Exploring the structural assembly of rice ADP-glucose pyrophosphorylase subunits using MD simulation.

ADP-glucose pyrophosphorylase plays a pivotal role as an allosteric enzyme, essential for starch biosynthesis in plants. The higher plant AGPase comparises of a pair of large and a pair of small subunits to form a heterotetrameric complex. Growing evidence indicates that each subunit plays a distinct role in regulating the underlying mechanism of starch biosynthesis. In the rice genome, there are four large subunit genes (OsL1-L4) and three small subunit genes (OsS1, OsS2a, and OsS2b). While the structural assembly of cytosolic rice AGPase subunits (OsL2:OsS2b) has been elucidated, there is currently no such documented research available for plastidial rice AGPases (OsL1:OsS1). In this study, we employed protein modeling and MD simulation approaches to gain insights into the structural association of plastidial rice AGPase subunits. Our results demonstrate that the heterotetrameric association of OsL1:OsS1 is very similar to that of cytosolic OsL2:OsS2b and potato AGPase heterotetramer (StLS:StSS). Moreover, the yeast-two-hybrid results on OsL1:OsS1, which resemble StLS:StSS, suggest a differential protein assembly for OsL2:OsS2b. Thus, the regulatory and catalytic mechanisms for plastidial AGPases (OsL1:OsS1) could be different in rice culm and developing endosperm compared to those of OsL2:OsS2b, which are predominantly found in rice endosperm.

Mapping of QTLs associated with yield and related traits under reproductive stage drought stress in rice using SNP linkage map.

BACKGROUND: Drought stress is a major constraint for rice production worldwide. Reproductive stage drought stress (RSDS) leads to heavy yield losses in rice. The prospecting of new donor cultivars for identification and introgression of QTLs of major effect (Quantitative trait locus) for drought tolerance is crucial for the development of drought-resilient rice varieties. METHODS AND RESULTS: Our study aimed to map QTLs associated with yield and its related traits under RSDS conditions. A saturated linkage map was constructed using 3417 GBS (Genotyping by sequencing) derived SNP (Single nucleotide polymorphism) markers spanning 1924.136 cM map length with an average marker density of 0.56 cM, in the F3 mapping population raised via cross made between the traditional ahu rice cultivar, Koniahu (drought tolerant) and a high-yielding variety, Disang (drought susceptible). Using the Inclusive composite interval mapping approach, 35 genomic regions governing yield and related traits were identified in pooled data from 198 F3 and F4 segregating lines evaluated for two consecutive seasons under both RSDS and irrigated control conditions. Of the 35 QTLs, 23 QTLs were identified under RSDS with LOD (Logarithm of odds) values ranging between 2.50 and 7.83 and PVE (phenotypic variance explained) values of 2.95-12.42%. Two major QTLs were found to be linked to plant height (qPH1.29) and number of filled grains per panicle (qNOG5.12) under RSDS. Five putative QTLs for grain yield namely, qGY2.00, qGY5.05, qGY6.16, qGY9.19, and qGY10.20 were identified within drought conditions. Fourteen QTL regions having ≤ 10 Mb QTL interval size were further analysed for candidate gene identification and a total of 4146 genes were detected out of these 2263 (54.63%) genes were annotated to at least one gene ontology (GO) term. CONCLUSION: Several QTLs associated with grain yield and yield components and putative candidate genes were identified. The putative QTLs and candidate genes identified could be employed to augment drought resilience in rice after further validation through MAS strategies.

Genetic analysis for detection of genes associated to drought tolerance in rice accessions belonging to north east India.

