Saurabh Pandey

Genome-Wide Dissection of Arabidopsis and Rice for the Identification and Expression Analysis of Glutathione Peroxidases Reveals Their Stress-Specific and Overlapping Response Patterns

Excessive generation of reactive oxygen species (ROS) due to environmental stresses critically effects plant development and productivity. Plants efficiently detoxify ROS by both non-enzymatic and enzymatic mechanisms. Plant glutathione peroxidases (GPXs) are non-haeme thiol peroxidases that catalyze the reduction of H2O2 (or organic hydroperoxides) to water or the respective alcohols using reduced glutathione or thioredoxin. Genome-wide analysis of the known GPXs from rice and Arabidopsis genomes revealed their gene structure, conserved motifs, localization and tissue-specific and/or organ-specific expression profiles in response to various abiotic stresses. Among the eight genes that encoded GPX proteins from Arabidopsis, AtGPX3 showed two alternate spliced forms that spread over four chromosomes. Five genes encoded for rice GPX proteins, while OsGPX1 showed three spliced variants that were distributed on five chromosomes. Utilizing the publicly available microarray and massively parallel signature sequencing (MPSS) data, the GPXs revealed stress-responsive, tissue-specific and/or organ-specific expression profiles. Presence of important cis-regulatory elements analyzed in the GPX promoter sequences revealed their overlapping or specific responsiveness to different abiotic stresses. Co-expression data of Arabidopsis GPX genes suggested that various protein kinase family members and stress-responsive proteins co-expressed with the GPX proteins. Transcript profile of rice GPX genes by qRT-PCR validated their functional roles in signal transduction and stress pathways. Results revealed that plant GPXs play a crucial role in response to stress and significantly contribute towards their growth and development.

Insilico Analysis of cis acting Regulatory Elements CAREs in Upstream Regions of Ascorbate Glutathione Pathway Genes from Oryza sativa

CAREs play an important role in plant stress tolerance by interacting with transcription factors and controlling the expression of many stress related potential genes. Recognition of promoters and their regulatory elements is one of the crucial challenges in biotechnology. In this study, we examine cis acting regulatory element in 5’ upstream regions (~1 kb) of Asc-Glu pathway genes such as SOD, APX, MDHAR, DHAR, and GR. The evolutionary relationships amongst these sequences were deciphered using MEGA v. 6.0. The promoter region these genes contain various cis acting regulatory elements such as MBS, DRE/C repeat, W box, HSE, TCA element, LTR, ABRE box, ARE box, Wun and DRE that have significant role in stress tolerance Asc-Glu promoter sequences analysis revealed their specific responsiveness or overlapping in various environment stress and significantly contribute toward plant growth and development.

Establishment of Efficient Regeneration System from Leaf Discs in LongPepper an Important Medicinal Plant (Piper longum L.)

Cultivation of Piper longum L. till recently was not very common; still it is extensively collected from the wild, threatening the very existence of the plant. Hence, it is alarming that there is an urgent need not only cultivate this plant but also develop conservation strategies. Conventional propagation is overwhelmed with problems of poor seed viability low percentage of germination, scanty and delayed rooting of vegetative cuttings indicating there is a need for alternative propagation methods. In vitro technique is an alternative approach to solve the problem. Therefore, the current research was aimed at developing a promising in vitro mass propagation protocol for Piper longum L. using leaf as explant. Murashige and Skoog medium was used throughout the experiment. For callus induction, MS medium supplemented with alone or in combination of IAA (1 to 2 mg/l) and BAP (1 to 2 mg/l) were used. Shoot induction was undertaken on MS medium supplemented with different concentrations and combinations of Kinetin and BAP (1 to 3 mg/l). Emerged shoots were transferred onto elongation medium supplemented with 2 mg/l Zeatin and 1 mg/l GA3. In vitro rooting was achieved on ½ MS medium+NAA 1 mg/l. Accordingly, the highest calli were induced on MS+1 mg/L IAA+1 mg/L BAP. Among the various treatments, the maximum percentage (91.50 ± 3.54) in vitro shooting was observed on MS+2 mg/L BAP+1 mg/L Kinetin. In vitro rooted shoots were successfully acclimatized in the greenhouse conditions. Therefore, it is possible to deduce that the current protocol is promising for in vitro mass propagation of Piper longum L. to solve the reproduction and cultivation problem of the plant.

Identification and expression analysis of DXS1 gene isolated from Aconitum balfourii Stapf.

In higher plants, two pathways are implicated in the synthesis of isoprenoids: the mevalonate pathway and methyl-d-erythritol 4-phosphate (MEP) pathway. In MEP pathway, 1-deoxy-d-xylulose-5-phosphate synthase (DXS) enzyme catalyzes the first committed step and it is recognized as a rate-limiting enzyme. In this study, a partial cDNA encoding DXS1 domain isolated from Aconitum balfourii Stapf. was cloned and characterized (AbDXS1). Analysis of DXS domain was performed and we found that AbDXS1 had extensive similarities to other DXS1 proteins. It comprised highly conserved DRAG and PSD domains. A comparative analysis of expression patterns for AbDXS1 using the already existing transcriptome profiles of model plants suggests its role in primary metabolism which needs to be validated further through functional genomics. These data would be helpful for exploring the functions of DXS and its isoforms in MEP pathway of Aconitum balfourii Stapf.

