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Open Access
Bioinformatics analysis of camalexin biosynthetic pathway and studies on defense responses of MAP2K4/MAP2K9/MAPK3 mutants during pathogenesis of Alternaria blight in Arabidopsis thaliana
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Gaur, Manu; Pandey, Dinesh
Alternaria blight is one of the most destructive fungal diseases which seriously hamper productivity of Brassica oilseeds in India. Alternaria brassicae is the main pathogen of the disease which produces chlorotic, necrotic toxins and phytohormones to cause the disease. These disease determinants have been known to affect various genes/ proteins of cell cycle and cell death pathways in susceptible Brassica plants to cause the disease and enable the pathogen to follow a semibiotrophic life style. It is being speculated that Alternaria brassicae pathogen affects highly conserved MAP kinase signal transduction pathway to cause the disease. In Arabidopsis , some of the MAP kinases including MPK3, MKK4, MKK9, are responsible for synthesis of ‘Camalexin’ which is one particular phytoalexin involved in mediating defense response against the necrotrophic fungal pathogen viz. ‘Botrytis cinerea’. In the present investigation, role of MPK3, MKK4 and MKK9 in triggering Camalexin based defense response of Brassica plant towards Alternaria brassicae was studied by using Arabidopsis mutants for these kinases and Bioinformatics analysis. Following the infection of Alternaria brassicae pathogen, disease index of mkk4, mkk9 and mpk3 mutant plants was observed to be more than that of wild type plants of Arabidopsis thaliana. These observations suggested increased susceptibility of mpk3, mkk4, mkk9 mutants of Arabidopsis for Alternaria blight due to decreased camalexin biosynthesis or differential expression of proteins. In order to study differential expression of proteins, the proteins were extracted from infected leaf samples of both wild type and mutant Arabidopsis plants and protein profiles were compared at initial, middle and late stage of infection by performing one dimensional SDS PAGE analysis. In wild and mutant plants of Arabidopsis, the protein extractability decreased as the disease progressed from early to middle stage due to degradation of host proteins and increased from middle to late stage due to synthesis of new proteins. SDS PAGE based analysis of proteins extracted from leaves of wild type and mutant Arabidopsis plants indicate differential expression of proteins in form of expression of unique prroteins and downregulation or upregulation of proteins during disease progression from early to late stage of pathogenesis in Arabidopsis mutants and wild type plants. MKK4/MKK9/MPK3 signalling module involved in biosynthesis of camalexin during defense response of B. rapa towards, Alternaria brassicae pathogen was elucidated with the help of Molecular modelling, docking, and protein-protein interaction analysis of MAP kinases retrieved from Brassica rapa genome. Network Biology based in silico approach was followed to identify linkages of MKK4, MKK9 and MPK3 with 51 known defense related genes through construction of PPI network. MPK3 was observed as major hub to which large numbers of defense regulated genes/proteins are connected. This suggests that MPK3 is largely responsible for activation of large number of defense related genes besides camalexin biosynthetic genes. These studies will open up new avenues for engineering defense pathway in Brassica for preventing losses caused by Alternaria blight disease.
