Genome­wide identification of CAMTA gene family members in rice (Oryza sativa L.) and in silico study on their versatility in respect to gene expression and promoter structure.

The calmodulin-binding transcription activator (CAMTA) is a family of transcriptional factors containing a cluster of calmodulin-binding proteins that can activate gene regulation in response to stresses. The presence of this family of genes has been reported earlier, though, the comprehensive analyses of rice CAMTA (OsCAMTA) genes, their promoter regions, and the proteins were not deliberated till date. The present report revealed the existence of seven CAMTA genes along with their alternate transcripts in five chromosomes of rice (Oryza sativa) genome. Phylogenetic trees classified seven CAMTA genes into three clades indicating the evolutionary conservation in gene structure and their association with other plant species. The in silico study was carried out considering 2 kilobases (kb) promoter regions of seven OsCAMTA genes regarding the distribution of transcription factor binding sites (TFbs) of major and plant-specific transcription factors whereas OsCAMTA7a was identified with highest number of TFbs, while OsCAMTA4 had the lowest. Comparative modelling, i.e., homology modelling, and molecular docking of the CAMTA proteins contributed the thoughtful comprehension of protein 3D structures and protein-protein interaction with probable partners. Gene ontology annotation identified the involvement of the proteins in biological processes, molecular functions, and localization in cellular components. Differential gene expression study gave an insight on functional multiplicity to showcase OsCAMTA3b as most upregulated stress-responsive gene. Summarization of the present findings can be interpreted that OsCAMTA gene duplication, variation in TFbs available in the promoters, and interactions of OsCAMTA proteins with their binding partners might be linked to tolerance against multiple biotic and abiotic cues.

Metagenomic Insights into Rhizospheric Microbiome Profiling in Lentil Cultivars Unveils Differential Microbial Nitrogen and Phosphorus Metabolism under Rice-Fallow Ecology.

The plant rhizosphere interfaces an array of microbiomes related to plant growth and development. Cultivar-specific soil microbial communities with respect to their taxonomic structure and specific function have not been investigated explicitly in improving the adaptation of lentil cultivars under rice-fallow ecology. The present study was carried out to decipher the rhizosphere microbiome assembly of two lentil cultivars under rice-fallow ecology for discerning the diversity of microbial communities and for predicting the function of microbiome genes related to nitrogen (N) and phosphorus (P) cycling processes deploying high-throughput whole (meta) genome sequencing. The metagenome profile of two cultivars detected variable microbiome composition with discrete metabolic activity. Cyanobacteria, Bacteroidetes, Proteobacteria, Gemmatimonadetes, and Thaumarchaeota were abundant phyla in the "Farmer-2" rhizosphere, whereas Actinobacteria, Acidobacteria, Firmicutes, Planctomycetes, Chloroflexi, and some incompletely described procaryotes of the "Candidatus" category were found to be robustly enriched the rhizosphere of "Moitree". Functional prediction profiles of the microbial metagenomes between two cultivars revealed mostly house keeping genes with general metabolism. Additionally, the rhizosphere of "Moitree" had a high abundance of genes related to denitrification processes. Significant difference was observed regarding P cycling genes between the cultivars. "Moitree" with a profuse root system exhibited better N fixation and translocation ability due to a good "foraging strategy" for improving acquisition of native P under the nutrient depleted rice-fallow ecology. However, "Farmer-2" revealed a better "mining strategy" for enhancing P solubilization and further transportation to sinks. This study warrants comprehensive research for explaining the role of microbiome diversity and cultivar-microbe interactions towards stimulating microbiome-derived soil reactions regarding nutrient availability under rice-fallow ecology.

Calmodulin-mediated signal transduction pathways in Arabidopsis are fine-tuned by methylation.

Calmodulin N-methyltransferase (CaM KMT) is an evolutionarily conserved enzyme in eukaryotes that transfers three methyl groups to a highly conserved lysyl residue at position 115 in calmodulin (CaM). We sought to elucidate whether the methylation status of CaM plays a role in CaM-mediated signaling pathways by gene expression analyses of CaM KMT and phenotypic characterization of Arabidopsis thaliana lines wherein CaM KMT was overexpressed (OX), partially silenced, or knocked out. CaM KMT was expressed in discreet spatial and tissue-specific patterns, most notably in root tips, floral buds, stamens, apical meristems, and germinating seeds. Analysis of transgenic plants with genetic dysfunction in CaM KMT revealed a link between the methylation status of CaM and root length. Plants with suppressed CaM methylation had longer roots and CaM KMT OX lines had shorter roots than wild type (Columbia-0). CaM KMT was also found to influence the root radial developmental program. Protein microarray analyses revealed a number of proteins with specificity for methylated forms of CaM, providing candidate functional intermediates between the observed phenotypes and the target pathways. This work demonstrates that the functionality of the large CaM family in plants is fine-tuned by an overarching methylation mechanism.