INTRODUCTION: The North East (NE) India is rich in biodiversity and also considered as the secondary centre for origin of rice. The NE rice accessions was characterized previously using genetic markers and morphological traits. Simultaneously, genome-wide association studies (GWAS) reveal significant marker-trait associations for the drought tolerance traits. METHODS AND RESULTS: The genetic diversity and population structure of 296 NE rice accessions were studied using 96,712 single nucleotide polymorphism (SNP) markers distributed across 12 chromosomes. The accessions were clustered into two major sub-groups (SG). A total of 91 accessions were assembled as SG1 and 114 accessions as SG2, while the remaining 91 were admixture genotypes. A total of 200 genotypes belonging to different groups were phenotyped for yield component traits under drought and control conditions. The GWAS was performed to identify significant marker-trait associations (MTAs). Consequently, 47 MTAs were detected under drought, exhibiting 0.02-9.95% of phenotypic variance (P.V.). Whereas 58 MTAs were discovered under control conditions, showing a 0.01-9.74% contribution to the phenotype. Through in-silico mining of QTLs, 2999 genes were identified. Among these; only 22 genes were directly associated with stress response. CONCLUSION: These QTLs/genes may be deployed for marker-assisted pyramiding to improve drought tolerance in popular drought susceptible rice varieties.

Molecular mapping of drought-responsive QTLs during the reproductive stage of rice using a GBS (genotyping-by-sequencing) based SNP linkage map.

BACKGROUND: In rice, drought stress at reproductive stage drastically reduces yield, which in turn hampers farmer's efforts towards crop production. The majority of the rice varieties have resistance genes against several abiotic and biotic stresses. Therefore, the traditional landraces were studied to identify QTLs/candidate genes associated with drought tolerance. METHODS AND RESULTS: A high-density SNP-based genetic map was constructed using a Genotyping-by-sequencing (GBS) approach. The recombinant inbred lines (RILs) derived from crossing 'Banglami × Ranjit' were used for QTL analysis. A total map length of 1306.424 cM was constructed, which had an average inter-marker distance of 0.281 cM. The phenotypic evaluation of F6 and F7 RILs were performed under drought stress and control conditions. A total of 42 QTLs were identified under drought stress and control conditions for yield component traits explaining 1.95-13.36% of the total phenotypic variance (PVE). Among these, 19 QTLs were identified under drought stress conditions, whereas 23 QTLs were located under control conditions. A total of 4 QTLs explained a PVE ≥ 10% which are considered as the major QTLs. Moreover, bioinformatics analysis revealed the presence of 6 candidate genes, which showed differential expression under drought and control conditions. CONCLUSION: These QTLs/genes may be deployed for marker-assisted pyramiding to improve drought tolerance in the existing rice varieties.

Comparative transcriptome profiling reveals differential defense responses among Alternaria brassicicola resistant Sinapis alba and susceptible Brassica rapa.

Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba, a close relative of Brassicas, and the highly susceptible Brassica rapa. The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa. Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola, which will aid in devising strategies for breeding resistant varieties of oilseed Brassica.

Identification and characterization of genes for drought tolerance in upland rice cultivar 'Banglami' of North East India.

INTRODUCTION: Rice is a major crop in Assam, North East (NE) India. The rice accessions belonging to NE India possess unique traits of breeder's interest, i.e., tolerant to biotic and abiotic stresses. In the present research programme, the stress responsive genes were identified within the QTLs associated with drought tolerance. The differential expression profiling of genes were performed under drought stress and control conditions. Thus, the 'candidate genes' associated with drought tolerance were recognised and may be deployed in a breeding programme. METHODS AND RESULTS: A drought-tolerant traditional rice cultivar, Banglami, was crossed with a high-yielding, drought-susceptible variety, Ranjit. The mapping population (F4) was raised through the single seed descent (SSD) method and used in QTL analysis. Under drought stress, a total of 4752 genes were identified through in-silico mining of QTLs. Among these, only 21 genes primarily associated with the stress response. The maximum of four stress-responsive genes were located within the QTLs, qNOG12.1 and qGY1.1. However, under control conditions, 2088 genes were identified, out of which, only 15 were categorised as the major stress responsive genes. The functional characterization of genes recognized 24 different types of proteins. Among these, peroxidase and heat shock proteins (Hsp) are the principal proteins encoded during stress. In addition to that, OsbZIP23, inorganic pyrophosphatase, universal stress protein, serine threonine kinase, NADPH oxidoreductase, and proteins belonging to the ABC1 family were also produced during stress condition. The differential expression profiling showed a profound expression pattern of three candidate genes under drought stress condition, i.e., OsI_32199 (Ascorbate peroxidase), OsI_37694 (Universal stress protein) and OsI_32167 (Heat shock protein 81 - 1). CONCLUSION: The novel candidate genes identified for drought tolerance, may be used in the breeding programme for the development of 'climate smart rice varieties'.