In-silico Characterization, Structural Modelling, Docking Studies andPhylogenetic Analysis of 5-Enolpyruvylshikimate-3-Phosphate SynthaseGene of Oryza sativa L.

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is one of the vital enzymes of the shikimate pathway which is involved in the biosynthesis of secondary metabolites and several amino acids. The multiple sequence alignment of these EPSPS protein sequences from different plants showed conserved regions at different stretches with maximum homology in amino acid residues. We revealed the homology model of Oryza sativa EPSPS (OsEPSPS) protein using the structure of E. coli EPSPS as template. The resulting model structure was refined by PROCHECK, RAMPAGE server, ProSA, Verify3D etc. that indicated the model structure is reliable. Ramachandran plot analysis showed that conformations for 94.3% of amino acid residues are within the most favoured regions. Through motif analysis, it was revealed that a conserved EPSPS domain is uniformly found in all EPSPS proteins irrespective of variable plant species suggesting its possible role in cellular and metabolic functions. The phylogenetic tree constructed revealed different clusters based on EPSPS in respect of bacteria, monocot and dicot plants. The interacting partners of the gene shows the importance of this gene family in regulating developmental and metabolic functions. The two conserved motifs LP(G/S)KSLSNRILLLAAL and LFLGNAGTAMRPL present in almost all EPSPS plant species may function as the catalytic domains of EPSPS enzymes and are supposed to contribute in the glyphosate binding site.

Structural modelling and phylogenetic analyses of PgeIF4A2 (Eukaryotic translation initiation factor) from Pennisetum glaucum reveal signature motifs with a role in stress tolerance and development

Eukaryotic translation initiation factor 4A (eIF4A) is an indispensable component of the translation machinery and also play a role in developmental processes and stress alleviation in plants and animals. Different eIF4A isoforms are present in the cytosol of the cell, namely, eIF4A1, eIF4A2, and eIF4A3 and their expression is tightly regulated in cap-dependent translation. We revealed the structural model of PgeIF4A2 protein using the crystal structure of Homo sapiens eIF4A3 (PDB ID: 2J0S) as template by Modeller 9.12. The resultant PgeIF4A2 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that showed the model structure is reliable with 77 % amino acid sequence identity with template. Investigation revealed two conserved signatures for ATP-dependent RNA Helicase DEAD-box conserved site (VLDEADEML) and RNA helicase DEAD-box type, Q-motif in sheet-turn-helix and α-helical region respectively. All these conserved motifs are responsible for response during developmental stages and stress tolerance in plants.

Molecular model of thylakoid membrane bound (SlAPX6) ascorbate peroxidase from Solanum lycopersicum

Ascorbate peroxidase (E 1.11.1.11) acts as primary key component of plant defense against photo protection and photo-oxidative stress. Chloroplastic (APX) located in the thylakoid membrane (tAPX) and stroma (sAPX) have been thought to be key regulators of intracellular levels of H2O2. Therefore, it is of interest to study thylakoid membrane bound SlAPX from Solanum lycopersicum (tomato, a fleshy fruit). However, a structure model is not yet solved for tomato thylakoid membrane bound SlAPX. Hence, a homology molecular model of SlAPX6 from S. lycopersicum was constructed using a template structure (PDB ID: 1APX) from Pisum sativum. The model was further assessed using accessible surface area (ASA) calculations to identify surface residues for further characterization of active site regions. We further characterized the active site regions in the enzyme for functional inference. This information provides insights for the understanding of photo protection and photo-oxidative stress tolerant in S. lycopersicum during flower development and fruit ripening.

Modeling and phylogenetic analysis of cytosolic ascorbate peroxidase (OsAPX1) from rice reveal signature motifs that may play a role in stress tolerance.

Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cycle that detoxifies reactive oxygen species in plants by catalyzing the conversion of hydrogen peroxide to water using ascorbate as a specific electron donor. Different APX isoforms are present in discrete subcellular compartments in rice and their expression is stress regulated. We revealed the homology model of OsAPX1 protein using the crystal structure of soybean GmAPX1 (PDB ID: 2XIF) as template by Modeller 9.12. The resultant OsAPX1 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that indicated the model structure is reliable with 83 % amino acid sequence identity with template, RMSD (1.4 Å), Verify3D (86.06 %), Zscores (-8.44) and Ramachandran plot analysis showed that conformations for 94.6% of amino acid residues are within the most favoured regions. Investigation revealed two conserved signatures for haeme ligand binding and peroxidase activity in the alpha helical region that may play a significant role during stress.