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The trypsin being universal enzyme forming family of proteases catalyzes the hydrolysis of proteins into amino acids and regenerates the serine hydroxyl an active site. The trypsin enzyme from D. saccharalis, uses sericin as its preferred substrate. Presence of catalytic triad (serine, aspartic acid and histidine) at the substrate binding site of this enzyme is very important for the catalytic activity. In the current study, the interacting mechanism between the substrate sericin protein and enzyme trypsin protein were explored by integrating various computational approaches including physico-chemical properties, biophysical properties, dynamics, gene ontology, molecular docking, protein - protein interactions, binding free energy calculation and structural motifs were studied. The evolutionary study performed by MEGA X showed that trypsin protein sequence (ALE15212.1) is closely related to cocoonase protein sequence (ADG26770.1) from Antheraea pernyi. 3-D models of trypsin and sericin proteins were predicted using I-TASSER and further validated by PROCHECK, and ProSAweb softwares. The predicted trypsin structure model was assigned E.C. no. 3.4.21.4 which refers hydrolytic mechanism. Gene Ontology predicted by QuickGO showed that trypsin has serine hydrolase activity (GO: 00017171), and part of proteolysis (GO: 0006508) as well as protein metabolic process (GO:0019538) actvity. Molecular docking studies between trypsin and sericin proteins were conducted by the HADDOCK 2.4 having best docked protein complex with Z-score - 1.9. 2D and 3D protein-protein interaction was performed with LIGPLOT

Background Flavonoids are a class of key polyphenolic secondary metabolites having various roles in plants, like in growth and development, stress defense, and reproduction. Dihydroflavonols are converted into flavonols by flavonol synthase (FLS) and this enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (ODD) superfamily. Millettia pinnata is important medicinal plant possessing various phytoconstituents involved in the treatment of various diseases. The FLS is involved in the synthesis of a natural flavonol 3,4′,5,7-tetrahydroxyflavone i.e., kaempferol. However, detailed information about FLS gene sequence and its characterization in M. pinnata is not available. Methods In the current investigation the full-length cDNA sequences of the FLS gene (MpFLS) was deduced by isolating RNA from M. pinnata leaf, cDNA prearaion, PCR based amplification, sequencing followed by sequence analysis. Results The full-length cDNA of MpFLS containing 952-bp open reading frame and encoding 310-amino-acid protein has been elucidated, and submitted to NCBI with accession number OM469017. Bioinformatics analysis of MpFLS protein showed 85% similarities with VuFLS (Vigna unguilata). Also, BLAST analysis of M. pinnata sequence exhibited maximum similarity with V. unguilata. Therefore, to find the possible functional characteristics of MpFLS, the genome wide investigation of this gene was performed in cowpea (V. unguilata) genome using Phytozome v13 database. Total 17 full length non-redundant VuFLSgenes were identified in V. unguilata. These genes and the corresponding proteins were examined for their physicochemical characteristics, chromosomal location, domain and motif architecture, and phylogeny. Phylogenetic and motif analysis revealed that all the proteins had the 2OG-FeII Oxy domain. The functional annotation analysis revealed their involvement in various biological processes and molecular processes. The majority of the genes at the subcellular level are found in the cytoplasm, followed by the nucleus. Conclusions Our research study provides information about FLS gene sequence in M. pinnata and its detailed characterization. Our result also revealed that MpFLSshows similarity in functional characteristics with VuFLS.

Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

Background Cocoonase is a serine protease present in sericigenous insects and majorly involved in dissolving of sericin protein allowing moth to escape. Cocoon structure is made up of sericin protein which holds fibroin filaments together. Cocoonase enzyme hydrolyzes sericin protein without harming the fibroin. However, until date, no detailed characterization of cocoonase enzyme and its presence in wild silk moth Antheraea mylitta has been carried out. Therefore, current study aimed for detailed characterization of amplified cocoonase enzyme, secondary and tertiary structure prediction, sequence and structural alignment, phylogenetic analysis, and computational validation. Several computational tools such as ProtParam, Iterative Threading Assembly Refinement (I-TASSER), PROCHECK, SAVES v6.0, TM-align, Molecular Evolutionary Genetics Analysis (MEGA) X, and Figtree were employed for characterization of cocoonase protein. Results The present study elucidates about the isolation of RNA, cDNA preparation, PCR amplification, and in silico characterization of cocoonase from Antheraea mylitta. Here, total RNA was isolated from head region of A. mylitta, and gene-specific primers were designed using Primer3 followed by PCR-based amplification and sequencing. The newly constructed 377-bp length sequence of cocoonase was subjected to in silico characterization. In silico study of A. mylitta cocoonase showed 26% similarity to A. pernyi strain Qing-6 cocoonase using Blastp and belongs to member of chymotrypsin-like serine protease superfamily. From phylogenetic study, it was found that A. mylitta cocoonase sequence is closely related to A. pernyi cocoonase sequence. Conclusions The present study revealed about the detailed in silico characterization of cocoonase gene and encoded protein obtained from A. mylitta head region. The results obtained infer the presence of cocoonase enzyme in the wild silkworm A. mylitta and can be used for cocoon degumming which will be a valuable and cost-effective strategy in silk industry.

