Effect of ACGT motif in spatiotemporal regulation of AtAVT6D, which improves tolerance to osmotic stress and nitrogen-starvation.

KEY MESSAGE: Plasma membrane-localized AtAVT6D importing aspartic acid can be targeted to develop plants with enhanced osmotic and nitrogen-starvation tolerance. AtAVT6D promoter can be exploited as a stress-inducible promoter for genetic improvements to raise stress-resilient crops. The AtAVT6 family of amino acid transporters in Arabidopsis thaliana has been predicted to export amino acids like aspartate and glutamate. However, the functional characterization of these amino acid transporters in plants remains unexplored. The present study investigates the expression patterns of AtAVT6 genes in different tissues and under various abiotic stress conditions using quantitative Real-time PCR. The expression analysis demonstrated that the member AtAVT6D was significantly induced in response to phytohormone ABA and stresses like osmotic and drought. The tissue-specific expression analysis showed that AtAVT6D was strongly expressed in the siliques. Taking together these results, we can speculate that AtAVT6D might play a vital role in silique development and abiotic stress tolerance. Further, subcellular localization study showed AtAVT6D was localized to the plasma membrane. The heterologous expression of AtAVT6D in yeast cells conferred significant tolerance to nitrogen-deficient and osmotic stress conditions. The Xenopus oocyte studies revealed that AtAVT6D is involved in the uptake of Aspartic acid. While overexpression of AtAVT6D resulted in smaller siliques in Arabidopsis thaliana. Additionally, transient expression studies were performed with the full-length AtAVT6D promoter and its deletion constructs to study the effect of ACGT-N24-ACGT motifs on the reporter gene expression in response to abiotic stresses and ABA treatment. The fluorometric GUS analyses revealed that the promoter deletion construct-2 (Pro.C2) possessing a single copy of ACGT-N24-ACGT motif directed the strongest GUS expression under all the abiotic conditions tested. These results suggest that Pro.C2 can be used as a stress-inducible promoter to drive a significant transgene expression.

Genome-wide analysis of AAAG and ACGT cis-elements in Arabidopsis thaliana reveals their involvement with genes downregulated under jasmonic acid response in an orientation independent manner.

Cis-regulatory elements are regions of noncoding DNA that regulate the transcription of neighboring genes. The study of cis-element architecture that functions in transcription regulation are essential. AAAG and ACGT are a class of cis-regulatory elements, known to interact with Dof and bZIP transcription factors respectively, and are known to regulate the expression of auxin response, gibberellin response, floral development, light response, seed storage proteins genes, biotic and abiotic stress genes in plants. Analysis of the frequency of occurrence of AAAG and ACGT motifs from varying spacer lengths (0-30 base pair) between these 2 motifs in both possible orientations-AAAG (N) ACGT and ACGT (N) AAAG, in the promoters and genome of Arabidopsis thaliana which indicated preferred orientation of AAAG (N) ACGT over ACGT (N) AAAG across the genome and in promoters. Further, microarray analysis revealed the involvement of these motifs in the genes downregulated under jasmonic acid response in an orientation-independent manner. These results were further confirmed by the transient expression studies with promoter-reporter cassettes carrying AAAG and ACGT motifs in both orientations. Furthermore, cluster analysis on genes with AAAG (N) ACGT and ACGT (N) AAAG motifs orientations revealed clusters of genes to be involved in ABA signaling, transcriptional regulation, DNA binding, and metal ion binding. These findings can be utilized in designing synthetic promoters for the development of stress-tolerant transgenic plants and also provides an insight into the roles of these motifs in transcriptional regulation.

Primer Designing for Amplifying an AT-Rich Promoter from Arabidopsis thaliana.

The aim of the present study is to optimize the PCR conditions required to amplify the promoter sequence of an amino acid transporter having an AT-rich base composition with a high number of tandem repeats. The present study also covers the key parameters that need to be kept in mind while designing primers. Results show that successful can be achieved by performing a 2-step PCR reaction at a lower extension temperature of 65 ̊C for an increased extension period of 1.5 min/kb, with MgCl2 concentration ranging from 2.5 to 3.0mM. The results also suggest that the DNA concentration of around 25-30 ng/µl was essential to achieve this amplification.