Niche differentiation of belowground microorganisms and their functional signatures in Assam type tea (Camellia sinensis var. assamica).

We employed an Illumina-based high-throughput metagenomics sequencing approach to unveil the rhizosphere and root endosphere microbial community associated with an organically grown Camellia population located at the Experimental Garden for Plantation Crops, Assam (India). The de novo assembled tea root endosphere metagenome contained 24,231 contigs (total 7,771,089 base pairs with an average length of 321 bps), while tea rhizosphere soil metagenome contained 261,965 sequences (total 230,537,174 base pairs, average length 846). The most prominent rhizobacteria belonged to the genera, viz., Bacillus (10.35%), Candidatus Solibacter (6.36%), Burkholderia (5.19%), Pseudomonas (3.9%), Streptomyces (3.52%), and Bradyrhizobium (2.77%), while the root endosphere was dominated by bacterial genera, viz., Serratia (46.64%), Methylobacterium (8.02%), Yersinia (5.97%), Burkholderia (2.05%), etc. The presence of few agronomically important bacterial genera, Bradyrhizobium, Rhizobium (each 0.93%), Sinorhizobium (0.34%), Azorhizobium, and Flavobacterium (0.17% each), was also detected in the root endosphere. KEGG pathway mapping indicated the presence of microbial metabolic pathway genes related to tyrosine metabolism, tryptophan metabolism, glyoxylate, and dicarboxylate metabolism which play important roles in endosphere activities, including survival, growth promotion, and host adaptation. The root endosphere microbiome also contained few important plant growth promoting traits related to phytohormone production, abiotic stress alleviation, mineral solubilization, and plant disease suppression.

In silico characterization and differential expression analysis of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) of Centella asiatica.

The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. Photochemical profiles, as well as pharmaceutical activities of Centella asiatica (L.), one of the most valuable medicinal plants, divulge the presence of secondary metabolites called Centellosides. Despite well-studied pharmaceutical activities, not much is known about the genes responsible for the synthesis of these compounds. In the present study, the full-length DXR gene sequence (JQ965955) of Centella submitted in NCBI was characterized using various bioinformatics tools and tissue specific differential expression studies were also carried out. The full-length CDNA of CaDXR contains an open reading frame (ORF) of 1425 bp which encodes a peptide of 474 amino acids. The molecular weight of this protein was found to be 51.5 kDa with isoelectric point of 6.33. The protein contains three conserved domain, namely NADPH (GSTGSIGT and LAAGSNV), substrate binding (LPADSEHSAI and NKGLEVIEAHY) and Cys-Ser-(Ala/Met/Val/Thr) cleavage-site domains. Phylogenetic studies of CaDXR sequence show close homology with DXR sequence of Angelica sinensis and Daucus carota subsp sativus as they all belong to Apiaceae family. In silico analysis predicted that CaDXR protein contains 21 α-helix and 11 ß-sheets and further DXR protein model was validated by Ramachandran plot analysis. The results of molecular dynamics (MD) simulations unveil dynamic stability of the proposed model and docking studies suggest that the NDP cofactor tightly binds in the active site of the protein with a strong network of hydrogen and hydrophobic interactions. The expression studies by semi-RT followed by qRT-PCR suggests that CaDXR is differentially expressed in different tissues (with maximal expression in the node and lowest in the roots). Thus, characterization and structure-function analysis of DXR gene in Centella facilitate us to understand not only the functions of DXR gene but also regulatory mechanisms involved in the MEP pathway in C. asiatica plant at the molecular level. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02723-w.