Detection and monitoring of viruses are essential for healthy plants and prosperity. Recent development in CRISPR/Cas system in diagnosis has open an avenue well suited for pathogen detection. Variety of CRISPR associated proteins are being discovered, suggesting array of application and detection strategies in diagnosis. Phytopathogenic viruses are diverse with respect to their nucleic acid compositions, which presents a challenge in developing a single device applicable for almost all viruses. The review describes about the efficient use of CRISPR/Cas Technology in diagnosis, such as SHERLOCK, DETECTR and SATORI. These methods are different in their characteristic to identify specific nucleic acids and processing the detectable signals. These technologies are in their infancy and lot of scope is there to develop commercial kits. Plant tissue culture-based industries, climate control green houses, indoor cultivation facilities etc. has been considered as few examples. This review will be beneficial for researchers seeking to develop detection mechanism based on CRISPR/Cas technology. The outcome in the form of cost-effective detection of viruses will be boon for agro-based industries, which are facing challenges through virus contamination.

Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, antidiabetic activities etc. It has been reported in many vegetables, fruits, herbs and medicinal plants. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols. Whereas flavonoid 3’-monooxygenase (F3’H, EC 1.14.14.82) catalyses the hydroxylation of dihydroflavonol, and flavonol. FLS is involved in the synthesis of the kaempferol whereas F3’H causes degradation of kaempferol. The present study aimed to analyse the binding affinity, stability and activating activity of enzyme FLS as well as inhibitory activity of enzyme F3’H involved in the enrichment of the kaempferol using the in-silico approaches. Computational study for physico-chemical properties, conserved domain identification, 3-D structure prediction and its validation, conservation analysis, molecular docking followed by molecular dynamics analysis of FLS and F3’H, protein-activator (FLS-LIG Complex) and protein-inhibitor (F3’H-LIG Complex) complexes have been performed. Other structural analyses like root mean square fluctuation (RMSF), root mean square deviation (RMSD), surface area solvent accessibility (SASA), radius of gyration (Rg), hydrogen bond analysis, principal component analysis (PCA), Poisson-Boltzmann analysis (MM_PBSA) and the dynamic cross correlation map (DCCM) analysis to explore the structural, functional and thermodynamic stability of the proteins and the complexes were also studied. The molecular docking result showed that FLS binds strongly with the activator ascorbate (CID _54670067) while F3’H binds with the inhibitor ketoconazole (CID_456201). The most powerful inhibitor (ketoconazole for F3’H) and activator (ascorbate for FLS) is determined by computing the thermodynamic binding free energy through MM_PBSA analysis. The current work provides wide-ranging structural and functional information about FLS and F3’H enzymes showing detailed molecular mechanism of kaempferol biosynthesis and its degradation and hence kaempferol enrichment. Finding of the present work opens up new possibilities for future research towards enrichment of kaempferol by using activator (ascorbate) for FLS and inhibitor (ketoconazole) for F3’H as well as for its large-scale production using in vitro approaches. Communicated by Ramaswamy H. Sarma

Cocoonase is known to digest the sericin protein that encapsulates the silkworm cocoon's fibroin protein. Silk fibroin and sericin are two types of proteins that make up silk, and accounts for around 20-30% of the overall cocoon weight. The aim of the study was to see the protein-protein interaction (PPI) and molecular dynamic study of sericin, cocoonase and protein-protein docked complex of silkworm by computational approaches. Here motif analysis, phylogenetic analysis, principal component analysis, root-mean-square deviation (RMSD), root mean square fluctuation, radius of gyration, structural and functional study of cocoonase and sericin as well as molecular docking study were carried out. The 33 amino acid residues of cocoonase shows interaction with 38 aa residues of sericin involving 4 disulphide bonds, 22 hydrogen bonds and 319 non-bonded contacts. The confirmational stability and flexibility of both the proteins as well as protein-protein complex were achieved at 70 ns of MD simulation study. RMSD-based data indicated that cocoonase is more stable than sericin and complex, and complex has a greater fluctuation with more compact (higher Rg) value than cocoonase and sericin, inferring higher conformational stability and flexibility of protein-protein complex than cocoonase and sericin. This study provides a new dimension for PPI study by computational approaches.Communicated by Ramaswamy H. Sarma.

Abiotic stress tolerance is a complex trait regulated by multiple genes and gene networks in plants. A range of abiotic stresses are known to limit rice productivity. Meta-transcriptomics has emerged as a powerful approach to decipher stress-associated molecular network in model crops. However, retaining specificity of gene expression in tolerant and susceptible genotypes during meta-transcriptome analysis is important for understanding genotype-dependent stress tolerance mechanisms. Addressing this aspect, we describe here "abiotic stress tolerant" (ASTR) genes and networks specifically and differentially expressing in tolerant rice genotypes in response to different abiotic stress conditions. We identified 6,956 ASTR genes, key hub regulatory genes, transcription factors, and functional modules having significant association with abiotic stress-related ontologies and cis-motifs. Out of the 6956 ASTR genes, 73 were co-located within the boundary of previously identified abiotic stress trait-related quantitative trait loci. Functional annotation of 14 uncharacterized ASTR genes is proposed using multiple computational methods. Around 65% of the top ASTR genes were found to be differentially expressed in at least one of the tolerant genotypes under different stress conditions (cold, salt, drought, or heat) from publicly available RNAseq data comparison. The candidate ASTR genes specifically associated with tolerance could be utilized for engineering rice and possibly other crops for broad-spectrum tolerance to abiotic stresses.