Promoter profiling of Arabidopsis amino acid transporters: clues for improving crops.

KEY MESSAGE: The review describes the importance of amino acid transporters in plant growth, development, stress tolerance, and productivity. The promoter analysis provides valuable insights into their functionality leading to agricultural benefits. Arabidopsis thaliana genome is speculated to possess more than 100 amino acid transporter genes. This large number suggests the functional significance of amino acid transporters in plant growth and development. The current article summarizes the substrate specificity, cellular localization, tissue-specific expression, and expression of the amino acid transporter genes in response to environmental cues. However, till date functionality of a majority of amino acid transporter genes in plant development and stress tolerance is unexplored. Considering, that gene expression is mainly regulated by the regulatory motifs localized in their promoter regions at the transcriptional levels. The promoter regions ( ~ 1-kbp) of these amino acid transporter genes were analysed for the presence of cis-regulatory motifs responsive to developmental and external cues. This analysis can help predict the functionality of known and unexplored amino acid transporters in different tissues, organs, and various growth and development stages and responses to external stimuli. Furthermore, based on the promoter analysis and utilizing the microarray expression data we have attempted to identify plausible candidates (listed below) that might be targeted for agricultural benefits.

Molecular docking studies of Indian variants of pathophysiological proteins of SARS-CoV-2 with selected drug candidates.

SARS-CoV-2 pandemic has recently made the entire world come to a standstill. The number of cases in the world, especially India, have been increasing exponentially. The need of the hour is to assimilate as much data as possible to fast track the pipeline of bringing in new therapeutic tools against this fatal virus. In this brief communication, we aim to throw light on the various variants of the proteins involved heavily in the pathophysiology of COVID-19, namely Spike protein, ACE2, GRP78, TMPRSS2 and NSP-12. We also portray the molecular docking studies of these proteins with specific drugs that are currently being associated with the same. In our brief study, we come across a few key findings. First of all the combinations of the variants of spike protein and ACE2 binding show overall 25% unfavourable ΔΔG. Second, NSP12 is the most mutation prone among all the NSPs of the SARS-CoV-2 genome and the most common mutations are P323L and A97V. Third, we discovered the variants found in the Indian subpopulation that have greater binding with the currently investigated drugs.

In vitro activity of reconstituted rubisco enzyme from Gloeobacter violaceus.

RuBisCO (Ribulose 1,5 bisphosphate carboxylase/oxygenase) by virtue of its dual specificity towards oxygen and carbon dioxide is an important rate-limiting step in photosynthesis and is believed to be the key factor for limited productivity of higher plants and algae. The photoautotrophic growth rate of cyanobacteria is a culmination of several factors including, rates of photosynthetic reactions, stress combating mechanisms and basic biomass generation metabolism in combination with optimal nutrient availability, irradiance, gaseous environment, etc. In case of cyanobacteria, the effect of RuBisCO in affecting the multiplication rate has been observed to show varied response. The current paper presents the RuBisCO activity of an early diverging cyanobacterium, Gloeobacter violaceus PCC 7421 and also compares the growth rates and RuBisCO activity of various cyanobacteria. A spectrophotometric estimation in a coupled enzyme assay system of the heterologous expressed G. violaceus PCC 7421 RuBisCO in E. coli, upon purification, revealed a carboxylation activity of LSu to be 5 nMol of phosphoglycerate min-1 mg-1 of protein, which is in coherence with the organism's slow growth rate. Further, the in vitro complementation of RbcL with RbcS in presence of RbcX of G. violaceus facilitated partial reconstitution of the protein and was hence found to cause a four-fold enhancement in its specific activity. The unique characteristics of the primitive cyanobacteria, such as, absence of thylakoids, lack of several photosystem constituting genes, slow carboxylation rate, pose limitation for its rapid multiplication. The RuBisCO carboxylation rate is observed as not the sole but an important parameter for obtaining optimal cell multiplication rates in photo-autotrophically multiplying cyanobacteria.

