Sandeep Arora

Modeling of the MAPK machinery activation in response to various abiotic and biotic stresses in plants by a system biology approach

Mitogen-Activated Protein Kinases (MAPKs) cascade plays an important role in regulating plant growth and development, generating cellular responses to the extracellular stimuli. MAPKs cascade mainly consist of three sub-families i.e. mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK), several cascades of which are activated by various abiotic and biotic stresses. In this work we have modeled the holistic molecular mechanisms essential to MAPKs activation in response to several abiotic and biotic stresses through a system biology approach and performed its simulation studies. As extent of abiotic and biotic stresses goes on increasing, the process of cell division, cell growth and cell differentiation slow down in time dependent manner. The models developed depict the combinatorial and multicomponent signaling triggered in response to several abiotic and biotic factors. These models can be used to predict behavior of cells in event of various stresses depending on their time and exposure through activation of complex signaling cascades.

In Silico Analysis of Expression Data for Identification of Genes Involved in Spatial Accumulation of Calcium in Developing Seeds of Rice

The calcium (Ca(2+)) transporters, like Ca(2+) channels, Ca(2+) ATPases, and Ca(2+) exchangers, are instrumental for signaling and transport. However, the mechanism by which they orchestrate the accumulation of Ca(2+) in grain filling has not yet been investigated. Hence the present study was designed to identify the potential calcium transporter genes that may be responsible for the spatial accumulation of calcium during grain filling. In silico expression analyses were performed to identify Ca(2+) transporters that predominantly express during the different developmental stages of Oryza sativa. A total of 13 unique calcium transporters (7 from massively parallel signature sequencing [MPSS] data analysis, and 9 from microarray analysis) were identified. Analysis of variance (ANOVA) revealed differential expression of the transporters across tissues, and principal component analysis (PCA) exhibited their seed-specific distinctive expression profile. Interestingly, Ca(2+) exchanger genes are highly expressed in the initial stages, whereas some Ca(2+) ATPase genes are highly expressed throughout seed development. Furthermore, analysis of the cis-elements located in the promoter region of the subset of 13 genes suggested that D of proteins play essential roles in regulating the expression of Ca(2+) transporter genes during rice seed development. Based on these results, we developed a hypothetical model explaining the transport and tissue specific distribution of calcium in developing cereal seeds. The model may be extrapolated to understand the mechanism behind the exceptionally high level of calcium accumulation seen in grains like finger millet.

Alleviation of ultraviolet-C-induced oxidative damage through overexpression of cytosolic ascorbate peroxidase

Experiments were conducted to investigate the relationship between ultraviolet (UV) C-induced oxidative damage and the activity of ascorbate peroxidase (APX), using transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana) plants overexpressing cytosolic APX gene (apx1). Transgenic plants having 2.3 fold higher total APX activity, as compared to the wild type plants, showed normal morphological characters. Exposure of 70-day-old plants to fixed intensity UV-C radiation caused an increase in the malondialdehyde (MDA) content in wild type as well as transgenic plants. However, the wild type plants showed significantly higher (p < 0.05) lipid peroxidation as compared to the transgenic plants. Higher proline accumulation was recorded in transgenic plants as compared to the wild type plants, after 24 hours of UV-C exposure. Although the ascorbate content decreased continuously with increasing exposure to UV-C radiation, yet the wild type plants exhibited higher ascorbate levels than the transgenic plants. A marked difference in H2O2 content, between the wild type and transgenic plants, was consistently observed up to 20 hours of UV-C exposure. A direct correlation of ascorbate, MDA and H2O2 levels was recorded with the extent of oxidative stress, signifying that these could be used as potential bio-marker molecules for oxidative stress. The results clearly demonstrate that overexpression of cytosolic APX can protect tobacco plants from UV-C-induced oxidative damage.

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.

Zinc sulfide nanoparticle mediated alterations in growth and anti-oxidant status of Brassica juncea

Abstract Studies were carried out to investigate the effect of zinc sulfide nanoparticles on growth and anti-oxidant markers in Brassica juncea. Growth of Brassica juncea seedlings was positively affected by zinc sulfide nanoparticle treatment, with a maximum increase of 49% in shoot dry weight, being recorded at 15 ppm concentration. Increased chlorophyll content and maximal sugar accumulation was also recorded at 15 ppm zinc sulfide nanoparticle treatment. The seedlings treated with 15 ppm and higher concentrations of zinc sulfide nanoparticles recorded increased reduced-glutathione levels as compared to the untreated seedlings. Higher growth indices of the treated seedlings were associated with improved antioxidant marker levels, recorded in terms of lower proline accumulation, and reduced hydrogen peroxide & lipid peroxidation levels. No significant change in total ascorbate content was recorded up to 15 ppm, while a 15% increase was recorded at 100 ppm zinc sulfide nanoparticle treatment. The reduced ascorbate content decreased by just 4.44% at 15 ppm zinc sulfide nanoparticle treatment, while the maximum reduced ascorbate level was recorded at 100 ppm. These observations indicate an alteration in the antioxidant status of the treated seedlings, which is responsible for improved growth profile of the seedlings treated with 15 ppm zinc sulfide nanoparticles, as compared to the seedlings treated with 100 ppm zinc sulfide nanoparticles. The results clearly indicate that zinc sulfide nanoparticles can be used to augment the growth of Brassica juncea seedlings, and this growth stimulatory effect is associated with alterations in antioxidant status of the treated seedlings.

Molecular cloning, in-silico characterization and functional validation of monodehydroascorbate reductase gene in Eleusine coracana

Ascorbic acid is a ubiquitous water soluble antioxidant that plays a critical role in plant growth and environmental stress tolerance. It acts as a free radical scavenger as well as a source of reducing power for several cellular processes. Because of its pivotal role in regulating plant growth under optimal as well as sub-optimal conditions, it becomes obligatory for plants to maintain a pool of reduced ascorbic acid. Several cellular processes help in maintaining the reduced ascorbic acid pool, by regulating its synthesis and regeneration processes. Current study demonstrates that monodehydroascorbate reductase is an important enzyme responsible for maintaining the reduced ascorbate pool, by optimizing the recycling of oxidized ascorbate. Cloning and functional characterization of this important stress inducible gene is of great significance for its imperative use in plant stress management. Therefore, we have cloned and functionally validated the role of monodehydroascorbate reductase gene (mdar) from a drought tolerant variety of Eleusine coracana. The cloned Ecmdar gene comprises of 1437bp CDS, encoding a 478 amino acid long polypeptide. The active site analysis showed presence of conserved Tyr348 residue, facilitating the catalytic activity in electron transfer mechanism. qPCR expression profiling of Ecmdar under stress indicated that it is an early responsive gene. The analysis of Ecmdar overexpressing Arabidopsis transgenic lines suggests that monodehydroascorbate reductase acts as a key stress regulator by modulating the activity of antioxidant enzymes to strengthen the ROS scavenging ability and maintains ROS homeostasis. Thus, it is evident that Ecmdar is an important gene for cellular homeostasis and its over-expression could be successfully used to strengthen stress tolerance in crop plants.

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.