Green synthesis of gold nanoparticles (GNPs) has received substantial attention, because nanoparticles are produced in an eco-friendly way using biomolecules present in plant extracts in a single step reaction. This research article highlights GNPs obtained using shade-dried leaf extracts of Millettia pinnata (L.) with aqueous auric chloride (HAuCl4) at ambient temperature. In the present study, GNPs with average particle size 37 nm in size were fabricated. Furthermore, the synthesis method to obtain stable and monodispersed GNPs was advanced by optimising enzyme concentration 100 μg/ml, pH 5.4, substrate concentration 0.45 mM and 12 h time of reaction. The confirmation of GNPs formation and characterisation was followed by UV-vis-absorption spectroscopy, dynamic light scattering (DLS), and zeta potential (ZP) for the analysis of shape, size, and stability, respectively. TEM images and powder XRD revealed the GNPs synthesis of spherical-shaped nanoparticles in the face-centred cubic arrangement. Cytotoxicity of GNPs was studied against A549 lung cancer cells with IC50 14.76 μg/ml and found lower as compared to doxorubicin IC50 11.23 μg/ml but significant enough to be used as a vehicle GNPs produced using green source can be used as significant therapeutic agents and drug delivery carriers.

Soil acidification exerts detrimental effects on rice plant leading to severe reduction in its yield. In the present study, we investigated the physiological, biochemical and microstructural changes in the leaves of rice cultivars, namely, Jhilli Dhan (JD) and Gora Dhan (GD), under varying pH conditions (pH 6.5, 5.5, 4.5 and 3.5). Seedlings were grown at varying pH levels for 14 days under controlled conditions. Root and shoot growth and chlorophyll content were found to be decreased with increasing acidity, whereas electrolyte leakage was increased with increasing acidity. Overall, seedling growth was significantly reduced at pH 3.5, while, it was maximum at pH 6.5 treatment, which might be the result of cumulative altered physiological parameters. Further, biochemical parameters, namely total soluble sugar (TSS), proline content and lipid peroxidation were found to be positively correlated with acidity. Microstructural changes were observed through Swept-Source Optical Coherence Tomography (SS-OCT) and Field Emission Scanning Electron Microscope (FESEM). The thickness between different layers of leaves was found to be disintegrating at low pH conditions and the thickness of parenchyma cells was reduced significantly. FESEM analysis revealed changes in characteristics of stomata under acidic stress. The understanding of physiological, biochemical and microstructural changes in rice leaves under varying pH conditions may help in developing rice with improved tolerance towards soil acidity stress.

not availableJ. bio-sci. 25: 77-81, 2017

The goal of this research was to explore the preliminary anticancer properties of five plants namely Calotropis procera, Moringa oleifera, Millettia pinnata, Basela alba and Euphorbia neriifolia available in Jharkhand which is used for the medicinal purpose by local tribes. In the present study, plant leaves from five species were collected, dried and extracted with solvents of increasing polarity, followed by assessment of their cytotoxicity in A549 non-small-cell lung cancer cells. In the antimicrobial assay, the methanol extract of the M. pinnata leaves exhibited comparatively higher zone of inhibition of 0.7 ± 0.20 cm against a Salmonella typhi culture than the other extracts. M. pinnata leaves extract also displayed the maximum percentage inhibition in the DPPH, 83.97 ± 0.01 FRAP, 193.14 ± 3.01 mM assays. Furthermore, the cytotoxicity of the chloroform (37.45 ± 1.04) and ethyl acetate extracts (34.20 ± 0.81) of M. pinnata against A549 cells was found relatively higher with respect to another extract. In contrast, a study with the L132 normal epithelial lung cell line revealed less toxicity from the chloroform extract (0.33 ± 0.19) compared to the ethyl acetate extract (6.65 ± 0.59). Based on these findings, phytochemical investigation on chloroform and ethyl acetate extract of M. pinnata was performed using UPLC-ESI–MS/MS analysis revealing the presence of β-sitosterol, lanceolatin B, karanjin, and stigmasterol. Congruently, a complete phytochemical and cytotoxic investigation of the M. pinnata extract constituents might infer the potency of this extract/s as anticancer, antioxidant and antimicrobial agents. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-019-1927-x) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) are one of the major cytoplasmic tools employed by the eukaryotes for post-transcriptional gene regulation. These ˜21 nt small non-coding RNA molecules are highly conserved among species which forms a base for identification of new miRNAs. In this study, we used previously known mature miRNAs to search their homologs in Arachis hypogaea ESTs. A total of 50 non-protein coding sequences showing homology with no more than 3 mismatches were folded back to hairpin stem-loop structures using mfold. These predicted structures were passed through strict filtration criteria to obtain 18 miRNAs, all of which were other than those reported in miRBase. Out of 18 miRNAs, 7 were found to be new. These miRNAs belonged to miR156, miR166, miR167, miR319, miR398, miR399, miR482 and miR1507 family. These miRNAs were found to target a total of 118 genes in Arabidopsis. These targets included disease resistant proteins, auxin responsive proteins, squamosa promoter binding like proteins, co-transporter protein, transposable element genes, NAD(P) binding protein and topoisomerase II. KEGG pathway analysis showed potential involvement of these miRNAs in regulating different pathways. Apart from miRNA and their targets, microRNA encoded peptides (miPEPs) for 14 miRNAs were also identified. These findings can be used in the appropriate manipulation of miRNAs and corresponding miPEPs that will be helpful towards the peanut crop improvement.