In Silico Analysis of CCGAC and CATGTG Cis-regulatory Elements Across Genomes Reveals their Roles in Gene Regulation under Stress.

Background: Plant yield closely depends on its environment and is negatively affected by abiotic stress conditions like drought, salinity, heat, and cold. Analysis of the stress-inducible genes in Arabidopsis has previously shown that CCGAC and CATGTG play a crucial role in controlling the gene expression through the binding of DREB/CBF and NAC TFs under various stress conditions, mainly drought and salinity. Methods: The pattern of these motifs is conserved, which has been analyzed in this study to find the mechanism of gene expression through spacer specificity, inter motif distance preference, functional analysis, and statistical analysis for four different plants, namely Oryza sativa, Triticum aestivum, Arabidopsis thaliana, and Glycine max. Results: The spacer frequency analysis has shown a preference for particular spacer lengths among four genomes. The spacer specificity at all the spacer lengths which predicts dominance of particular base pairs over others, was analyzed to find the preference of the sequences in the flanking region. Functional analysis on stress-regulated genes for saline, osmotic, and heat stress clearly shows that these motif frequencies with inter motif distance (0-30) in the promoter region of Arabidopsis are highest in genes which are upregulated by saline and osmotic stress and downregulated by heat stress. Conclusion: Microarray data were analyzed to confirm the role of both motifs in stress response pathways. Transcription factors seem to prefer larger motif size with repeated CCGAC and CATGTG elements. The common preference for one spacer was further validated through Box and Whisker's statistical analysis.

Co-expression network analysis of protein phosphatase 2A (PP2A) genes with stress-responsive genes in Arabidopsis thaliana reveals 13 key regulators.

Abiotic and biotic stresses adversely affect plant growth and development and eventually result in less yield and threaten food security worldwide. In plants, several studies have been carried out to understand molecular responses to abiotic and biotic stresses. However, the complete circuitry of stress-responsive genes that plants utilise in response to those environmental stresses are still unknown. The protein phosphatase 2A (PP2A) gene has been known to have a crucial role in abiotic and biotic stresses; but how it regulates the stress response in plants is still not known completely. In this study, we constructed gene co-expression networks of PP2A genes with stress-responsive gene datasets from cold, drought, heat, osmotic, genotoxic, salt, and wounding stresses to unveil their relationships with the PP2A under different conditions of stress. The graph analysis identified 13 hub genes and several influential genes based on closeness centrality score (CCS). Our findings also revealed the count of unique genes present in different settings of stresses and subunits. We also formed clusters of influential genes based on the stress, CCS, and co-expression value. Analysis of cis-regulatory elements (CREs), recurring in promoters of these genes was also performed. Our study has led to the identification of 16 conserved CREs.

Glutathione S-transferase: a versatile protein family.

Glutathione-S transferase (GST) is a most ancient protein superfamily of multipurpose roles and evolved principally from gene duplication of an ancestral GSH binding protein. They have implemented in diverse plant functions such as detoxification of xenobiotic, secondary metabolism, growth and development, and majorly against biotic and abiotic stresses. The vital structural features of GSTs like highly divergent functional topographies, conserved integrated architecture with separate binding pockets for substrates and ligand, the stringent structural fidelity with high Tm values (50º-60º), and stress-responsive cis-regulatory elements in the promoter region offer this protein as most flexible plant protein for plant breeding approaches, biotechnological applications, etc. This review article summarizes the recent information of GST evolution, and their distribution and structural features with emphasis on the assorted roles of Ser and Cys GSTs with the signature motifs in their active sites, alongside their recent biotechnological application in the area of agriculture, environment, and nanotechnology have been highlighted.

Role of Chromatin Architecture in Plant Stress Responses: An Update.

