Stacking geometry for non‐canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion‐corrected DFT‐D study

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson‐Crick base pairs, different orientations of the bases to form hydrogen‐bonded non‐canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non‐canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non‐canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non‐canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll‐slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 328–338, 2015.     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.     

A biologically inspired measure for coexpression analysis

Two genes are said to be coexpressed if their expression levels have a similar spatial or temporal pattern. Ever since the profiling of gene microarrays has been in progress, computational modeling of coexpression has acquired a major focus. As a result, several similarity/distance measures have evolved over time to quantify coexpression similarity/dissimilarity between gene pairs. Of these, correlation coefficient has been established to be a suitable quantifier of pairwise coexpression. In general, correlation coefficient is good for symbolizing linear dependence, but not for nonlinear dependence. In spite of this drawback, it outperforms many other existing measures in modeling the dependency in biological data. In this paper, for the first time, we point out a significant weakness of the existing similarity/distance measures, including the standard correlation coefficient, in modeling pairwise coexpression of genes. A novel measure, called BioSim, which assumes values between -1 and +1 corresponding to negative and positive dependency and 0 for independency, is introduced. The computation of BioSim is based on the aggregation of stepwise relative angular deviation of the expression vectors considered. The proposed measure is analytically suitable for modeling coexpression as it accounts for the features of expression similarity, expression deviation and also the relative dependence. It is demonstrated how the proposed measure is better able to capture the degree of coexpression between a pair of genes as compared to several other existing ones. The efficacy of the measure is statistically analyzed by integrating it with several module-finding algorithms based on coexpression values and then applying it on synthetic and biological data. The annotation results of the coexpressed genes as obtained from gene ontology establish the significance of the introduced measure. By further extending the BioSim measure, it has been shown that one can effectively identify the variability in the expression patterns over multiple phenotypes. We have also extended BioSim to figure out pairwise differential expression pattern and coexpression dynamics. The significance of these studies is shown based on the analysis over several real-life data sets. The computation of the measure by focusing on stepwise time points also makes it effective to identify partially coexpressed genes. On the whole, we put forward a complete framework for coexpression analysis based on the BioSim measure.     

Oxidant induced injury of erythrocyte—Role of green tea leaf and ascorbic acid

Oxidant and free radical-generating system were used to promote oxidative damage in erythrocytes. Among the oxidants used, phenylhydrazine represents one of the most investigated intracellular free radical-generating probes, which in the presence of haemoglobin autooxidises and give rise to hydroxyl radical, a marker for cellular damage. Erythrocyte, as a single cell, is a good model to be used for studying the haemolytic mechanism of anaemia. Our present investigations reveal increased lipid peroxidation of erythrocyte using phenylhydrazine as well as other oxygen-generating systems (hydrogen peroxide, iron with hydrogen peroxide). It has further been observed that not only lipid peroxidation, phenylhydrazine causes significant elevation in methemoglobin formation, catalase activity and turbidity, in the above system, which are the typical characteristics of haemolytic anaemia. However, exogenous administration of green tea leaf extract and ascorbic acid as natural antioxidants and free radical scavengers were shown to protect separately increased lipid peroxidation caused by phenylhydrazine, though the degree of protection is more in case of green tea leaf extract than ascorbic acid. Results suggest that oxidative damage in vivo due to haemolytic disease may be checked to some extent by using natural antioxidants. (Mol Cell Biochem 276: 205–210, 2005)