Gaining access to sequence and structure information of telomere-binding proteins helps in understanding the essential biological processes involve in conserved sequence-specific interaction between DNA and the proteins. Rice telomere-binding protein (RTBP1) and Nicotiana glutinosa telomere repeat binding factor (NgTRF1) are helix-turn-helix motif type of proteins that plays role in telomeric DNA protection and length regulation. Both the proteins share same type of domain, but till now there is very less communication on the in silico studies of these complete proteins. Here we intend to do a comparative study between two proteins through modeling of the complete proteins, physiochemical characterization, MD simulation and DNA-protein docking. I-TASSER and CLC protein work bench was performed to find out the protein 3D structure as well as the different parameters to characterize the proteins. MD simulation was completed by GROMOS forcefield of GROMACS for 10 ns of time stretch. The simulated 3D structures were docked with template DNA (3D DNA modeled through 3D-DART) of TTTAGGG conserved sequence motif using HADDOCK Web server. By digging up all the facts about the proteins, it was revealed that around 120 amino acids in the tail part were showing a good sequence similarity between the proteins. Molecular modeling, sequence characterization and secondary structure prediction also indicate the similarity between the protein's structure and sequence. The result of MD simulation highlights on the RMSD, RMSF, Rg, PCA and energy plots which also conveys the similar type of motional behavior between them. The best complex formation for both the proteins in docking result also indicates for the first interaction site which is mainly the helix3 region of the DNA-binding domain. The overall computational analysis reveals that RTBP1 and NgTRF1 proteins display good amount of similarity in their physicochemical properties, structure, dynamics and binding mode.

Living beings are dependent on green energy for their survival and crops are playing most potential role in it. Sudden changes, i.e., biotic and abiotic stresses, are always a big factor affecting the productivity of crops, and in this adverse environmental conditions, it is very much imperative to find the alternate strategies for increasing crop productivity and to feed the increasing worldwide populations. We have always the knowledge of obtaining the best cultivar as earlier the farmers used to select the healthy seedlings for agriculture. Gregor Johann Mendel’s work Mendelian inheritance gives us a platform to use the conventional methods with scientific and validated models for better yields of crops. Therefore, initiatives need to be taken to tackle this problem. In recent years advanced biotechnological approaches, tools, and techniques have played important role. With the use of these advanced technologies researchers are able to do whole genome sequencing, annotation, prediction, and validation of gene expression, prediction of possible function as well as their involvement in various metabolic pathways, and identification of the presence of set of nucleic acid sequences (motif) in the promoter region as well as their protein-DNA interaction. Efforts has also been done to develop the genetically modified crop having important characters and to give good yield under adverse conditions.

Small heat shock proteins (sHSP) are molecular chaperons which play a key role in protein homeostasis under stress conditions. Point mutation of aspartic acid (D) substitution for asparagine (N) at residue 11 (D11N) in HSP16.9B protein was predicted in HSP16.9B gene in wheat. However, its impact on protein function and structural consequences has not been explored. In this study, we examined the effect of point mutation using molecular modelling and molecular dynamics (MD) simulations. Moreover, point mutation induced addition of beta-sheet before the mutation position in the mutant protein. Three-dimensional homology protein modelling, structure validation, and molecular dynamics were carried out to investigate the conformational transitions and dynamics of the HSP16.9B protein due to D11N non-synonymous single nucleotide polymorphism (nsSNP). The MD results indicated that the stability of the mutant protein structure during entire simulation runs. Altogether, our investigation will provide useful understanding related to structural and functional basis of HSP16.9.

Kerria lacca (Kerr) is commercially harnessed for lac resin, which is principally an ester complex of aleuritic acid (9,10,16-trihydroxyhexadecanoic acid) and jalaric acid. The present study is an attempt made to identify the possible pathways involved in the biosynthesis of lac resin. It is proposed that acetyl-CoA is the common precursor for the biosynthesis of aleuritic acid and sesquiterpenic acids (jalaric acid). Prenyltransferases are involved in the biosynthesis of sesquiterpenes; hydroxylation of hexadecanoic acid, after chain elongation, appears to occur through the action of cytochrome P450 enzymes. Two related genes as proposed above were identified and sequenced. The diurnal rhythm of resin secretion and protein concentrations were also studied and correlated for ascertaining the active secretory phase.