Sessile plants possess an assembly of signaling pathways that perceive and transmit environmental signals, ultimately resulting in transcriptional reprogramming. Histone is a key feature of chromatin structure. Numerous histone-modifying proteins act under different environmental stress conditions to help modulate gene expression. DNA methylation and histone modification are crucial for genome reprogramming for tissue-specific gene expression and global gene silencing. Different classes of chromatin remodelers including SWI/SNF, ISWI, INO80, and CHD are reported to act upon chromatin in different organisms, under diverse stresses, to convert chromatin from a transcriptionally inactive to a transcriptionally active state. The architecture of chromatin at a given promoter is crucial for determining the transcriptional readout. Further, the connection between somatic memory and chromatin modifications may suggest a mechanistic basis for a stress memory. Studies have suggested that there is a functional connection between changes in nuclear organization and stress conditions. In this review, we discuss the role of chromatin architecture in different stress responses and the current evidence on somatic, intergenerational, and transgenerational stress memory.

Genome wide analysis of W-box element in Arabidopsis thaliana reveals TGAC motif with genes down regulated by heat and salinity.

To design, synthetic promoters leading to stress-specific induction of a transgene, the study of cis-regulatory elements is of great importance. Cis-regulatory elements play a major role in regulating the gene expression spatially and temporally at the transcriptional level. The present work focuses on one of the important cis-regulatory element, W-box having TGAC as a core motif which serves as a binding site for the members of the WRKY transcription factor family. In the present study, we have analyzed the occurrence frequency of TGAC core motifs for varying spacer lengths (ranging from 0 to 30 base pairs) across the Arabidopsis thaliana genome in order to determine the biological and functional significance of these conserved sequences. Further, the available microarray data was used to determine the role of TGAC motif in abiotic stresses namely salinity, osmolarity and heat. It was observed that TGAC motifs with spacer sequences like TGACCCATTTTGAC and TGACCCATGAATTTTGAC had a significant deviation in frequency and were thought to be favored for transcriptional bindings. The microarray data analysis revealed the involvement of TGAC motif mainly with genes down-regulated under abiotic stress conditions. These results were further confirmed by the transient expression studies with promoter-reporter cassettes carrying TGAC and TGAC-ACGT variant motifs with spacer lengths of 5 and 10.

Optimization of PCR conditions for amplifying an AT-rich amino acid transporter promoter sequence with high number of tandem repeats from Arabidopsis thaliana.

OBJECTIVE: The aim of the present study is to optimize the PCR conditions required to amplify the promoter sequence of an amino acid transporter having an AT-rich base composition with a high number of tandem repeats. RESULT: Results show that successful amplification can be achieved by performing a 2-step PCR at a lower extension temperature of 65 °C for an increased extension period of 1.5 min/kb, with MgCl2 concentration ranging from 2.5 to 3.0 mM. The results also suggest that the DNA concentration of about 25-30 ng/µl was essential to achieve this amplification.

Regulatory components of carbon concentrating mechanisms in aquatic unicellular photosynthetic organisms.

This review provides an insight into the regulation of the carbon concentrating mechanisms (CCMs) in lower organisms like cyanobacteria, proteobacteria, and algae. CCMs evolved as a mechanism to concentrate CO2 at the site of primary carboxylating enzyme Ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco), so that the enzyme could overcome its affinity towards O2 which leads to wasteful processes like photorespiration. A diverse set of CCMs exist in nature, i.e., carboxysomes in cyanobacteria and proteobacteria; pyrenoids in algae and diatoms, the C4 system, and Crassulacean acid metabolism in higher plants. Prime regulators of CCM in most of the photosynthetic autotrophs belong to the LysR family of transcriptional regulators, which regulate the activity of the components of CCM depending upon the ambient CO2 concentrations. Major targets of these regulators are carbonic anhydrase and inorganic carbon uptake systems (CO2 and HCO3- transporters) whose activities are modulated either at transcriptional level or by changes in the levels of their co-regulatory metabolites. The article provides information on the localization of the CCM components as well as their function and participation in the development of an efficient CCM. Signal transduction cascades leading to activation/inactivation of inducible CCM components on perception of low/high CO2 stimuli have also been brought into picture. A detailed study of the regulatory components can aid in identifying the unraveled aspects of these mechanisms and hence provide information on key molecules that need to be explored to further provide a clear understanding of the mechanism under study.

Towards combinatorial transcriptional engineering.

