Browsing by Author "Wankhede, Dhammaprakash Pandhari"

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Arsenic stress activates MAP kinase in rice roots and leaves
(Elsevier, 2011) Rao, Kudupudi Prabhakara; Vani, Gubbala; Kumar, Kundan; Wankhede, Dhammaprakash Pandhari; Misra, Mohit; Gupta, Meetu; Sinha, Alok Krishna
The toxic metalloid arsenite has become a potential threat to rice growing regions leading to serious contamination in food chain. In the present study effect of different physiological concentration of arsenite that is toxic and triggers the molecular events were evaluated in rice seedlings. Along with severe effect on the growth of rice seedling, production of reactive oxygen species (ROS) and nitric oxide (NO) in arsenite treated rice roots was also observed. Activation of a 42kDa mitogen activated protein kinase (MAPK/MPK) by arsenite was observed in rice leaves and 42 and 44kDa in roots in dose dependent manner. The activated MAPK could be immunoprecipitated with anti-phospho-tyrosine antibody, 4G10. The kinetic of MAPK activation by arsenite was found to be dose dependent. Transcript analysis of MAPK family and immunokinase assay in arsenite treated rice seedling revealed significant level of induction in OsMPK3 transcripts in leaves and OsMPK3, OsMPK4 transcripts in roots. Among MAPK kinase (MKKs) gene family, OsMKK4 transcripts were found to be induced in arsenite treated rice leaves and roots. In-silico homology modeling and docking analysis supported OsMPK3-OsMKK4 interaction. The data indicates that arsenite stress is transduced through MAPK signaling cascade in rice.
Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae
(Taylor & Francis, 2013) Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna
Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tolerance to abiotic stresses. Yeast cells transformed with CrMPK3 was found to show enhanced tolerance to UV and heat stress. Comparison of CrMPK3 and SLT2, a MAPK from yeast shows high-sequence identity particularly at conserved domains. Additionally, heat stress is also shown to activate a 43 kDa MAP kinase, possibly CrMPK3 in C. roseus leaves. These findings indicate the role of CrMPK3 in stress-induced MIA accumulation as well as in stress tolerance.
CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids
(BMC, 2012) Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Jaggi, Monika; Singh, Pallavi; Jalmi, Siddhi Kashinath; Raghuram, Badmi; Sheikh, Arsheed Hussain; Sinha, Alok Krishna
Mitogen activated protein kinase (MAPK) cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA) produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress.
Effect of loss of T-DNA genes on MIA biosynthetic pathway gene regulation and alkaloid accumulation in Catharanthus roseus hairy roots
(Springer, 2010) Taneja, Jyoti; Jaggi, Monika; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna
Hairy roots are generated by integration of T-DNA in host plant genome from root inducing (Ri) plasmid of Agrobacterium rhizogenes and have been utilized for production of secondary metabolites in different plant systems. In Catharanthus roseus, hairy roots are known to show different morphologies, growth patterns, and alkaloid contents. It is also known that during transformation, there is a differential loss of a few T-DNA genes. To decipher the effect of loss of T-DNA genes on the various aspects of hairy roots, ten hairy root clones were analyzed for the presence or absence of T-DNA genes and its implications. It was found that the loss of a few ORFs drastically affects the growth and morphological patterns of hairy roots. The absence of T(R)-DNA from hairy roots revealed increased transcript accumulation and higher alkaloid concentrations, whereas callusing among hairy root lines led to decreased transcript and alkaloid accumulation. Significantly higher expression of MIA biosynthetic pathway genes and low abundance of regulator transcripts in hairy root clones in comparison with non-transformed control roots were also observed. This study indicates that it is not only the integration of T-DNA at certain region of host plant genome but also the presence or absence of important ORFs that affects the expression patterns of MIA biosynthetic pathway genes, regulators, and accumulation of specific alkaloids.
Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum
(Springer, 2013) Wankhede, Dhammaprakash Pandhari; Biswas, Dipul Kumar; Rajkumar, Subramani; Sinha, Alok Krishna
Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.
Involvement of mitogen activated protein kinase kinase 6 in UV induced transcripts accumulation of genes in phytoalexin biosynthesis in rice
(Springer, 2013) Wankhede, Dhammaprakash Pandhari; Kumar, Kundan; Singh, Pallavi; Sinha, Alok Krishna
BACKGROUND: Ultra violet radiation leads to accumulation of phytoalexins (PA) in rice (Oryza sativa) which are typically accumulated when the plants are infected with rice blast pathogen Magnaporthe oryzae. Although extensive works have been done in elucidating phytoalexin biosynthesis, UV stress signal transduction leading to accumulations of rice phytoalexin is largely unknown.
Rice mitogen activated protein kinase kinase and mitogen activated protein kinase interaction network revealed by in-silico docking and yeast two-hybrid approaches
(PLOS, 2013) Wankhede, Dhammaprakash Pandhari; Misra, Mohit; Singh, Pallavi; Sinha, Alok Krishna
Protein-protein interaction is one of the crucial ways to decipher the functions of proteins and to understand their role in complex pathways at cellular level. Such a protein-protein interaction network in many crop plants remains poorly defined owing largely to the involvement of high costs, requirement for state of the art laboratory, time and labour intensive techniques. Here, we employed computational docking using ZDOCK and RDOCK programmes to identify interaction network between members of Oryza sativa mitogen activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK). The 3-dimentional (3-D) structures of five MAPKKs and eleven MAPKs were determined by homology modelling and were further used as input for docking studies. With the help of the results obtained from ZDOCK and RDOCK programmes, top six possible interacting MAPK proteins were predicted for each MAPKK. In order to assess the reliability of the computational prediction, yeast two-hybrid (Y2H) analyses were performed using rice MAPKKs and MAPKs. A direct comparison of Y2H assay and computational prediction of protein interaction was made. With the exception of one, all the other MAPKK-MAPK pairs identified by Y2H screens were among the top predictions by computational dockings. Although, not all the predicted interacting partners could show interaction in Y2H, yet, the harmony between the two approaches suggests that the computational predictions in the present work are reliable. Moreover, the present Y2H analyses per se provide interaction network among MAPKKs and MAPKs which would shed more light on MAPK signalling network in rice.
Signal convergence through the lenses of MAP kinases: paradigms of stress and hormone signaling in plants
(Springer, 2013) Kumar, Kundan; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna
Common mechanisms plants use to translate the external stimuli into cellular responses are the activation of mitogen-activated protein kinase (MAPK) cascade. These MAPK cascades are highly conserved in eukaryotes and consist of three subsequently acting protein kinases, MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK) and MAP kinase (MAPK) which are linked in various ways with upstream receptors and downstream targets. Plant MAPK cascades regulate numerous processes, including various environmental stresses, hormones, cell division and developmental processes. The number of MAPKKs in Arabidopsis and rice is almost half the number of MAPKs pointing important role of MAPKKs in integrating signals from several MAPKKKs and transducing signals to various MAPKs. The cross talks between different signal transduction pathways are concentrated at the level of MAPKK in the MAPK cascade. Here we discussed the insights into MAPKK mediated response to environmental stresses and in plant growth and development.