Open Access
Delineating the redox signaling network under oxidative stress through over expression of Ecapx1 in Eleusine coracana
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-12) Pande, Anjali; Arora, Sandeep
Global warming has emerged as one of the biggest threats to agriculture in the last two decades. In arid and semi-arid regions, where temperatures are already close to the physiological maxima for crops, higher temperatures may increase the incidence of heat stress in crops; negatively affecting the growth and productivity. Under such conditions, development of climate resilient crops is a dire need. As reactive oxygen species are a common denominator in the effect of various environmental stresses, therefore it is important to understand the signaling events in plants that are implicated in activating the anti-oxidative defense in plants and providing redox homeostasis. Towards this objective, we successfully developed Exapx1 over-expressing E. coracana plants. The transgenic lines were developed by using Agrobacterium mediated transformation method. The transgenic lines with increased ascorbate peroxidase activity where used to delineate the redox sensing mechanisms that are involved in providing protection from oxidative stress. The transgenic lines were morphologically similar to the wild type plants and had similar PS-II activity. Increased expression of apx1 gene in the transgenic lines could induce higher expression of superoxide dismutase and monodehydroascorbate reductase genes, under stress; but no significant increase in dehydroascorbate reductase and glutathione reductase gene expression was recorded. Over-expression of Ecapx1 gene could selectively (and not universally) induce the expression and activity of other anti-oxidant enzymes of the AsA-GSH pathway, indicting the existence of a cross talk or signaling mechanism in plants, for coordinated expression of these enzymes. On exposure to stress, the transgenic lines accumulated lesser H2O2 as compared to the wild type plants and also had lower proline levels. An effective control over H2O2 levels (through Ecapx1 over-expression) prevented undue activation of specific antioxidative enzymes like DHAR and GR, while helping to maintain the redox balance of the cells under stress. Incidentally, the transgenic lines had higher reduced to total ascorbate ratio, than the wild type plants, that was responsible for sensing the increased influx of ROS and activate the cellular anti-oxidative defense network, through down-stream signaling.
Open Access
Effect of zno and fe2o3 nanoparticles as released from an alternate nutrient sources in in vitro culture media on various biomolecules Picrorhiza kurroa Royle ex Benth
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-12) Bameta, Alka; Gaur, A.K.
Genus Picrorhiza and their different species from varied habitat have been recognized and proven for various ailments, significant contribution from India has a potential due to the enormous biodiversity with special reference to native species collected from either from cultivated at various micro and macro environment or otherwise. Picrorhiza kurroa Royle ex Benth is one of the precious herbs of genus Picrorhiza. Several iridoid glycosides are present in this plant which includes their derivatives having various pharmacophore, are being subjected for their utilization in several modern and traditional medicines systems to cure number of ailments since ancient time. The plant is endemic and has been vastly explored. At present it is well recognized for hepatoprotection. It is essential to apply biotechnological approaches to enhance the production of various pharacophore either by extracts and their purifications or semi synthesis from in vitro as well as in vivo. Application of nanoscience related technologies from the bio mass through various culturing practices in various environments, have provided versatility with the secondary metabolites. During the present research work, various attempts have been made to optimized protocols for in vitro regeneration of Picrorhiza kurroa along with the effect of ZnO and Fe2O3 nanoparticles as an alternate nutrient source to recognize some facts about the synthesis of secondary metabolites, antioxidative enzymes (SOD, CAT, POX, APOX) and expression of rate limiting genes ((CAM, HMGR, DXS, EPSPS, GDPS, PAL)) involved in the synthesis of Picroside II. Nanoparticles study results indicated that it might be possible that IPP for biogenesis of different iridoid glycoside and their derivatives under application of their precursor the mevalonate, non mevalonate and phenyl-propanoid pathways might enhance the Picroside II. ZnO and Fe2O3 nanoparticles have been proved better method for eliciting their effect in order to enhance picrosides production.