Understanding the thermal response of rice eukaryotic transcription factor eIF4A1 towards dynamic temperature stress: insights from expression profiling and molecular dynamics simulation.

Eukaryotic translation initiation factors (eIFs) are the group of regulatory proteins that are involved in the initiation of translation events. Among them, eIF4A1, a member of the DEAD-box RNA helicase family, participates in a wide spectrum of activities which include, RNA splicing, ribosome biogenesis, and RNA degradation. It is well known that ATP-binding and subsequent hydrolysis activities are crucial for the functionality of such helicases. Although the stress-responsive upregulation of eIF4A1 has been reported in plants during stress, it is difficult to anticipate the functionality of the corresponding protein product. Therefore, to understand the activity of eIF4A1 in rice in response to temperature stress, we first conducted an expression analysis of the gene and further investigated the structural stability of the eIF4A1-ATP/Mg2+ complex through molecular dynamics (MD) simulations at different temperature conditions (277 K, 300 K, and 315 K). Our results demonstrated a three to fourfold increased expression of rice eIF4A1 both in root and shoot at 42 °C compared to control. Furthermore, the MD simulation portrayed strong ATP/Mg2+ binding at a higher temperature in comparison to control and cold temperature. Overall, the increased expression pattern of eIF4A1 and strong ATP/Mg2+ binding at higher temperature indicated the heat stress-tolerant capacity of the gene in rice. The results from our study will help in understanding the activity of gene and guide the researchers for screening of novel stress inducible candidate genes for the engineering of temperature stress tolerant plants.Communicated by Ramaswamy H. Sarma.

Combined CADD and Virtual Screening to Identify Novel Nonpeptidic Falcipain-2 Inhibitors.

BACKGROUND: Plasmodium falciparum is the most dangerous and widespread diseasecausing species of malaria. Falcipain-2 (FP2) of Plasmodium falciparum, is a potential target for antimalarial chemotherapy since it is involved in an essential cellular function such as hemoglobin degradation during the parasite's life cycle. However, despite their central role in the life cycle of the parasite, no commercial drug targeting Falcipain-2 has been developed to date. Prior efforts to develop peptide-based drugs against Plasmodium have been futile due to their susceptibility to being degraded by host enzymes. OBJECTIVE: Here, we report computer-aided drug design of new nonpeptidic inhibitors against FP2, which are likely to be safe from degradation by host enzymes. METHODS: We have virtually screened for the probable FP2 inhibitors from the PubChem database by submitting the well-equilibrated 3-D structure of FP2. Furthermore, virtual screenings and dockings were carried out using PyRx and Discovery Studio. RESULTS: We found 15 top-ranking molecules with carbaldehyde pharmacophore having a good fit with the target protein. Based on the C-Docker values, the top 4 hits (PubChem 44138738, Pub- Chem 20983198, PubChem 20983081 and PubChem 28951461) for FP2 were identified. These four hits have been observed to bound to the active cleft of the protein. Moreover, their complexes were also found to be stable from the RMSD and Radius of Gyration analysis. CONCLUSION: The selected compounds 2-(benzylamino)-8-methylquinoline-3-carbaldehyde (Pub- Chem44138738), 6-bromo-2-(3,4-dihydro-1H-isoquinolin-2-yl)quinoline-3-carbaldehyde (Pub- Chem 20983198), 2-(3,4-dihydro-1H-isoquinolin-2-yl)-6-ethylquinoline-3-carbaldehyde(PubChem 20983081)and 2-[benzyl(methyl)amino]quinoline-3-carbaldehyde (PubChem 28951461) may be the starting point for further modification as a new type of nonpeptidic drug for malaria disease.

