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Item Transcription factor OsNF-YB9 regulates reproductive growth and development in rice(Springer Nature Publishing AG, 2019) Das, Sweta; Parida, Swarup K.; Agarwal, Pinky; Tyagi, Akhilesh K.Nuclear Factor-Y (NF-Y) family of transcription factors takes part in many aspects of growth and development in eukaryotes. They have been classifed into three subunit classes, namely, NF-YA, NF-YB and NF-YC. In plants, this transcription factor family is much diverged and takes part in several developmental processes and stress. We investigated NF-Y subunit genes of rice (Oryza sativa) and found OsNF-YB9 as the closest homologue of LEAFY COTYLEDON1. OsNF-YB9 delayed the heading date when ectopically expressed in rice. Expression of several heading date regulating genes such as Hd1, Ehd1, Hd3a and RFT1 were altered. OsNF-YB9 overexpression also resulted in morphological defects in the reproductive organs and led to pseudovivipary. OsNF-YB9 interacted with MADS1, a key regulator of foral development. This NF-Y subunit acted upstream to several transcription factors as well as signalling proteins involved in brassinosteroid and gibberellic acid metabolism and cell cycle. OsNF-YB9 and OsNF-YC12 interacted in planta and the latter also delayed heading in rice upon overexpression suggesting its involvement in a similar pathway. Our data provide new insights into the rice heading date pathway integrating these OsNF-Y subunit members to the network. These features can be exploited to improve vegetative growth and yield of rice plants in future.Item CRISPR-Cas9 directed genome engineering for enhancing salt stress tolerance in rice(Elsevier B.V., 2019) Farhat, Sufia; Jain, Neha; Singh, Nisha; Sreevathsa, Rohini; Dash, Prasanta K.; Rai, Rhitu; Yadav, Sandeep; Kumar, Pramod; Sarkar, Ananda K.; Jain, Ajay; Singh, Nagendra K.; Rai, VandnaCrop productivity in rice is harshly limited due to high concentration of salt in the soil. To understand the intricacies of the mechanism it is important to unravel the key pathways operating inside the plant cell. Emerging state-of-the art technologies have provided the tools to discover the key components inside the plant cell for salt tolerance. Among the molecular entities, transcription factors and/or other important components of sensing and signaling cascades have been the attractive targets and the role of NHX and SOS1 transporters amply described. Not only marker assisted programs but also transgenic approaches by using reverse genetic strategies (knockout or knockdown) or overexpression have been extensively used to engineer rice crop. CRISPR/Cas is an attractive paradigm and provides the feasibility for manipulating several genes simultaneously. Here, in this review we highlight some of the molecular entities that could be potentially targeted for generating rice amenable to sustain growth under high salinity conditions by employing CRISPR/Cas. We also try to address key questions for rice salt stress tolerance other than what is already known.Item Development of efficient protocol for rice transformation overexpressing MAP kinase and their effect on root phenotypic traits(Springer Nature, 2019) Singh, Pallavi; Ara, Hussain; Tayyeba, Sumaira; Pandey, Chandana; Sinha, Alok KrishnaExhaustive studies on mitogen-activated protein kinase (MAPK) have reported the importance in regulating a variety of responses during plant growth and development. In particular, the potential MAPK genes, MPK3 and MPK6, seem to regulate a plethora of responses, conferring tolerance to varied abiotic, biotic, and developmental stimuli. This makes both MPK3 and MPK6 potential targets for further studies. It would be an important concern to overexpress and knock out these pivotal proteins and then, in turn, to monitor the plant response which is expected to correlate action of a gene to a trait in cellular and organismal contexts. However, overexpression of MAPK genes has remained a puzzle in plants. In the present study, we report the generation of stable transgenic lines overexpressing OsMPK3 in indica and japonica cultivars and OsMPK6 in japonica cultivar under the control of an inducible promoter. We also establish the crucial steps and troubleshooting for each of the indicated rice transformation medium components. Later, we study the potential role of these MAPKs in high-throughput analysis of root system architectural (RSA) traits. It was observed that OsMPK6 overexpression lines had a more robust and spread out root architectural system while OsMPK3 overexpression lines had a typical bushy phenotype.Item Possible role of plant MAP kinases in the biogenesis and transcription regulation of rice microRNA pathway factors(Elsevier B.V., 2018) Badmi, Raghuram; Sheikh, Arsheed Hussain; Bhagat, Prakash Kumar; Verma, Deepanjali; Noryang, Stanzin; Sinha, Alok KrishnaSignalling pathways play vital roles as determinants of almost all the molecular processes inside a eukaryotic cell. They are more often considered to be the link between extracellular and intracellular environmental cues. Gene silencing pathways have emerged to be involved in regulation of stress responses and developmental processes. However, very little is known about the crosstalk between signalling and silencing pathways and their influence on each other. The present work describes the effects of general protein kinase inhibitors and specific mitogen activated protein kinase (MAPK) pathway inhibitors on the components of microRNA pathway in rice. The kinase inhibitors significantly reduced the activities of miRNA biogenesis complex and changed the transcript expression of miRNA pathway factors. This study suggests a possible regulation of microRNA machinery by plant kinases and MAP kinases in particular.Item Rice actin binding protein RMD controls