Transcriptome analysis reveals key pathways and candidate genes controlling seed development and size in ricebean (Vigna umbellata)
(Frontiers Media S.A., 2022) Verma, Sachin Kumar; Mittal, Shikha; Gayacharan; Wankhede, Dhammaprakash Pandhari; Parida, Swarup K.; Chattopadhyay, Debasis; Prasad, Geeta; Mishra, Dwijesh Chandra; Joshi, Dinesh Chandra; Singh, Mohar; Singh, Kuldeep; Singh, Amit Kumar
Ricebean (Vigna umbellata) is a lesser known pulse with well-recognized potential. Recently, it has emerged as a legume with endowed nutritional potential because of high concentration of quality protein and other vital nutrients in its seeds. However, the genes and pathways involved in regulating seed development and size are not understood in this crop. In our study, we analyzed the transcriptome of two genotypes with contrasting grain size (IC426787: large seeded and IC552985: small seeded) at two different time points, namely, 5 and 10 days post-anthesis (DPA). The bold seeded genotype across the time points (B5_B10) revealed 6,928 differentially expressed genes (DEGs), whereas the small seeded genotype across the time point (S5_S10) contributed to 14,544 DEGs. We have also identified several candidate genes for seed development-related traits like seed size and 100-seed weight. On the basis of similarity search and domain analysis, some candidate genes (PHO1, cytokinin dehydrogenase, A-type cytokinin, and ARR response negative regulator) related to 100-seed weight and seed size showed downregulation in the small seeded genotype. The MapMan and KEGG analysis confirmed that auxin and cytokinin pathways varied in both the contrasting genotypes and can therefore be the regulators of the seed size and other seed development-related traits in ricebeans. A total of 51 genes encoding SCF TIR1/AFB , Aux/IAA, ARFs, E3 ubiquitin transferase enzyme, and 26S proteasome showing distinct expression dynamics in bold and small genotypes were also identified. We have also validated randomly selected SSR markers in eight accessions of the Vigna species (V. umbellata: 6; Vigna radiata: 1; and Vigna mungo: 1). Cross-species transferability pattern of ricebean-derived SSR markers was higher in V. radiata (73.08%) than V. mungo (50%). To the best of our knowledge, this is the first transcriptomic study conducted in this crop to understand the molecular basis of any trait. It would provide us a comprehensive understanding of the complex transcriptome dynamics during the seed development and gene regulatory mechanism of the seed size determination in ricebeans.
UV‐B activates a ‘group A’ mitogen activated protein kinase in Oryza sativa
(Springer, 2016) Wankhede, Dhammaprakash Pandhari; Singh, Pallavi; Jaggi, Monika; Rao, Kudupudi Prabhakara; Raina, Susheel Kumar; Sinha, Alok Krishna
Increased level of Ultra violet-B radiation at earth’s surface has several deleterious consequences for plants and ecosystems. Higher UV-B level affects crop plants in several ways and also gives penalty in terms of crop yield. With changing climatic conditions it is crucial to elucidate signal transduction pathway involved in UV-B stress. Here, involvement of mitogen activated protein kinases (MAPKs) in UV-B stress was investigated in rice (Oryza sativa). Transcripts profiling of all 15 rice MAPKs have shown UV-B induced expressions of a few MAPK genes including OsMPK3, OsMPK6, OsMPK4, OsMPK17-1, OsMPK17-2, OsMPK20-1, OsMPK20-4 and OsMPK20-5. In-gel kinase assay as well as immuno-kinase assay showed activation of single MAPK of size ~45 kDa in response to UV-B treatment. Further UV-B responsive activity of rice MAPKs, OsMPK3, OsMPK4 and OsMPK6 was checked using antibodies for the respective orthologs in Arabidopsis which indicated activation of OsMPK3 in UV-B stress. Additionally, GST: OsMPK3 protein showed UV responsive phosphorylation when incubated with crude protein extract from UV exposed plants in an in-vitro phosphorylation assay. These results indicate activation of OsMPK3 in UV-B stress. Further, possible involvement of UV-B responsive OsWRKY89 downstream of MAPK cascade was studied. It was observed that staurosporin and other specific chemical inhibitors of MAPK could attenuate UV-B induced expression of OsWRKY89. OsMPK3 was also found to interact with OsWRKY89 in yeast two-hybrid assay, although the interaction was weak in nature. The present work gives an account of MAPKs in UV-B stress in rice.