Mechanism of interaction of an endofungal bacterium Serratia marcescens D1 with its host and non-host fungi.

Association of bacteria with fungi is a major area of research in infection biology, however, very few strains of bacteria have been reported that can invade and reside within fungal hyphae. Here, we report the characterization of an endofungal bacterium Serratia marcescens D1 from Mucor irregularis SS7 hyphae. Upon re-inoculation, colonization of the endobacterium S. marcescens D1 in the hyphae of Mucor irregularis SS7 was demonstrated using stereo microscopy. However, S. marcescens D1 failed to invade into the hyphae of the tested Ascomycetes (except Fusarium oxysporum) and Basidiomycetes. Remarkably, Serratia marcescens D1 could invade and spread over the culture of F. oxysporum that resulted in mycelial death. Prodigiosin, the red pigment produced by the Serratia marcescens D1, helps the bacterium to invade fungal hyphae as revealed by the increasing permeability in fungal cell membrane. On the other hand, genes encoding the type VI secretion system (T6SS) assembly protein TssJ and an outer membrane associated murein lipoprotein also showed significant up-regulation during the interaction process, suggesting the involvement of T6SS in the invasion process.

Insights into the mode of flavin mononucleotide binding and catalytic mechanism of bacterial chromate reductases: A molecular dynamics simulation study.

Enzymes from natural sources protect the environment via complex biological mechanisms, which aid in reductive immobilization of toxic metals including chromium. Nevertheless, progress was being made in elucidating high-resolution crystal structures of reductases and their binding with flavin mononucleotide (FMN) to understand the underlying mechanism of chromate reduction. Therefore, herein, we employed molecular dynamics (MD) simulations, principal component analysis (PCA), and binding free energy calculations to understand the dynamics behavior of these enzymes with FMN. Six representative chromate reductases in monomeric and dimeric forms were selected to study the mode, dynamics, and energetic component that drive the FMN binding process. As evidenced by MD simulation, FMN prefers to bind the cervix formed between the catalytic domain surrounded by strong conserved hydrogen bonding, electrostatic, and hydrophobic contacts. The slight movement and reorientation of FMN resulted in breakage of some crucial H-bonds and other nonbonded contacts, which were well compensated with newly formed H-bonds, electrostatic, and hydrophobic interactions. The critical residues aiding in tight anchoring of FMN within dimer were found to be strongly conserved in the bacterial system. The molecular mechanics combined with the Poisson-Boltzmann surface area binding free energy of the monomer portrayed that the van der Waals and electrostatic energy contribute significantly to the total free energy, where, the polar solvation energy opposes the binding of FMN. The proposed proximity relationships between enzyme and FMN binding site presented in this study will open up better avenues to engineer enzymes with optimized chromate reductase activity for sustainable bioremediation of heavy metals.

Molecular characterization and sequence analyses of Banana bunchy top virus infecting banana cultivar Jahaji (Dwarf Cavendish) in Assam, India.

Several isolates of Banana bunchy top virus (BBTV) have been reported worldwide. They are members of either the Pacific Indian Ocean (PIO) or the South East Asian (SEA) group. However, there is only one completely sequenced isolate published from the northeastern part of India till date. Therefore, we obtained the complete sequences of all the six genomic components of a BBTV isolate from the northeastern Indian state of Assam. The isolate was named as BBTV-As-JOR, and its genome showed the presence of the reported conserved motifs. Nevertheless, like other Indian BBTV isolate, the major common regions in DNA-R and DNA-U3 of BBTV-As-JOR had deletions of 26 and 36 nucleotides, respectively. Phylogenetic analysis based on 312 sequences of BBTV DNA-R classified BBTV-As-JOR as a member of the PIO group; similar phylogenetic patterns were also found with the other genomic segments. Analysis with Recombination Detection Program revealed two intra-segment recombination events involving DNA-C of geographically distinct BBTV isolates. On the other hand, DNA-U3 and DNA-N were found to be involved in few inter-segment recombination events in BBTV-As-JOR. This is the first report of a BBTV isolate from Assam and also of another PIO isolate from the region (the other isolate, BBTV-Umiam, was much closer to the SEA group). The detected possible recombinants could emerge as a major future threat for the banana cultivations in the country considering the asexual nature of propagation of banana crop.