Open Access
Genotyping-by-sequencing and functional genomics approaches for identification and validation of SNP markers/genes associated with grain calcium trait in finger millet
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-07) Sharma, Divya; Anil Kumar
Finger millet (Eleusine coracana (L.) Gaertn), holds immense agricultural and economic importance due to its richness in calcium and its proteins being good source of essential amino acids. Hence, research towards such indigenous, mineral nutrient rich crop will surely help in accomplishing the challenges of mineral malnutrition. Markertrait studies in finger millet could be useful in the detection of markers associated with high grain calcium and, eventually, in selection of superior genotypes. With an aim to identify markers linked to grain yeild or its component traits and calcium content, initially phenotyping was done for fourteen agro-morphological traits and grain calcium content amongst finger millet global germplasm collection at two different environments viz. Almora and Pantnagar. Analysis of variance for both the environments resulted in highly significant differences among accessions for most of the traits, which showed the existence of sufficient variability. Also the wide range of variation in the agronomic performance of the accessions suggested that these traits could be considered as good candidates for marker-trait associations. The calcium content varied from 53mg/100g seed to 454mg/100g seed with an average of 277mg/ 100 g seed at E1 and from 42mg/100g seed to 466mg/100g seed with an average of 274mg/ 100 g seed at E2, which indicates presence of sufficient variability for grain calcium trait amongst the selected finger millet genotypes. Further, to study the genetic makeup of 202 diverse finger millet accessions Genotyping by Sequencing (GBS) analysis was done, which resulted in a genome wide set of several thousand SNPs identified within every accession. Genetic diversity and population structure analysis was conducted on a natural collection of 202 finger millet genotypes. Neighbour joining cluster analysis grouped the population into 3 clusters ‘A’, ‘B’ and ‘C’ based on their geographical location. The major cluster A consisted of broadly all the Indian genotypes whereas cluster B and C comprised of exotic genotypes. A majority of accessions of Asian origin showed their tendency to cluster together with accessions of African lowland countries, which fits the hypothesis that finger millet was first domesticated in African highlands and from there it was introduced to India, thereby proving the origin of finger millet. There was good congruence between the phylogenetic tree and the population structure identified using STRUCTURE software. SNPs through GBS were used for association mapping to identify reliable marker(s) linked to grain yield or its component traits and calcium content. Five SNP markers showed homology to candidate genes of Oryza sativa (Rice) and Setaria italica, which play an important role in flowering, maturity and grain yield. Two potential SNP markers were found to be significantly associated to grain calcium content. The orthologous regions of the two identified SNP markers were found in cereal crops like Setaria italica encoding genes for Calmodulin Binding Protein (CBP) and CBL-Interacting Protein Kinase 7(CIPK7), which might play important role in grain calcium accumulation process. Promoter analysis of these genes revealed that they contain many stress responsive elements. Since, finger millet is a drought tolerant crop and also accumulate high amounts of calcium, the promoter region of these genes contain stress responsive elements, thus imparting stress tolerance to finger millet along with high grain calcium content as calcium ions play a central role in stress signaling. Differential expression analysis by qPCR showed that the two genes (EcCBP and EcCIPK7) were highly expressed in high calcium genotypes as compared to medium and low calcium genotypes. EcCIPK7 gene identified in the present study might be involved in regulating the activity of CaX exchanger and consequently increases the calcium storage in vacuoles, thus forming the basis of high grain calcium accumulation in finger millet. Since calmodulin (CaM) expresses abundantly in developing spikes and thus regulates the Ca2+ ATpase transporter located in vacuolar membrane, therefore, higher expression of EcCBP gene in later stages of spike development might be responsible for its interaction with CaM and might be responsible for higher accumulation of calcium in finger millet grains. Therefore, these genes could be considered as strong candidates for higher grain yield, Ca accumulation and its further use for Ca bio-fortification. However, exploration of its spatial distribution within seed, over expression and knockout studies will help in understanding the exact role of these genes in high seed calcium accumulation. Since, there is very limited information on genetic analysis of calcium content in finger millet grains, results from the present study would provide a fundamental basis for future research on genetic improvement of calcium content in finger millet and other crops.
Open Access
Overexpression and downregulation of dof1 transcription factor for elucidating its role in carbon and nitrogen metabolism in Eleusine coracana and Arabidopsis thaliana
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Kashyap, Anamika; Singh, B.R.