Versatility of germin-like proteins in their sequences, expressions, and functions.

Germin-like proteins (GLPs) are evolutionary conserved ubiquitous plant glycoproteins belonging to the cupin superfamily. A large number of GLP family members have been identified from different higher and lower plant species, and those have been classified into different subfamilies. Although three histidine residues (H) and one glutamate residue (E) in germin box B and C were conserved among all the GLP subfamily members, how the sequences of one subfamily member differ from the other is unclear. Progress in the field of genomics, transcriptomics, and proteomics has made it possible to understand the variation at gene level among different GLP members from diverse genera and also their biological significances. GLPs from different plant species were found to have various enzymatic properties including oxalate oxidase (OxO), superoxide dismutase (SOD), ADP glucose pyrophosphatase/phosphodiesterase (AGPPase), and polyphenol oxidase (PPO) activities. 'Omics' study demonstrated the expression as well as involvement of GLP family members in almost every part of higher plants as well as in lower plants. Additionally, GLPs from different species were reported to be involved in biotic as well as abiotic stresses and also in the growth and development. This review describes the present research status of GLPs from different plant species, their expressions, and functional significances. Sequence variation was detected among GLP subfamily members at the amino acid level, and based on the sequence variation and phylogenetic analyses, two new GLP subfamilies have been proposed in this review.

DNA-encoded libraries via late-stage functionalization strategies: a review.

The hit finding strategy in drug discovery has undergone a tremendous change in the past decade with the advent of DNA-encoded libraries with diverse chemical libraries. The miniaturization of the assays has enabled high-throughput screening on diverse targets to identify binders as a starting point for medicinal chemistry campaign. The diverse chemical space that can be accessed through DEL provides a unique opportunity to explore new chemistries on DNA. This review highlights the metal-mediated synthetic pathways that allow late-stage functionalisation of DNA strands to access such DEL libraries. Critical analysis of the literature and the methods employed has been done to allow readers to understand the usefulness, as well as the limitations of these protocols.

The Identification of GPR52 Agonist HTL0041178, a Potential Therapy for Schizophrenia and Related Psychiatric Disorders.

HTL0041178 (1), a potent GPR52 agonist with a promising pharmacokinetic profile and exhibiting oral activity in preclinical models, has been identified. This molecule was the outcome of a judicious molecular property-based optimization approach, focusing on balancing potency against metabolic stability, solubility, permeability, and P-gp efflux.

Novel Macrocyclic Antagonists of the CGRP Receptor Part 2: Stereochemical Inversion Induces an Unprecedented Binding Mode.

The diastereomeric macrocyclic calcitonin gene-related peptide (CGRP) antagonists HTL0029881 (3) and HTL0029882 (4), in which the stereochemistry of a spiro center is reversed, surprisingly demonstrate comparable potency. X-ray crystallographic characterization demonstrates that 3 binds to the CGRP receptor in a precedented manner but that 4 binds in an unprecedented, unexpected, and radically different manner. The observation of this phenomenon is noteworthy and may open novel avenues for CGRP receptor antagonist design.

Novel Macrocyclic Antagonists of the Calcitonin Gene-Related Peptide Receptor: Design, Realization, and Structural Characterization of Protein-Ligand Complexes.

A series of macrocyclic calcitonin gene-related peptide (CGRP) receptor antagonists identified using structure-based design principles, exemplified by HTL0028016 (1) and HTL0028125 (2), is described. Structural characterization by X-ray crystallography of the interaction of two of the macrocycle antagonists with the CGRP receptor ectodomain is described, along with structure-activity relationships associated with point changes to the macrocyclic antagonists. The identification of non-peptidic/natural product-derived, macrocyclic ligands for a G protein coupled receptor (GPCR) is noteworthy.

TypiCal but DeliCate Ca++re: Dissecting the Essence of Calcium Signaling Network as a Robust Response Coordinator of Versatile Abiotic and Biotic Stimuli in Plants.

Plant growth, development, and ultimately crop productivity are largely impacted by the interaction of plants with different abiotic and biotic factors throughout their life cycle. Perception of different abiotic stresses, such as salt, cold, drought, heat, and heavy metals, and interaction with beneficial and harmful biotic agents by plants lead to transient, sustained, or oscillatory changes of [calcium ion, Ca2+]cyt within the cell. Significant progress has been made in the decoding of Ca2+ signatures into downstream responses to modulate differential developmental and physiological responses in the whole plant. Ca2+ sensor proteins, mainly calmodulins (CaMs), calmodulin-like proteins (CMLs), and others, such as Ca2+-dependent protein kinases (CDPKs), calcineurin B-like proteins (CBLs), and calmodulin-binding transcription activators (CAMTAs) have played critical roles in coupling the specific stress stimulus with an appropriate response. This review summarizes the current understanding of the Ca2+ influx and efflux system in plant cells and various Ca2+ binding protein-mediated signal transduction pathways that are delicately orchestrated to mitigate abiotic and biotic stresses. The probable interactions of different components of Ca2+ sensor relays and Ca2+ sensor responders in response to various external stimuli have been described diagrammatically focusing on established pathways and latest developments. Present comprehensive insight into key components of the Ca2+ signaling toolkit in plants can provide an innovative framework for biotechnological manipulations toward crop improvability in near future.

