Project description:Purpose: To identify high temperature, sailinity and drought-responsive miRNAs at genome wide level in B. juncea var varuna. Results: In this study four small RNA libraries viz. B. juncea control (BJC), B. juncea high temperature stressed (BJH), B. juncea salinity stressed (BJS) and B. juncea drought stressed (BJD) were prepared and sequenced. With the help of UEA sRNA workbench software package 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for B. juncea, we generated and assembled its genome sequence at a very low coverage. Using the generated sequence and other publically available Brassica genomic/transcriptomic resources as mapping reference, 126 novel (not reported so far in any plant species) were discovered for the first time in B. juncea. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of a few selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in B. rapa to gain insights into functional roles manifested by B. juncea miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions. Conclusions: We have generated in this study, the first comprehensive abiotic stress influenced small RNA dataset in B. juncea. The combinatorial approach of NGS and computational methods led to the discovery of 51 conserved and 126 novel miRNAs. The present study provides a holistic view of B. juncea miRNAome under conditions of high temperature, salinity and drought. The catalogue of miRNA sequences, their expression and putative targets, generated in this study can be utilized to design crop improvement strategies in B. juncea and related species. Total four small RNA libraries were prepared and sequenced independently [B. juncea control (BJC), B. juncea high temperature stressed (BJH), B. juncea salinity stressed (BJS) and B. juncea drought stressed (BJD)] on Illumina GAIIx

Project description:In low rainfall regions soils are naturally conditioned with frequent co-occurrence of salinity and alkalinity. Plant salinity responses both at physiological and molecular level have been extensively researched. However, effects of the combined treatment of alkaline salinity that could greatly reduce plant growth and the mechanisms responsible for tolerance remain indeterminate. In Brassica juncea, large reductions in biomass and increased leaf Na+ concentration under alkaline salinity indicates that the combined treatment had greater negative effect than salinity on both growth and the physiological responses of the plant. To determine molecular mechanisms potentially controlling adaptive tolerance responses to salinity and alkaline salinity, the moderately tolerant genotype NDR 8501 was further investigated using microarray analysis. The transcripts of treated leaf tissues verses those of the untreated control sample were analysed after prolonged stress of four weeks. In total, 528 salinity responsive and 1245 alkaline salinity responsive genes were indentified and only 101 genes were expressed jointly in either of the two treatments. Transcription of 37% more genes involved in response to alkaline salinity than salinity alone, which suggests the increased impact and severity of the combined stress on the plant, indicating the transcription of a far greater number of genes likely involved in mitigation and damage control. Transcription of KUP2 and KUP7 genes involved in potassium homeostasis under salinity alone and NHX1 and ENH1 genes for ion (K+ and Na+) homeostasis under alkaline salinity, clearly demonstrated that different genes and genetic pathways are involved in response to each stress. They further provide supporting evidence for the physiological responses that occur in the plant, with massive reprogramming of the transcriptome leading to partial ion exclusion, shuttling and compartmentation. Salinity and alkaline salinity induced gene expression in Brassica juncea leaf was measured at 4 weeks of prolonged treatment of 50 mM NaCl alone and combined with 2.5 mM HCO3- versus non-stressed control. A single experiment was conducted using Brassica juncea genotype NDR 8501 at a single time point (fours weeks after treatment) with three replications per treatment.

