springer, 2019) Choudhary, S; Thakur, S; Bhardwaj, Pankaj
    The discovery of small RNAs has offered exciting opportunities in manipulating gene expression. The non-coding RNAs cause target gene inactivation at the transcriptional, post-transcriptional or translational level. In addition to the default silencing approach, they provide another mode of gene regulation by transitivity. Here, gradual amplification in effector RNAs number allows regulation of genes other than the original target and causes the outspread of silencing from its origin to aid a robust response. Unlike the short-range cell-to-cell movement of silencing signal (through plasmodesmata), little is known of the mediators of systemic silencing (usually through phloem). Through the present review, we combine the reports available so far to better understand the characteristics of secondary silencing, factors involved, and summarize the instances where it has been employed in plants. Understanding the molecular mechanism behind transitivity has led to the designing of efficient transgenes for targeted gene inactivation, utilized in silencing of a multigene family, and in the field of functional genomics. Studies uncovering the origin of distinct secondary silencing pathways in plants have been exploited for developing artificial RNA silencing methods such as hairpin RNA, artificial microRNA, intrinsic direct repeat, inverted repeat, artificial trans-acting siRNA, phased siRNA, and virus-induced gene silencing. The techniques have facilitated the spread of traits such as pathogenic resistance or alter plant growth and development features. The mechanism of reprogramming in the silencing machinery and the consequent genetic manipulation through transitive RNA is still not completely understood and its exploitation in crop improvement programmes is still in a developing phase. © 2019, Springer Nature B.V.