Dof1 is a transcription factor belonging to one of the well characterized plant specific transcription factor family Dof, presented on chromosome1. Dof1 was first recognized in maize and by its over expression in Arabidopsis thaliana it was revealed that dof1 is involved in the regulation of genes which play important role in carbon/nitrogen metabolism like phosphoenolpyruvate carboxylase (PEPC), pyruvate kinase (PK), citrate synthase (CS), and isocitrate dehydrogenase (ICDH), GS (Glutamate Synthetase) and GOGAT (Glutamine oxoglutarate aminotransferase). In our laboratory, full-length gene sequence of Dof1 was cloned and sequenced from finger millet genotype. Upon comparing the EcDof1 protein sequence with Dof1 of other cereals revealed high sequence similarity with rice Dof1. Real time expression analysis of Dof1 has been carried out in three genotypes of finger millet differing in grain protein content revealed that expression of Dof1 is higher for high protein genotype as compare to low protein genotype. Hence, Dof1 appears to be the key regulator in the expression of genes involved in carbon and nitrogen metabolism. Molecular cloning and characterization of dof1, from a high protein genotype of Eleusine coracana (GE-3885), was done to elucidate the role of Dof1 transcription factor. Dof1 was cloned in expression vector for its overexpression and downregulation in Eleusine coracana and Arabidopsis thaliana. Physiological and biochemical analysis of dof1 gene in transgenic lines was done to validate its role in carbon and nitrogen metabolism. Prior to this, the high protein genotype of Eleusine coracana was studied to see the expression of dof1 along with different genes involved in carbon and nitrogen assimilation under different treatments of carbon and nitrogen. And also, to transform finger millet with dof1 transcription factor (cis-trangenic), complete plant regeneration protocol was standardized via direct organogenesis and somatic embryogenesis using mature seeds as explants. Functional analysis of the transgenic plants clearly reveals that Dof1 transcription factor controls the expression of genes involved in carbon assimilation or organic acid metabolism including PEPC and PK. No significant increase in expression of GS, GOGAT and NiR was observed in Dof1transgenic plants but there was an increase in both chlorophyll and protein content in them. These results indicated that, Dof1 is indirectly involved in assimilation of nitrogen by enhancing the amount of carbon metabolites/intermediates which are needed for assimilation. Hence, Dof1 transcription factor could be targeted for transforming various plants to generate nitrogen use efficient crops.
Open Access
Comparative expression analysis of defense-related genes in both wild and transgenic Brassica juncea (var.) Varuna harboring mapk3 gene in response to infection by Albugo candida
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-05) Modak, Annayasa; Gohar Taj
Mustard and Rapeseed are the third leading source of vegetable oil in the world after soybean and oil palm. According to Department of Agriculture & Farmers welfare 2017-2018 annual production of Rapeseed and mustard is 75.40 Lakh tones. Production in India is still not enough to meet the domestic requirement. The potential yield of Brassica is very high but there is a wide gap between the potential and realized yield of Brassica and it is largely because of biotic & abiotic stresses. White rust is one of the most important disease of Brassica and it is caused by pathogen Albugo candida.Even 60% reduction in yield of Brassica juncea is due to its high susceptibility to white rust. Plants defend themselves against most potent pathogens through various defense mechanisms. Activation of MAPK signalling cascade is one of major defense pathways by which extracellular stimuli are transduced into intracellular responses in all the eukaryotes. MAPK cascade also play key role in regulation of various defense related genes in response to pathogens. Therefore our investigation was to analyse expression level of potential MAPK cascade and defense related genes in wild and transgenic Brassica juncea (var.) Varuna harboring overexpressed MAPK3 gene to access tolerance against Albugo candida. Lower disease index in transgenic Brassica suggests its tolerance to Albugo candida. Higher expression level of MAPK6 than MAPK3 in the transgenic Brassica suggests that MAPK6 mimics a portion of MAPK3 signalling pathway and work in a direct cascade for production of defense related proteins. Overexpression of MAPK3 gene was observed in downregulating the expression of MAPK4 in transgenic Brassica juncea . The expression of WRKY33 and WRKY29 was also analysed in the present study. Expression levels of both WRKY33 and WRKY29 were found to be higher in transgenic plant overexpressing MAPK3. Expression of 3 Defense related genes (OASTL-B, ACD2, and CSD2) were found higher in transgenic plants horboring overexpressed MAPK3 gene & expression level of CYP20-3 was found higher in wild Brassica plant. The defense related genes (OASTL-B, ACD2, CSD2, and CYP20-3) were also subjected to In-silico study. Insilico study showed the Secondary structure, tertiary structure and putative phosphorylation sites of these 4 defense related genes & also derived protein-protein interaction of potential MAPKs cascade and WRKY (29, 33) with OASTL-B, ACD2, CSD2 & CYP20-3.
Open Access
Molecular analysis of Osdof 1 transcription factor in response to nitrogen and abiotic stress treatments in rice (Oryza sativa L.)
