Articles written in Journal of Biosciences

    • High level expression of soybean trypsin inhibitor gene in transgenic tobacco plants failed to confer resistance against damage caused byHelicoverpa armigera

      Ashis Kumar Nandi Debabrata Basu Sampa Das Soumitra K Sen

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      Helicoverpa armigera is a major pest of many tropical crop plants. Soybean trypsin inhibitor (SBTI) was highly effective against the proteolytic activity of gut extract of the insect. SBTI was also inhibitory to insect growth when present in artificial diet. The gene coding for SBTI was cloned from soybean (Glycine max, CVBirsa) and transferred to tobacco plants for constitutive expression. Young larvae ofH. armigera, fed on the leaves of the transgenic tobacco plants expressing high level of SBTI, however, maintained normal growth and development. The results suggest that in certain cases the trypsin inhibitor gene(s) may not be suitable candidates for developing insect resistant transgenic plants.

    • Down-regulation of OsSAG12-1 results in enhanced senescence and pathogen-induced cell death in transgenic rice plants

      Subaran Singh Mrunmay Kumar Giri Praveen Kumar Singh Adnan Siddiqui Ashis Kumar Nandi

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      Senescence is a highly regulated process accompanied by changes in gene expression. While the mRNA levels of most genes decline, the mRNA levels of specific genes (senescence associated genes, SAGs) increase during senescence. Arabidopsis SAG12 (AtSAG12) gene codes for papain-like cysteine protease. The promoter of AtSAG12 is SA-responsive and reported to be useful to delay senescence by expressing cytokinin biosynthesis gene isopentenyltransferase specifically during senescence in several plants including Arabidopsis, lettuce and rice. The physiological role of AtSAG12 is not known; the homozygous atsag12 mutant neither fails to develop senescence-associated vacuoles nor shows any morphological phenotype. Through BLAST search using AtSAG12 amino acid sequences as query, we identified a few putative homologues from rice genome (OsSAGs; Oryza sativa SAGs). OsSAG12-1 is the closest homologue of AtSAG12 with 64% similar amino acid composition. Expression of OsSAG12-1 is induced during senescence and pathogen-induced cell death. To evaluate the possible role of OsSAG12-1 we generated RNAi transgenic lines in Japonica rice cultivar TP309. The transgenic lines developed early senescence at varying levels and showed enhanced cell death when inoculated with bacterial pathogen Xanthomonas oryzae pv.oryzae. Our results suggest that OsSAG12-1 is a negative regulator of cell death in rice.

    • Arabidopsis FLOWERING LOCUS D influences systemic-acquiredresistance-induced expression and histone modifications of WRKY genes

      Vijayata Singh Shweta Roy Deepjyoti Singh Ashis Kumar Nandi

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      A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections – a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that FLOWERING LOCUS D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD’s involvement in epigenetic regulation of SAR.

    • Identification of plant defence regulators through transcriptional profiling of Arabidopsis thaliana cdd1 mutant

      Swadhin Swain Nidhi Singh Ashis Kumar Nandi

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      A sustainable balance between defence and growth is essential for optimal fitness under pathogen stress. Plants activate immune response at the cost of normal metabolic requirements. Thus, plants that constitutively activate defence are deprived of growth. Arabidopsis thaliana mutant constitutive defence without defect in growth and development1 (cdd1) is an exception. The cdd1 mutant is constitutive for salicylic acid accumulation, signalling, and defence against biotrophic and hemibiotrophic pathogens, without having much impact on growth. Thus, cdd1 offers an ideal genetic background to identify novel regulators of plant defence. Here we report the differential gene expression profile between cdd1 and wild-type plants as obtained by microarray hybridization. Expression of several defence-related genes also supports constitutive activation of defence in cdd1. We screened T-DNA insertion mutant lines of selected genes, for resistance against virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Through bacterial resistance, callose deposition and pathogenesis-associated expression analyses, we identified four novel regulators of plant defence. Resistance levels in the mutants suggest that At2g19810 and [rom] At5g05790 are positive regulators, whereas At1g61370 and At3g42790 are negative regulators of plant defence against bacterial pathogens.

    • The rice OsSAG12-2 gene codes for a functional protease that negatively regulates stress-induced cell death


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      Senescence is the final stage of plant development. Although expression of most of the genes is suppressed duringsenescence, a set of genes referred as senescence-associated genes (SAGs) is induced. Arabidopsis thaliana SAG12(AtSAG12) is one such gene that has been mostly studied for its strict association with senescence. AtSAG12 encodes apapain-like cysteine protease, expressed predominantly in senescence-associated vacuoles. Rice genome containsmultiple AtSAG12 homologues (OsSAGs). OsSAG12-1, the closest structural homologue of AtSAG12, is a negativeregulator of developmental and stress-induced cell death. Proteolytic activity has not been established for any SAG12homologues in vitro. Here, we report that OsSAG12-2, the second structural homologue of AtSAG12 from rice, codesfor a functional proteolytic enzyme. The recombinant OsSAG12-2 protein produced in Escherichia coli undergoesautolysis to generate a functional protease. The matured OsSAG12-2 protein shows 27% trypsin-equivalent proteolyticactivity on azocasein substrate. Dark-induced senescence activates OsSAG12-2 expression. Down-regulation of

      OsSAG12-2 in the transgenic artificial miRNA lines results in enhanced salt- and UV-induced cell death, even thoughit does not affect cell viability in the stress-free condition. Our results show that OsSAG12-2 codes for a functionalprotease that negatively regulates stress-induced cell death in rice.

    • Rice MYC2 (OsMYC2) modulates light-dependent seedling phenotype, disease defence but not ABA signalling


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      Arabidopsis MYC2 (AtMYC2) is a bHLH class transcription factor that mediates light-dependent seedling development,disease defence, JA and ABA signalling. AtMYC2 gene modulates hypocotyl elongation and expression of chlorophyll A/Bbinding protein 1 (CAB1) and rubisco small subunit protein1 (RBCS1) under blue light. The atmyc2 mutants are resistantagainst virulent bacterial pathogens. MYC2 orthologues from several crop plants have been characterized. The rice geneOs10g42430 has been referred earlier as OsMYC2 and has been shown to promote expression of JA-inducible genes.However, the role of OsMYC2 in seedling development under ABA, dark or light of specific wavelengths was not known.It was also not known whether OsMYC2 complements AtMYC2 function in Arabidopsis. We show here that expression ofOsMYC2 in the atmyc2 mutant of Arabidopsis complements the blue-light-mediated defects in hypocotyl elongation andexpression of CAB1 and RBCS1. We generated multiple transgenic rice lines for over-expression and RNAi-mediatedsuppression of OsMYC2. In agreement with AtMYC2 function, OsMYC2 over-expression and RNAi lines showedenhanced and suppressed seedling growth compared to WT plants respectively under blue light, and showed little effectunder white light or dark. In agreement with the negative regulatory role of AtMYC2 in disease defence, the RNAi linesshowed enhanced resistance against bacterial pathogen Xanthomonas oryzae pv oryzae. However, in contrast to AtMYC2function, OsMYC2 influences seedling development under red light and show no effect in ABA-mediated seed germination.Thus, the results suggest evolutionarily conserved as well as the distinct role of OsMYC2 in comparison with AtMYC2.

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