Revealing shared differential co-expression profiles in rice infected by virus from reoviridae and sequiviridae group.

Differential co-expression is a cutting-edge approach to analyze gene expression data and identify both shared and divergent expression patterns. The availability of high-throughput gene expression datasets and efficient computational approaches have unfolded the opportunity to a systems level understanding of functional genomics of different stresses with respect to plants. We performed the meta-analysis of the available microarray data for reoviridae and sequiviridae infection in rice with the aim to identify the shared gene co-expression profile. The microarray data were downloaded from ArrayExpress and analyzed through a modified Weighted Gene Co-expression Network Analysis (WGCNA) protocol. WGCNA clustered the genes based on the expression intensities across the samples followed by identification of modules, eigengenes, principal components, topology overlap, module membership and module preservation. The module preservation analysis identified 4 modules; salmon (638 genes), midnightblue (584 genes), lightcyan (686 genes) and red (562 genes), which are highly preserved in both the cases. The networks in case of reoviridae infection showed neatly packed clusters whereas, in sequiviridae, the clusters were loosely connected which is due to the differences in the correlation values. We also identified 83 common transcription factors targeting the hub genes from all the identified modules. This study provides a coherent view of the comparative aspect of the expression of common genes involved in different virus infections which may aid in the identification of novel targets and development of new intervention strategy against the virus.

Mechanism Underlying Heat Stability of the Rice Endosperm Cytosolic ADP-Glucose Pyrophosphorylase.

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme's regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.

Identification and validation of plant miRNA from NGS data-an experimental approach.

miRNAs are class of endogenously initiated noncoding RNAs, which are most critical in gene expression and regulation at posttranscriptional level. They do so either by cleavage of the target mRNA or by translational repression. miRNAs are being given enough attention in recent years because of its role in myriad developmental processes including tumorogenesis and host-pathogen interaction. Advent of Next Generation Sequencing (NGS) technology and computational approach made it possible to pinpoint the precise role of miRNA and their target. Identification of miRNAs and their target has several approaches depending on efficiency, cost and time. The present review summarizes the developments in the field of plant miRNA w.r.t. to experimental approaches that are being followed to identify and validate the miRNAs and their targets.

Bacillus megaterium adapts to acid stress condition through a network of genes: Insight from a genome-wide transcriptome analysis.

RNA-seq analysis of B. megaterium exposed to pH 7.0 and pH 4.5 showed differential expression of 207 genes related to several processes. Among the 207 genes, 11 genes displayed increased transcription exclusively in pH 4.5. Exposure to pH 4.5 induced the expression of genes related to maintenance of cell integrity, pH homeostasis, alternative energy generation and modification of metabolic processes. Metabolic processes like pentose phosphate pathway, fatty acid biosynthesis, cysteine and methionine metabolism and synthesis of arginine and proline were remodeled during acid stress. Genes associated with oxidative stress and osmotic stress were up-regulated at pH 4.5 indicating a link between acid stress and other stresses. Acid stress also induced expression of genes that encoded general stress-responsive proteins as well as several hypothetical proteins. Our study indicates that a network of genes aid B. megaterium G18 to adapt and survive in acid stress condition.

An Insight Into Structure, Function, and Expression Analysis of 3-Hydroxy-3-Methylglutaryl-CoA Reductase of Cymbopogon winterianus.