crown root angle in response to external phosphate(Springer Nature, 2018) Huang, Guoqiang; Liang, Wanqi; Sturrock, Craig J.; Pandey, Bipin K.; Giri, Jitender; Mairhofer, Stefan; Wang, Daoyang; Muller, Lukas; Tan, Hexin; York, Larry M.; Yang, Jing; Song, Yu; Kim, Yu-Jin; Qiao, Yang; Xu, Jian; Kepinski, Stefan; Bennett, Malcolm J.; Zhang, DabingRoot angle has a major impact on acquisition of nutrients like phosphate that accumulate in topsoil and in many species; low phosphate induces shallower root growth as an adaptive response. Identifying genes and mechanisms controlling root angle is therefore of paramount importance to plant breeding. Here we show that the actin-binding protein Rice Morphology Determinant (RMD) controls root growth angle by linking actin filaments and gravity-sensing organelles termed statoliths. RMD is upregulated in response to low external phosphate and mutants lacking of RMD have steeper crown root growth angles that are unresponsive to phosphate levels. RMD protein localizes to the surface of statoliths, and rmd mutants exhibit faster gravitropic response owing to more rapid statoliths movement. We conclude that adaptive changes to root angle in response to external phosphate availability are RMD dependent, providing a potential target for breeders.Item Differential expression of salt-responsive genes to salinity stress in salt-tolerant and salt-sensitive rice (Oryza sativa L.) at seedling stage(Springer Nature, 2018) Singh, Vijayata; Singh, Ajit Pal; Bhadoria, Jyoti; Giri, Jitender; Singh, Jogendra; T. V., Vineeth; Sharma, P. C.The understanding of physio-biochemical and molecular attributes along with morphological traits contributing to the salinity tolerance is important for developing salt-tolerant rice (Oryza sativa L.) varieties. To explore these facts, rice genotypes CSR10 and MI48 with contrasting salt tolerance were characterized under salt stress (control, 75 and 150 mM NaCl) conditions. CSR10 expressed higher rate of physio-biochemical parameters, maintained lower Na/K ratio in shoots, and restricted Na translocation from roots to shoots than MI48. The higher expression of genes related to the osmotic module (DREB2A and LEA3) and ionic module (HKT2;1 and SOS1) in roots of CSR10 suppresses the stress, enhances electrolyte leakage, promotes the higher compatible solute accumulation, and maintains cellular ionic homeostasis leading to better salt stress tolerance than MI48. This study further adds on the importance of these genes in salt tolerance by comparing their behaviour in contrasting rice genotypes and utilizing specific marker to identify salinity-tolerant accessions/donors among germplasm; overexpression of these genes which accelerate the selection procedure precisely has been shown.Item Characterization of mediator complex and its associated proteins from rice(Springer, 2017) Samanta, Subhasis; Thakur, Jitendra K.The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.Item Lectin protein kinase is induced in plant roots in response to the endophytic fungus, Piriformospora indica(Springer, 2017) Nivedita; Verma, Praveen K.; Upadhyaya, Kailash C.The symbiotic association of Piriformospora indica provides growth promotion leading to increased biomass as well as enhanced plant tolerance to biotic and abiotic stresses in a variety of plant species. Since various protein kinases are thought to be involved in mutual signaling in symbiotic interactions, we analyzed transcript profile of rice protein kinase genes during the infection process by real-time PCR. A rice L-type lectin protein kinase (OslecRK) and Arabidopsis AtLecRK genes are highly upregulated in roots during the initial stage of symbiotic association. In order to investigate the role of LecRK gene in root colonization, the Arabidopsis mutant line (Atlecrk) of rice homolog was used which shows the restricted intracellular penetration of the P. indica hyphae. The results indicate that LecRK might be a crucial factor in perception and recognition during plant–P. indica association by regulating fungal invasion.Item Functional involvement of a mitogen activated protein kinase module, OsMKK3-OsMPK7-OsWRK30 in mediating resistance against Xanthomonas oryzae in rice(Nature Publishing Group, 2016) Jalmi, Siddhi Kashinath; Sinha, Alok KrishnaMitogen-activated protein kinases (MAPKs) are highly conserved signaling modules in eukaryotes, transmitting signals from upstream receptor to downstream target by phosphorelay mechanism. Here we report involvement of a poorly characterized group C MAPK of rice namely, OsMPK7 along with its upstream MAPK kinase, OsMKK3 and downstream target, OsWRKY30 during Xanthomonas oryzae infection, a causal agent of leaf blight disease in rice. X. oryzae infection resulted in induction of OsMPK7 and OsMKK3. OsMKK3 was found to physically interact and phosphorylate OsMPK7. Overexpression of OsMPK7 and OsMKK3, individually and in combinations resulted in inhibition of disease symptoms caused by X. oryzae, however silencing of OsMPK7 resulted in disease susceptibility. Furthermore, OsWRKY30 was identified as downstream target of OsMPK7 through protein-protein interaction techniques and was found to be a positive regulator of defence response against X. oryzae pathogen. The overexpression of OsMKK3-OsMPK7 upregulated genes involved in pathogenesis, cell wall structure maintenance and cell metabolism indicating possible mechanism of disease resistance. These leaves also showed restricted movement of the pathogen from the point of infection to uninfected area. Taken together, this work suggests a positive involvement of OsMKK3-OsMPK7-OsWRKY30 module in imparting disease resistance against X. oryzae infection in rice.Item 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 KrishnaIncreased 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.