The functional role and commercial importance of insect pigments have been studied for well over a century. They are classified into those synthesized by insects, which include anthraquinones, aphins, pterins, tetrapyrroles, ommochromes, melanins and papiliochromes, and those sequestered from their host plants, the antioxidative carotenoids and water-soluble flavonoids. They can also be categorized into those that are produced by cyclization of linear precursors, e.g. anthraquinones, aphins and tetrapyrroles and those derived from cyclic precursors such as pterins, ommochromes, melanins and anthocyanins. Anthraquinones and aphins are derived by cyclization of linear polyketides via successive condensation of simple carboxylic acid metabolites and occur in two major Superfamilies of Hemiptera, the Coccoidea and Aphidoidae, respectively. Ommochromes, tetrapyrroles and melanins are derived from different amino acid precursors, tryptophan, glycine and tyrosine, respectively. Apart from providing body colouration, ommochromes are visual pigments, melanins act as a protectant against UV and tetrapyrroles facilitate oxygen transport to cells. Papiliochromes are synthesized using both, the essential amino acids tyrosine and tryptophan. Pterins are derived from guanosine triphosphate (GTP) and are also present in ommatidia of eyes. The sequestered pigments, anthocyanins and carotenoids, are synthesized from phenylalanine and by condensation of two isoprene units, respectively, in plants. The biosyntheses of chemochromes in insects are governed by a complex set of enzymes, pathways and genetics. This review provides a comprehensive understanding of the molecules that are not only responsible for the striking colours but also provide functional benefits for insects. The commercially important pigments are also discussed.

Objective: Certain anthraquinone compounds are effectively used for treatment of cancer. The present study describes the inhibitory activity of lac dye, a mixture of polyhydroxy anthraquinone compounds (Laccaic acid A, B, C, D and E), produced by the Indian lac insect, Kerria lacca (Kerr). Materials and Methods: In vitro testing for anticancer activity of lac dye was done at four concentrations (10, 20, 40, 80 μg/ml) on ten human malignant cell lines including six human leukemia cell lines, U973, Raji, K562, Jurkat, MOLT 4 and HL60 using SRB assay. Results and Conclusion: Both crimson and yellow lac dye inhibits proliferation of Human leukemia cell lines, Raji, U937, K562, HL60 and Jurkat, therefore can be considered as a potential anticancer agent for leukemia.

Citronella oil is the main product of Java citronella grass (Cymbopogon winterianus Jowitt) rich in geraniol and citronellol, widely used in mosquito repellents and perfumeries. The age of the plant plays a key role in oil composition and its yield such that young leaves have lesser oil content than the mature leaves. Also, a remarkable difference between fresh and dried leaves regarding oil yield is observed. The various methods of extracting essential oils from citronella grass with respect to yield (%) were studied. Average percent yield in the manual extraction and hydro-distillation procedure was 0.8 and 1 % respectively, which was better as compared to steam distilled oil (0.7 %). The chromatographic analysis of essential oils with respect to standards geraniol and citronellol were studied by high performance thin layer chromatography (HPTLC) with n-hexane and ethyl acetate (3:2) as mobile phase followed by its separation on plates. The developed plates showed geraniol, citronellol and citronellal as major bands. The analysis of all extracted oil samples by means of electrospray ionization-mass spectrometry (ESI-MS) in the positive ion mode showed rapid mass fingerprints of constituents present in the samples according to the observed mass of standards. Furthermore, the analysis of vibrational spectra was accomplished with Fourier transform infra-red spectroscopy (FTIR) specifying all the functional groups as major peaks confirming all of them as monoterpene alcohols with conjugated double bonds. Thus, HPTLC, ESI-MS and FTIR studies evidenced that the two essential oil components were majorly present in the methanol extract suggesting methanol as a good extractant in the manual extraction process.

Identification of genes that are coexpressed across various tissues and environmental stresses is biologically interesting, since they may play coordinated role in similar biological processes. Genes with correlated expression patterns can be best identified by using coexpression network analysis of transcriptome data. In the present study, we analyzed the temporal-spatial coordination of gene expression in root, leaf and panicle of rice under drought stress and constructed network using WGCNA and Cytoscape. Total of 2199 differentially expressed genes (DEGs) were identified in at least three or more tissues, wherein 88 genes have coordinated expression profile among all the six tissues under drought stress. These 88 highly coordinated genes were further subjected to module identification in the coexpression network. Based on chief topological properties we identified 18 hub genes such as ABC transporter, ATP-binding protein, dehydrin, protein phosphatase 2C, LTPL153 - Protease inhibitor, phosphatidylethanolaminebinding protein, lactose permease-related, NADP-dependent malic enzyme, etc. Motif enrichment analysis showed the presence of ABRE cis-elements in the promoters of > 62% of the coordinately expressed genes. Our results suggest that drought stress mediated upregulated gene expression was coordinated through an ABA-dependent signaling pathway across tissues, at least for the subset of genes identified in this study, while down regulation appears to be regulated by tissue specific pathways in rice.