The modular nature of the transcriptional unit makes it possible to design robust modules with predictable input-output characteristics using a 'parts- off a shelf' approach. Customized regulatory circuits composed of multiple such transcriptional units have immense scope for application in diverse fields of basic and applied research. Synthetic transcriptional engineering seeks to construct such genetic cascades. Here, we discuss the three principle strands of transcriptional engineering: promoter and transcriptional factor engineering, and programming inducibilty into synthetic modules. In this context, we review the scope and limitations of some recent technologies that seek to achieve these ends. Our discussion emphasizes a requirement for rational combinatorial engineering principles and the promise this approach holds for the future development of this field.

ICRF-193, an anticancer topoisomerase II inhibitor, induces arched telophase spindles that snap, leading to a ploidy increase in fission yeast.

ICRF-193 [meso-4,4-(2,3-butanediyl)-bis(2,6-piperazinedione)] is a complex-stabilizing inhibitor of DNA topoisomerase II (topo II) that is used as an effective anticancer drug. ICRF-193 inhibits topo II catalytic activity in vitro and blocks nuclear division in vivo. Here, we examined the effects of ICRF-193 treatment on chromatin behavior and spindle dynamics using detailed live mitotic cell analysis in the fission yeast, Schizosaccharomyces pombe. Time-lapse movie analysis showed that ICRF-193 treatment leads to an elongation of presumed chromatin fibers connected to kinetochores during mid-mitosis. Anaphase spindles begin to arch, and eventually spindle poles come together abruptly, as if the spindle snapped at the point of spindle microtubule overlap in telophase. Segregating chromosomes appeared as elastic clumps and subsequently pulled back and merged. The snapped spindle phenotype was abolished by microtubule destabilization after thiabendazole treatment, accompanied by unequal chromosome segregation or severe defects in spindle extension. Thus, we conclude that ICRF-193-treated, unseparated sister chromatids pulling toward opposite spindle poles produce the arched and snapped telophase spindle. ICRF-193 treatment increased DNA content, suggesting that the failure of sister chromatids to separate properly in anaphase, causes the spindle to break in telophase, resulting in polyploidization.

PP2C-like Promoter and Its Deletion Variants Are Induced by ABA but Not by MeJA and SA in Arabidopsis thaliana.

Gene expression is mediated through interaction between cis regulatory elements and its cognate transcription factors. Cis regulatory elements are defined as non-coding DNA sequences that provide the binding sites for transcription factors and are clustered in the upstream region of genes. ACGT cis regulatory element is one of the important cis regulatory elements found to be involved in diverse biological processes like auxin response, salicylic acid (SA) response, UV light response, ABA response and jasmonic acid (JA) response. We identified through in silico analysis that the upstream region of protein phosphatase 2C (PP2C) gene has a distinct genetic architecture of ACGT elements. In the present study, the activation of the full length promoter and its deletion constructs like 900 base pair, 500 base pair, 400 base pair and NRM (Nathji Rajesh Mehrotra) were examined by stable transformation in Arabidopsis thaliana using ß-glucuronidase as the reporter gene. Evaluation of deletion constructs of PP2C-like promoter was carried out in the presence of phytohormones like abscisic acid (ABA), SA and JA. Our result indicated that the full length and 900 base pair promoter-reporter constructs of PP2C-like promoter was induced in response to ABA but not to methyl jasmonate and SA.

Role of habitat and great oxidation event on the occurrence of three multisubunit inorganic carbon-uptake systems in cyanobacteria.