Functional role of rice germin-like protein1 in regulation of plant height and disease resistance.

The functional role of rice (Oryza sativa) germin-like protein1 (OsGLP1) was elucidated through development of transgenic plants involving endogenous gene silencing in rice and heterologous gene expression in tobacco. Usually, the single copy OsGLP1 gene in rice plant was found to be expressed predominantly in green vegetative tissues. The transgenic rice lines showed significant reduction in endogenous OsGLP1 expression due to 26nt siRNA-mediated gene silencing, displayed semi-dwarfism and were affected seriously by fungal diseases, compared to the untransformed plant. Structural homology modeling predicted a superoxide dismutase (SOD) domain in OsGLP1 protein which upon over-expression in transgenic tobacco plant clearly documented SOD activity. Our observations on the maintenance of cell dimension, cell wall-associated localization particularly in the sub-epidermal tissues and the SOD activity of OsGLP1 could explain its functional role in regulation of plant height and disease resistance in rice plant.

Transgenically expressed rice germin-like protein1 in tobacco causes hyper-accumulation of H2O2 and reinforcement of the cell wall components.

Our recent report documented that the rice germin-like protein1 (OsGLP1), being a cell wall-associated protein involves in disease resistance in rice and possesses superoxide dismutase (SOD) activity as recognized by heterologous expression in tobacco. In the present study, the transgenic tobacco plants were analyzed further to decipher the detailed physiological and biochemical functions of the OsGLP1 and its associated SOD activity. The transgenic tobacco lines expressing SOD-active OsGLP1 showed tolerance against biotic and abiotic stresses mitigated by hyper-accumulating H(2)O(2) upon infection by fungal pathogen (Fusarium solani) and treatment to chemical oxidizing agent (ammonium persulfate), respectively. Histological staining revealed enhanced cross-linking of the cell wall components in the stem tissues of the transgenic plants. Fourier transform infrared spectroscopy (FTIR) analysis of the biopolymer from the stem tissues of the transgenic and untransformed plants revealed differential banding pattern of the spectra corresponding to various functional groups. Our findings demonstrate that the OsGLP1 with its inherent SOD activity is responsible for hyper-accumulation of H(2)O(2) and reinforcement of the cell wall components.

In-silico study of biotic and abiotic stress-related transcription factor binding sites in the promoter regions of rice germin-like protein genes.

Germin-like proteins (GLPs) are involved in biotic and abiotic stress tolerance in different plant species. Rice (Oryza sativa L.) genome contains about 40 GLP family member proteins in nine chromosomes. Although some of the rice GLP (OsGLP) promoters have been studied through in silico analysis as well as experimentally, studies regarding the distribution pattern of the biotic and abiotic stress associated transcription factor binding sites (TFbs) in the promoter regions of OsGLP genes have not been attempted thoroughly. Several transcription factors (TFs) namely NAC, WRKY, bHLH, bZIP, MYB and AP2/ERF act as major TFs concerned with biotic as well as abiotic stress responses across various plant species. In the present study the in silico analysis was carried out using the 1.5 kilobases (kb) promoter regions from 40 different OsGLP genes for the presence of NAC, WRKY, bHLH, bZIP, MYB and AP2/ERF TFbs in it. Among various OsGLP gene promoters, OsGLP8-11 was found to contain highest number of tested TFbs in the promoter region whereas the promoter region of OsGLP5-1 depicted least amount of TFbs. Phylogenetic study of promoter regions of different OsGLP genes revealed four different clades. Our analyses could reveal the evolutionary significance of different OsGLP gene promoters. It can be presumed from the present findings as well as previous reports that OsGLP gene duplications and subsequent variations in the TFbs in OsGLP gene promoter regions might be the consequences of neofunctionalization of OsGLP genes and their promoters for biotic and abiotic stress tolerance in rice.

Exon inclusion is dependent on predictable exonic splicing enhancers.

We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18:1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.

Sex-oriented research on dioecious crops of Indian subcontinent: an updated review.

A number of dioecious species are grown across India and some of those plants play a crucial role in the agro-based economy of the country. The diagnosis of sex is very difficult in the dioecious plant prior flowering wherein sex identification at the seedling stage is of great importance to breeders as well as farmers for crop improvement or production purpose. A comprehensive approach of sex determination comprising morphological, biochemical, cytological and molecular attributes is a must required for gender differentiation in dioecious plant species. In the present review, we highlighted the economical, medicinal as well as industrial importance of most of the dioecious species extensively grown in Indian subcontinent. In addition to that, the cytogenetic, genetic as well as molecular information in connection to their sex determination were critically discussed in this review.