An ubiquitous molecular chaperon, small heat shock proteins (sHSP) maintain protein homeostasis under stress conditions. Single nucleotide polymorphism was predicted in HSP16.9B gene but so far its impact on protein structure has not been extensively studied. Keeping this point in mind, we applied computational methods and performed molecular dynamics simulation to examine the effect of aspartic acid substitution for asparagine at 11th position (D11N) in HSP16.9B. Furthermore, the secondary structural analysis revealed an addition of beta sheet before the mutation point in the mutant protein. Three dimensional protein structure modeling, validation of structures and molecular dynamics were performed to study the mechanism of the non-synonymous single nucleotide polymorphism on structural changes. The root mean square deviation (RMSD) result showed the stability of the mutated structure throughout simulations. Moreover, root mean square fluctuation (RMSF) of atoms and Hydrogen-bond patterns further supported our results.

Peanut (Arachis hypogaea L.) ranks fifth among the world oil crops and is widely grown in India and neighbouring countries. Due to its large and unknown genome size, studies on genomics and genetic modification of peanut are still scanty as compared to other model crops like Arabidopsis, rice, cotton and soybean. Because of its favourable cultivation in semi-arid regions, study on abiotic stress responsive genes and its regulation in peanut is very much important. Therefore, we aim to identify and annotate the abiotic stress responsive candidate genes in peanut ESTs. Expression data of drought stress responsive corresponding genes and EST sequences were screened from dot blot experiments shown as heat maps and supplementary tables, respectively as reported by Govind et al. (2009). Some of the screened genes having no information about their ESTs in above mentioned supplementary tables were retrieved from NCBI. A phylogenetic analysis was performed to find a group of utmost similar ESTs for each selected gene. Individual EST of the said group were further searched in peanut ESTs (1,78,490 whole EST sequences) using stand alone BLAST. For the prediction as well as annotation of abiotic stress responsive selected genes, various tools (like Vec-Screen, Repeat Masker, EST-Trimmer, DNA Baser, WISE2 and I-TASSER) were used. Here we report the predicted result of Contigs, domain as well as 3D structure for HSP 17.3KDa protein, DnaJ protein and Type 2 Metallothionein protein.

Flooding is one of the threatening abiotic stresses in recent global warming. In order to understand flooding-caused low oxygen stress response at molecular level, microarray-linked isolation of the hypoxia inducible genes were conducted. Seventeen genes that were up-regulated by the factor of more than 3 fold, were confirmed as hypoxia inducible. Among them, a CCCH-type zinc finger protein gene, OsCCCH-Zn-1, was further characterized due to its novelty as a hypoxia-inducible zinc finger gene as well as its significant induction by hypoxia stress. OsCCCH-Zn-1 was also up-regulated by submergence, ABA and drought stresses. In the normal growth condition, OsCCCH-Zn-1 was expressed in the flag leaf sheath, highest internode and developing seeds. In rice, at least 12 CCCH-type zinc finger protein genes were retrieved by in silico analysis. Among these, we found that the zinc finger genes OsCCCH-Zn-1, -2, -6 were induced by hypoxia stress.

Telomere is a nucleoprotein complex that plays important role in stability and their maintenance and consists of random repeats of species specific motifs. In budding Saccharomyces cerevisiae, Repressor Activator Protein 1 (Rap1) is a sequence specific protein that involved in transcriptional regulation. Rap1 consist of three active domains like N-terminal BRCT-domain, DNA-binding domain and C-terminal RCT-domain. In this study the unknown 3D structure of Myb-type domain (having 61 residues) within DNAbinding domain was modeled by Modeller7, and verified using different online bioinformatics tools (ProCheck, WhatIf, Verify3D). Dynamics of Myb-type domain of Rap1was carried out through simulation studies using GROMACS software. Time dependent interactions among the molecules were analyzed by Root Mean Square Deviation (RMSD), Radius of Gyration (Rg) and Root Mean Square Fluctuation (RMSF) plots. Motional properties in reduced dimension were also performed by Principal Component Analysis (PCA). Result indicated that Rap1 interacts with DNA major groove through its Helix Turn Helix motifs. Helix 3 was rigid, less amount of fluctuation was found as it interacts with DNA major groove. Helix2 and N-terminal having considerable fluctuation in the time scale.

Long-term (30 d) effects of 100, 200, 300, and 400 mM NaCl on photosystem 2 (PS 2)-mediated electron transport activity and content of D1 protein in the thylakoid membranes of chrysanthemum (Dendranthema grandiflorum) cultured in vitro at low irradiance 20 µmol(photon) m−2 s−1 were investigated. 100 mM NaCl increased contents of chlorophylls (Chl) a and b, carotenoids (Car; xanthophylls + carotenes), and the ratio of Chl a/b, and Car/Chl a+b. However, further increase in NaCl concentration led to the significant reduction in the contents of Chl a, and Chl b, and increase in the ratio of Chl a/b and Car/Chl a+b. NaCl treatment decreased the PS 2-mediated electron transport activity and contents of various thylakoid membrane polypeptides including D1 protein.