The oxygenase reaction catalyzed by RuBisCO became an issue only after the evolution of the oxygenic photosynthesis in cyanobacteria. Several strategies were developed by autotrophic organisms as an evolutionary response to increase oxygen levels to help RuBisCO maximize its net carboxylation rate. One of the crucial advancements in this context was the development of more efficient inorganic carbon transporters which could help in increasing the influx of inorganic carbon (Ci) at the site of CO2 fixation.We conducted a survey to find out the genes coding for cyanobacterial Ci transporters in 40 cyanobacterial phyla with respect to transporters present in Gloeobacter violaceous PCC 7421, an early-diverging cyanobacterium. An attempt was also made to correlate the prevalence of the kind of transporter present in the species with its habitat. Basically, two types of cyanobacterial inorganic carbon transporters exist, i.e. bicarbonate transporters and CO2-uptake systems. The transporters also show variation in context to their structure as some exist as single subunit proteins (BicA and SbtA), while others exist as multisubunit proteins (namely BCT1, NdhI3 and NdhI4). The phylogeny and dist ribution of the former have been extensively studied and the present analysis provides an insight into the latter ones. The in silico analysis of the genes under study revealed that their distribution was greatly influenced by the habitat and major environmental changes such as the great oxidation event (GOE) in the course of their evolution.

Developments and challenges in biodiesel production from microalgae: A review.

The imminent depletion of fossil fuels and the surging global demand for renewable energy have led to the search for nonconventional energy sources. After a few decades of trial and error, the world is now testing the sources of the third generation of fossil fuels, which contain for most parts microalgae. With more than 80% oil content, being adaptable in growth parameters and highly versatile, microalgae are highly promising sources of biofuels in the present time. The present article makes a sweeping attempt to highlight the various methods employed for cultivation of microalgae, techniques to harvest and extract biomass from huge algal cultures, as well as their downstream production and processing procedures. The advantages, limitations, and challenges faced by each of them have been described to some extent. Major concerns pertaining to biofuels are supposed to be their environmental sustainability and economic viability along with their cost effectiveness. This would require a great deal of empirical data on existing systems and a great deal of optimization to generate a more robust one. We have concluded our article with a SWOT analysis of using algae for biodiesel production in a tabulated form.

In Silico Analysis of the Endonuclease III Protein Family Identifies Key Residues and Processes During Evolution.

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to genomic DNA molecules. DNA repair processes significantly overcome DNA damage and restore the normal nucleotide sequence and DNA structure. This study focuses on the evolution of the endonuclease III gene/protein family, which plays a key role in the base excision repair pathway. We analyzed 463 homologs of the endonuclease III protein and compared them with the corresponding gene and 16S/18S rRNA sequences to understand the evolutionary processes of this protein family. The sequence analysis and comparison reveal consensus sequence motifs within the ENDO3c and iron-sulfur cluster loop domains that are functionally and structurally important. On the basis of phylogenetic analysis, we propose an evolutionary model of the endonuclease III protein family. Horizontal gene transfer was identified as the key event among bacteria, archaea, and eukaryotic organisms that occurred during the evolution of the endonuclease III gene family among bacteria, archaea, and eukaryotic organisms. This analysis may be exploited to achieve a better prediction of the endonuclease III family gene/protein in unannotated organisms or families of organisms that are completely sequenced as well as in those for which sequencing is ongoing.

Abscisic acid and abiotic stress tolerance - different tiers of regulation.

Abiotic stresses affect plant growth, metabolism and sustainability in a significant way and hinder plant productivity. Plants combat these stresses in myriad ways. The analysis of the mechanisms underlying abiotic stress tolerance has led to the identification of a highly complex, yet tightly regulated signal transduction pathway consisting of phosphatases, kinases, transcription factors and other regulatory elements. It is becoming increasingly clear that also epigenetic processes cooperate in a concerted manner with ABA-mediated gene expression in combating stress conditions. Dynamic stress-induced mechanisms, involving changes in the apoplastic pool of ABA, are transmitted by a chain of phosphatases and kinases, resulting in the expression of stress inducible genes. Processes involving DNA methylation and chromatin modification as well as post transcriptional, post translational and epigenetic control mechanisms, forming multiple tiers of regulation, regulate this gene expression. With recent advances in transgenic technology, it has now become possible to engineer plants expressing stress-inducible genes under the control of an inducible promoter, enhancing their ability to withstand adverse conditions. This review briefly discusses the synthesis of ABA, components of the ABA signal transduction pathway and the plants' responses at the genetic and epigenetic levels. It further focuses on the role of RNAs in regulating stress responses and various approaches to develop stress-tolerant transgenic plants.