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Khushboo, Manjari Mala; Singh, B.R.
Abiotic stress such as draught (water deficeiency), excessive watering , extreme temperature (such as cold ,frost, and heat ), salinity , and poor nitrogen use efficiency are major limiting factor which give negative impact to growth , yield and seed quality. Rice become more sensitive toward draught during- booting( most critical stage), tillering and primordial stage of its growth , inspite of its sensitivity the poor nitrogen uptake by the rice i.e only 30-40% of the applied nitrogen fertilizer which drastically effect the yield of the crop. Due to this reason , in last 40 year , the amount of synthetic nitrogen (N) applied to crop has risen dramatically, from 12T g/year to 104T g/year resulting in increase in yield but with considerable impacts on global environment because plants consume much less than half of the applied , means more then half of the fertilizer are leach into surrounding lake , ponds , river and cause environmental problem .Taking consideration of these problem,the aim of the study was determination of change in physiological parameter viz. plant height , leaf area and root length , biochemical parameter viz. total chlorophyll content, proline content and total soluble sugar ,transcription factor viz. dof1 , and lastly transporter AMT1;1 was studied. Change in this parameter directly effect the yield of the crop. When plant suffer to any abiotic stress the injury which observe firstly via naked is on leave. Stress due to salt greatly influence the leaf area while stress due to nitrogen deficiency mainly effect on chlorophyll. After a period of stress duration root physiology also get affected ,upto certain concentration of nitrogen deficiency ,elongation in root length was observe but in case of salinity stress shrinkage in root length was observe .Increase in proline (as compare to other amino acid) and sugar concentration can be taken as biochemical parameter for plant under stress . By accumulation of these things ossmoticum of cell get influence, which help the plant to overcome during stress condition. However increase in proline is more influence during stress as compare to free sugar. After this different morphological and biochemical parameter, dof1 expression was analysed to know its relation with nitrogen .The expression fold of Dof1 decreases with decrease in concentration of nitrogen.
Open Access
Assessment of immunogenic potential of Calcium phosphate adjuvanted nanoparticles of outer membrane proteins of Salmonella Typhi
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Singh, Yashpal; Saxena, Mumtesh Kumar
Typhoid is an important acute systemic illness affecting human beings globally and especially in developing countries of Asia and Africa. The disease leads to millions of deaths and heavy economic loss annually worldwide. The causative agent of typhoid is Salmonella enterica serovar Typhi. Antibiotics for treatment are available but most of them are becoming outdated day by day because of development of multiple drug resistance in Salmonella. Currently available vaccines have their own limitations. Therefore, there is a need to develop better vaccine. Outer membrane proteins have proven their immune potential as well as Calcium phosphate nanoparticles in DNA vaccines as adjuvant have exhibited their potential in provoking efficient cell mediated and humoral immune response. In present study, the calcium phosphate adjuvanted nanoparticles of outer membrane proteins of Salmonella Typhi (CaP-NPs-Omp complex) were assessed for its efficacy as alternative vaccine candidates for typhoid. The prepared vaccine (CaP-NPsOmp complex) was characterized and tested in mice model. The safety of vaccine was evaluated by toxicity analysis in rats. On characterization of CaP-NPs-Omp complex, particles were found to be in the size range of 20-50 nm with high stability and purity. CaP-NPs-Omp complex produced effective cell-mediated and humoral immune response along with protective immunity. On toxicity analysis, CaP-NPs-Omp complex was found to be safe. On the basis of our findings we could presume that Calcium phosphate nanoparticles encapsulating Omps of S.Typhi may turn out to be an alternative option for a safe, efficient and cost effective vaccine against Salmonella Typhi.