Effects of various stress treatments such as NaCl, hydrogen peroxide, hydroxyl free radical, and high irradiance (HI, 1 000 µmol m−2 s−1) on the photosystem (PS) 2 mediated electron transport rate and the degradation of D1 protein in the thylakoid membranes of barley were studied. The applied stresses caused significant reduction in the PS 2-mediated electron transport and a degradation of D1 protein that was highest during the HI-treatment. Presence of 2,6-dichlorophenol indophenol (DCPIP), which is an artificial electron acceptor from water, significantly minimizes the HI-induced deleterious effect on the PS 2-mediated electron transport rate, disarrangement of PS machinery, and degradation of the D1 protein. HI in the absence of an acceptor resulted in production of reactive oxygen species due to electron transfer to oxygen.

MYB transcription factor (TF) is one of the largest TF families and regulates defense responses to various stresses, hormone signaling as well as many metabolic and developmental processes in plants. Understanding these regulatory hierarchies of gene expression networks in response to developmental and environmental cues is a major challenge due to the complex interactions between the genetic elements. Correlation analyses are useful to unravel co-regulated gene pairs governing biological process as well as identification of new candidate hub genes in response to these complex processes. High throughput expression profiling data are highly useful for construction of co-expression networks. In the present study, we utilized transcriptome data for comprehensive regulatory network studies of MYB TFs by “top-down” and “guide-gene” approaches. More than 50% of OsMYBs were strongly correlated under 50 experimental conditions with 51 hub genes via “top-down” approach. Further, clusters were identified using Markov Clustering (MCL). To maximize the clustering performance, parameter evaluation of the MCL inflation score (I) was performed in terms of enriched GO categories by measuring F-score. Comparison of co-expressed cluster and clads analyzed from phylogenetic analysis signifies their evolutionarily conserved co-regulatory role. We utilized compendium of known interaction and biological role with Gene Ontology enrichment analysis to hypothesize function of coexpressed OsMYBs. In the other part, the transcriptional regulatory network analysis by “guide-gene” approach revealed 40 putative targets of 26 OsMYB TF hubs with high correlation value utilizing 815 microarray data. The putative targets with MYB-binding cis-elements enrichment in their promoter region, functional co-occurrence as well as nuclear localization supports our finding. Specially, enrichment of MYB binding regions involved in drought-inducibility implying their regulatory role in drought response in rice. Thus, the co-regulatory network analysis facilitated the identification of complex OsMYB regulatory networks, and candidate target regulon genes of selected guide MYB genes. The results contribute to the candidate gene screening, and experimentally testable hypotheses for potential regulatory MYB TFs, and their targets under stress conditions.

Small non-coding RNAs (sRNAs) namely microRNAs (miRNAs) and trans-acting small interfering RNAs (tasi-RNAs) play a crucial role in post-transcriptional regulation of gene expression and thus the control plant development and stress responses. In order to identify drought-responsive miRNAs and tasi-RNAs in sorghum, we constructed small RNA libraries from a drought tolerant (M35-1) and susceptible (C43) sorghum genotypes grown under control and drought stress conditions, and sequenced by Illumina Genome Analyzer IIx. Ninety seven conserved and 526 novel miRNAs representing 472 unique miRNA families were identified from sorghum. Ninety six unique miRNAs were found to be regulated by drought stress, of which 32 were up- and 49 were down-regulated (fold change ≥ 2 or ≤ -2) at least in one genotype, while the remaining 15 miRNAs showed contrasting drought-regulated expression pattern between genotypes. A maximum of 17 and 18 miRNAs was differentially regulated under drought stress condition in the sensitive and tolerant genotypes, respectively. These results suggest that genotype dependent stress responsive regulation of miRNAs may contribute, at least in part, to the differential drought tolerance of sorghum genotypes. We also identified two miR390-directed TAS3 gene homologs and the auxin response factors as tasi-RNA targets. We predicted more than 1,300 unique target genes for the novel and conserved miRNAs. These target genes were predicted to be involved in different cellular, metabolic, response to stimulus, biological regulation, and developmental processes. Genome-wide identification of stress-responsive miRNAs, tasi-RNAs and their targets identified in this study will be useful in unravelling the molecular mechanisms underlying drought stress responses and genetic improvement of biomass production and stress tolerance in sorghum.