Citronella (Cymbopogon winterianus) is one of the richest sources of high-value isoprenoid aromatic compounds used as flavour, fragrance, and therapeutic elements. These isoprenoid compounds are synthesized by 2 independent pathways: mevalonate pathway and 2-C-methyl-d-erythritol-4-phosphate pathway. Evidence suggests that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a rate-controlling enzyme for the synthesis of variety of isoprenoids. This study reports the isolation, characterization, and tissue-specific expression analysis of HMGR from citronella. The modelled HMGR is a class I type of HMGR enzyme with 3-domain architecture. The active site comprises a cofactor (nicotinamide adenine dinucleotide phosphate) and the substrate-binding motifs. The real-time and quantitative reverse transcription-polymerase chain reaction results revealed equal expression level in both leaf sheath and root tissue. The results from our study shall be a valuable resource for future molecular intervention to alter the metabolic flux towards improvement of key active ingredient in this important medicinal plant.

Molecular Modeling and Dynamics Simulation Analysis of KATNAL1 for Identification of Novel Inhibitor of Sperm Maturation.

BACKGROUND: Hormone based birth control often causes various side effects. A recent study revealed that temporary infertility without changing hormone levels can be attained by inhibiting Katanin p60 ATPase-containing subunit A-like 1 protein (KATNAL1) which is critical for sperm maturation in the testes. OBJECTIVE: This study aimed at attaining the most energetically stable three dimensional (3D) structure of KATNAL1 protein using comparative modeling followed by screening of a ligand library of known natural spermicidal compounds for their binding affinity with KATNAL1. This in turn may inhibit the development of mature sperm in the seminiferous epithelium. METHOD: A series of computational techniques were used for building the 3D structure of KATNAL1 which was further optimized by molecular dynamics (MD) simulation. For revealing the ATP binding mode of KATNAL1, docking study was carried out using the optimized model obtained from the MD simulation. The docking study was also employed to test the binding efficiency of the ligand library. RESULTS: Molecular docking study confirmed the ATP binding of KATNAL1 with various hydrophobic and hydrogen bond interactions. Binding efficiency of the ligand library suggested that calotropin, a cardenolide of Calotropis procera showed the highest binding efficiency against the target protein without toxicity. MD simulation of the docked complex validated the results of the docking study. CONCLUSION: This study revealed the ATP binding mode of KATNAL1 and identified calotropin as a potential lead molecule against it showing high binding efficiency with good bioavailability and no mutagenicity. Further in vitro and in vivo bioassay of calotropin could facilitate the development of novel non-hormonal male-specific contraceptive in near future.

Genome wide transcriptome profiling reveals differential gene expression in secondary metabolite pathway of Cymbopogon winterianus.

Advances in transcriptome sequencing provide fast, cost-effective and reliable approach to generate large expression datasets especially suitable for non-model species to identify putative genes, key pathway and regulatory mechanism. Citronella (Cymbopogon winterianus) is an aromatic medicinal grass used for anti-tumoral, antibacterial, anti-fungal, antiviral, detoxifying and natural insect repellent properties. Despite of having number of utilities, the genes involved in terpenes biosynthetic pathway is not yet clearly elucidated. The present study is a pioneering attempt to generate an exhaustive molecular information of secondary metabolite pathway and to increase genomic resources in Citronella. Using high-throughput RNA-Seq technology, root and leaf transcriptome was analysed at an unprecedented depth (11.7 Gb). Targeted searches identified majority of the genes associated with metabolic pathway and other natural product pathway viz. antibiotics synthesis along with many novel genes. Terpenoid biosynthesis genes comparative expression results were validated for 15 unigenes by RT-PCR and qRT-PCR. Thus the coverage of these transcriptome is comprehensive enough to discover all known genes of major metabolic pathways. This transcriptome dataset can serve as important public information for gene expression, genomics and function genomics studies in Citronella and shall act as a benchmark for future improvement of the crop.