Project description:Engineered abiotic stress resistance is an important target for increasing agricultural productivity.There are concerns, however, regarding possible ecological impacts of transgenic crops. In contrast to the first wave of transgenic crops, many abiotic stress resistance genes can initiate complex downstream changes. Transcriptome profiling has been suggested as a comprehensive non-targeted approach to examine secondary effects. We compared phenotypic and transcriptomic effects of constitutive expression of genes intended to confer salt stress tolerance by three different mechanisms: a transcription factor, CBF3/DREB1a; a metabolic gene, M6PR, for mannitol biosynthesis; and the Na+/H+ antiporter, SOS1. Transgenic CBF3, M6PR, and SOS1 Arabidopsis thaliana were grown together in the growth chamber, greenhouse and field. In the absence of salt, M6PR and SOS1 lines performed comparably to wild type; CBF3 lines exhibited dwarfing as reported previously. All three transgenes conferred fitness advantage when subjected to 100 mM NaCl in the growth chamber. CBF3 and M6PR affected transcription of numerous abiotic stress- related genes as measured by Affymetrix microarray analysis. M6PR additionally modified expression of biotic- and oxidative- stress genes. Transcriptional effects of SOS1 in the absence of salt were smaller and primarily limited to redox-related genes. The extent of transcriptome change, however, did not correlate with effects on growth and reproduction. Thus, magnitude of global transcriptome differences may not predict phenotypic differences upon which environment and selection act to influence fitness. These observations have implications for interpretation of transcriptome analyses in the context of risk assessment and emphasize importance of evaluation within a phenotypic context. Both transgenic plants and relative WT plants were grown in the growth chamber in the absence and presence of salt stress. Plants from 20 days after sowing (6 days after salt treatment) were used for RNA extraction and hybridization on Affymetrix microarrays. There were two biological replicates for each genotype and salt treatment combination.

Project description:Purpose: To identify high temperature, sailinity and drought-responsive miRNAs at genome wide level in B. juncea var varuna. Results: In this study four small RNA libraries viz. B. juncea control (BJC), B. juncea high temperature stressed (BJH), B. juncea salinity stressed (BJS) and B. juncea drought stressed (BJD) were prepared and sequenced. With the help of UEA sRNA workbench software package 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for B. juncea, we generated and assembled its genome sequence at a very low coverage. Using the generated sequence and other publically available Brassica genomic/transcriptomic resources as mapping reference, 126 novel (not reported so far in any plant species) were discovered for the first time in B. juncea. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of a few selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in B. rapa to gain insights into functional roles manifested by B. juncea miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions. Conclusions: We have generated in this study, the first comprehensive abiotic stress influenced small RNA dataset in B. juncea. The combinatorial approach of NGS and computational methods led to the discovery of 51 conserved and 126 novel miRNAs. The present study provides a holistic view of B. juncea miRNAome under conditions of high temperature, salinity and drought. The catalogue of miRNA sequences, their expression and putative targets, generated in this study can be utilized to design crop improvement strategies in B. juncea and related species.

Project description:Arabidopsis ecotypes of Sha and Ler showed differences in tolerance to salinity stress. A previous study indicated that a premature stop codon resulting in a truncated Response to ABA and Salt 1 (RAS1) protein in Sha contributes to the increased salt tolerance relative to Ler ecotype. Sha exhibited higher germination rates and longer roots on MS plate, presumably due to the decreased ABA sensitivity in Sha. More Sha plants also survived in soil after salt treatment with relatively lower electrolyte leakage when compared to Ler. Transcriptome analysis revealed that expression levels of many genes were changed between Sha and Ler ecotypes and by salt treatments. About 500 transcripts were commonly changed by at least one salinity effect and one ecotype effect, and 171 of them were co-regulated by all four comparisons. Transcripts involved in redox, secondary metabolism, auxin metabolism, photosynthesis, cell wall, and protein synthesis were mainly down-regulated by salinity effects, while transposable element genes, microRNA and antisense sequences, histone superfamily genes, and biotic stress related genes were significantly changed by Sha ecotype effects and only slightly by salinity. Several metabolic pathways such as stress, TCA, hormone/lipid/secondary metabolism, redox, development, and GO terms involved in stress, oxidation, and defense response were enriched by both salinity and ecotype effects. Ninety-five highly inducible genes were identified as candidates of RAS1 target genes and the functions involved hormone metabolism, biotic stress, RNA, DNA synthesis, protein metabolism, cell, and microRNA metabolism. All these results indicated that the Sha ecotype was possibly preconditioned to abiotic stress relative to Ler through regulation of signaling pathways and stress responsive gene expression. These comparative transcriptomic and analytical results also confirm the complexity of ABA responses and salt stress tolerance mechanisms, and they suggest additional targets for improving tolerance.

Dataset Information

De Novo Assembly and Characterization of Stress Transcriptome in a salinity-Tolerant Variety CS52 of Brassica juncea

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