Organic reactions in water: an efficient zinc-mediated stereoselective synthesis of (E)- and (Z)-trisubstituted alkenes using unactivated alkyl halides.

[reaction: see text] Treatment of the acetyl derivatives of the Baylis-Hillman adducts 3-hydroxy-2-methylene-alkanoates and 3-hydroxy-2-methylene-alkanenitriles with unactivated alkyl halides in the presence of Zn in saturated aqueous NH(4)Cl solution at room temperature afforded (2E)-2-substituted-alk-2-enoates in the first case and (2Z)-2-substituted-alk-2-enenitriles with high (Z)-selectivity in the second case.

Synapsis alters RAG-mediated nicking at Tcrb recombination signal sequences: implications for the "beyond 12/23" rule.

At the Tcrb locus, Vß-to-Jß rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the "beyond 12/23" rule (B12/23 rule). Previous work showed that Vß recombination signal sequences (RSSs) do not recombine with Jß RSSs because Jß RSSs are crippled for either nicking or synapsis. This result raised the following question: how can crippled Jß RSSs recombine with Dß RSSs? We report here that the nicking of some Jß RSSs can be substantially stimulated by synapsis with a 3'Dß1 partner RSS. This result helps to reconcile disagreement in the field regarding the impact of synapsis on nicking. Furthermore, our data allow for the classification of Tcrb RSSs into two major categories: those that nick quickly and those that nick slowly in the absence of a partner. Slow-nicking RSSs can be stimulated to nick more efficiently upon synapsis with an appropriate B12/23 partner, and our data unexpectedly suggest that fast-nicking RSSs can be inhibited for nicking upon synapsis with an inappropriate partner. These observations indicate that the RAG proteins exert fine control over every step of V(D)J cleavage and support the hypothesis that initial RAG binding can occur on RSSs with either 12- or 23-bp spacers (12- or 23-RSSs, respectively).

Engineered plant virus resistance.

Virus diseases are among the key limiting factors that cause significant yield loss and continuously threaten crop production. Resistant cultivars coupled with pesticide application are commonly used to circumvent these threats. One of the limitations of the reliance on resistant cultivars is the inevitable breakdown of resistance due to the multitude of variable virus populations. Similarly, chemical applications to control virus transmitting insect vectors are costly to the farmers, cause adverse health and environmental consequences, and often result in the emergence of resistant vector strains. Thus, exploiting strategies that provide durable and broad-spectrum resistance over diverse environments are of paramount importance. The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Genetic engineering offers various options for introducing transgenic virus resistance into crop plants to provide a wide range of resistance to viral pathogens. This review examines the current strategies of developing virus resistant transgenic plants.

An intergenic region shared by At4g35985 and At4g35987 in Arabidopsis thaliana is a tissue specific and stress inducible bidirectional promoter analyzed in transgenic arabidopsis and tobacco plants.

On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications.

Discovery, design and synthesis of Y-shaped peroxisome proliferator-activated receptor δ agonists as potent anti-obesity agents in vivo.

We have discovered and demonstrated the in vitro and in vivo PPARδ-selective activity of novel Y-shaped agonists. These compounds activated hPPARδ with EC(50) values between 1 and 523 nM. Surprisingly, compounds 10a, 11d, 11e and 11f were the most potent and most selective hPPARδ agonists with 10(4)-fold selectivity over the other two subtypes, namely, hPPARα and hPPARγ. The PPARδ ligands 10a, 11e and 11f showed good bioavailability and in vivo efficacy.

Comparative lipid profiling of two endophytic fungal isolates--Colletotrichum sp. and Alternaria sp. having potential utilities as biodiesel feedstock.

Lipid accumulation abilities of two endophytic fungal isolates - Colletotrichum sp. and Alternaria sp. grown under optimum and nutrient-stress conditions were investigated and compared. Significant variations in lipid contents, ranging from 30% to 58% of their dry biomass were found in liquid culture using various carbon sources. Since, >50% of the total lipid was estimated to be neutral lipid for both the fungal species, predicted biodiesel properties were theoretically calculated based upon the determined fatty acid profiles; and the values were found to be comparable to those of commonly used plant oils for biodiesel production. The two endophytes grew successfully on the combined rice straw and wheat bran as substrate that was degraded by their secretory enzymes including cellulase [1.21-2.51 FPU/g dry substrate (gds)] in solid state fermentation and produced substantial amount of lipid (60.32-84.30 mg/gds). Our study highlights the potential utilities of these two novel endophytic fungi as biodiesel feedstock.