Open Access
A study on newly developed wheat-Aegilops kotschyi derivative lines, putative genes for iron homeostasis and EMS mutants for biofortification of hexaploid wheat with iron and zinc
(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-06) Naveen Kumar; Sundip Kumar
Micronutrients (Fe and Zn) malnutrition affects more than 2 billion people worldwide. Wheat is one of the most important staple crops, which provides more than 40% calories at different parts of the globe. Modern cultivars of wheat have lower concentration and limited variability of grain micronutrients in comparison to its wild relatives. The biofortified line of wheat (3P-3-2) developed through wide hybridization among hexaploid wheat and Aegilops kotschyi 396 followed by pollen irradiation was observed to have at par yield with three of the wheat checks namely PBW343(Lr24+GPCB1), PBW703/698 and HD2967(Yr40+Lr57). This line has shown more than 35% (39.7 mg/Kg) increase in Fe over PBW343(Lr24+GPCB1) (29.33 mg/Kg) and more than 23% (34.41 mg/Kg) increase in Zn over PBW343(Lr24+GPCB1) (27.76 mg/Kg). The overall impact of this high concentration of micronutrients is directly based on their bioavailability which is influenced by antinutritional (tannins and phytic acid) and nutritional (beta-carotene and ascorbic acid) factors. Hence, hexaploid wheat, its wild relatives, wheat-Ae kotschyi derivative lines and advanced biofortified lines were studied to analyze the bioavailability of Fe and Zn. The higher contents of ascorbic acid was estimated in Aegilops longissima (1.29 ppm) (Acc. 3507) and biofortified line S-944-2 (1.112 ppm) and the higher contents of beta-carotene was estimated in Triticum monococcum 473 (6.89 ppm), Aegilops kotschyi-393 (6.86), S-934-2 (5.28 ppm) and S-934-3 (5.29 ppm). The lower contents of phytic acid was observed in Aegilops longissima (Acc. 3507) (10.86 mg/g), Aegilops kotschyi (Acc. 3502) (12.52 mg/g) and biofortified line (R-10) (11.96 mg/g) and the lowest contents of tannic acid were observed in Ae. dicoccoides (Acc. W-744) (9.27 ppm) and the biofortified line S-933-3 (11.19 ppm). To explore the iron homeostasis genes diversity in hexaploid wheat and its progenitors, the insilico characterization of iron homeostasis gene was done. In-silico characterization results indicates the presence of five NAS genes each from the Triticum urartu, Aegilops speltoides and Aegilops tauschii and the presence of 20 NAS genes in hexaploid wheat Chinese spring on group 2, 3, 4, 5 and 6 of chromosomes. Three of the six NAAT genes were characterized as NAAT1 and other three were as NAAT2 on long arm of chromosomes of group 1 while 2 genes (TuNAAT1 and TuNAAT2) from Triticum urartu and one gene each from Aegilops speltoides (AespelNAAT2) and Aegilops tauschii (AeTauNAAT1). Three DMAS genes were identified in wheat cultivar Chinese spring which were present on the group 4 chromosome, while single gene was identified each from Triticum urartu, Aegilops speltoides and Aegilops tauschii. The in-silico characterization of NAAT and DMAS genes indicated variation in length of these genes. The variation in homologous genes are more likely due variation in the length of intron sequences. Further gene expression studies of three of these genes in hexaploid wheat (Chinese spring) indicates that the iron deficiency and age of the plant directly regulates the expression of these three (TaNAAT, TaDMAS and TaNAS9A) genes in wheat roots while TaIDEF2 was found to be post transcriptionally regulated. From the newly developed mutant population in wheat cultivar DPW 621-50, we have identified a the putative glyphosate tolerant plant, a putative lodging tolerant plant (Lt-1) and other mutants for various traits which may further be helpful in mapping genes for these traits. In addition to the above mentioned traits the mutant’s population was also used to isolate the mutants containing higher contents of minerals (Ca, Cu, Fe and Zn) in grains. The mutant line M-152-P-1 has 131.17% (60.77 ppm) Zn, 112% (89.09 ppm) Fe over the parent cultivar (DPW 621-50). The mutant line DWF-21 and Bus-2-P-2 has 86.76% (206.36 ppm) and 85.20 % (204.63 ppm) increase in their Ca contents in comparison to DPW 621-50 while lines M-9-P-4 and DWF- 47 has 110.5 % and 104.80 % in Cu in comparison to the control.

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