Six months old in vitro-grown Anoectochilus formosanus plantlets were transferred to ex-vitro acclimation under low irradiance, LI [60 µmol(photon) m−2 s−1], intermediate irradiance, II [180 µmol(photon) m−2 s−1], and high irradiance, HI [300 µmol(photon) m−2 s−1] for 30 d. Imposition of II led to a significant increase of chlorophyll (Chl) b content, rates of net photosynthesis (P N) and transpiration (E), stomatal conductance (g s), electron transfer rate (ETR), quantum yield of electron transport from water through photosystem 2 (ΦPS2), and activity of ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBPCO, EC 4.1.1.39). This indicates that Anoectochilus was better acclimated at II compared to LI treatment. On the other hand, HI acclimation led to a significant reduction of Chl a and b, P N, E, g s, photochemical quenching, dark-adapted quantum efficiency of open PS2 centres (Fv/Fm), probability of an absorbed photon reaching an open PS2 reaction centre (Fv′/Fm′), ETR, ΦPS2, and energy efficiency of CO2 fixation (ΦCO2/ΦPS2). This indicates that HI treatment considerably exceeded the photo-protective capacity and Anoectochilus suffered HI induced damage to the photosynthetic apparatus. Imposition of HI significantly increased the contents of antheraxanthin and zeaxanthin (ZEA), non-photochemical quenching, and conversion of violaxanthin to ZEA. Thus Anoectochilus modifies its system to dissipate excess excitation energy and to protect the photosynthetic machinery.

During the development of the globular embryos via heart-shaped, torpedo-shaped, and cotyledonary embryos into plantlets, contents of chlorophyll (Chl) a and b and carotenoids, and activity of ribulose-1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) were investigated. In the solid cultivation (SC) the contents of Chl a, Chl b, Chl a/b, and total pigments increased up to plantlet stage. In the liquid cultivation (LC), contents of Chl a, Chl b, and total pigments increased till the torpedo-shaped stage, but decreased with the further development up to plantlets stage. During SC, RuBPC activity increased up to the torpedo-shaped embryo stage, but in the LC RuPBC activity increased continuously with the progress in the developmental stages. The correlations between Chl a and RuBPC activity on the SC and LC were negative, r = −0.26 and −0.56, respectively.

Drought effect on two dimensional iso-electric focusing/sodium dodecyl sulfate-poly acrylamide gel electrophoretic (2-D IEF/SDS-PAGE) protein pattern was studied in Anoectochilus formosanus leaves under ex vitro condition. Spectrophotometery analysis revealed that under drought condition protein content decreased significantly. Similarly, 2-D IEF/SDS-PAGE and PDQuest software analysis indicated that out of 17 protein spots, expression of 15 protein spots were quantitatively down-regulated and 2 spots were up-regulated. Moreover, 11 protein spots were qualitatively up-regulated while 6 were down-regulated.

Excessive photon effects on the photosynthetic pigments and violaxanthin de-epoxidase activity in the xanthophyll cycle of two-month-old spinach leaves were investigated. The leaves were exposed to an excessive photon flux of 1300 μmol m −2 s −1 for 24 h. Photosynthetic pigments such as chlorophylls and carotenoids were quantified and purified by high-performance liquid chromatography. The results showed that the high irradiance induced the dynamic conversion of photosynthetic pigments, and that a higher ratio of zeaxanthin/total chlorophyll was built up. The reactions with photosystem 2 (PS2) particles supplemented with violaxanthin de-epoxidase and Tween 20 at 30 °C for 3 h resulted in a decrease in violaxanthin and an increase in antheraxanthin and zeaxanthin, as compared to reactions carried out in the absence of violaxanthin de-epoxidase, using PS2 particles isolated from normal and high irradiance-illuminated leaves. Lutein was taken as an internal standard among xanthophylls because it remained constant. In the high irradiance-illuminated leaf, for the reactions of PS2 particles in the absence of violaxanthin de-epoxidase and in the presence of violaxanthin de-epoxidase supplemented with Tween 20, their de-epoxidation indexes were similar to the reaction determined in the presence of violaxanthin de-epoxidase from the leaves that were kept at the light intensity of 15 μmol m −2 s −1 , reflecting the promotive effects of excessive photon as well as Tween 20 on violaxanthin de-epoxidase activity.

Drought effects on photosynthesis, stomatal conductance, transpiration, reducing sugar, total sugar, starch, protein, phosphoenolpyruvate carboxylase, superoxide dismutase, and peroxidase activity were studied on Doritaenopsis. Five-month-old in vitro cultured Doritaenopsis orchids were transplanted under ex vitro condition in a greenhouse for 8 months. Four nutrient solutions, i.e. 1/2S, 1S, 3/2S and 2S with the electrical conductivity of 0.9, 1.4, 1.9 and 2.4 dS m−1, respectively, were supplied in ebb and flood irrigation system. Imposition of drought was done by withholding the supply of nutrient solution. Under light and dark condition photosynthesis, stomatal conductance and transpiration decreased with increasing the duration of drought. Rewatering after 15 days of drought resulted in sudden revival from the stress. In drought condition reducing sugar content increased while total sugar and starch content decreased. With the imposition of drought protein content increased at 1/2S nutrient solution while decreased maximally at 3/2S. In control condition peroxidase and superoxide dismutase activity was maximum at 2S and 1/2S, respectively. Imposition of drought increased the phosphoenolpyruvate carboxylase and peroxidase activity while superoxide dismutase activity was decreased at 1S nutrient solution.