Investigating the role of miR156 network in abiotic stress tolerance in alfalfa
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Medicago Sativa (alfalfa)
TISSUE(S): Leaf
SUBMITTER: 
Justin Renaud
LAB HEAD: Justin Renaud
PROVIDER: PXD019560 | Pride | 2020-10-16
REPOSITORIES: Pride
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Project description:Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. The application of genomic approaches would advance development of alfalfa as a cellulosic feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. These ESTs were de novo assembled into 132,153 unique sequences. By combining the de novo assembled ESTs (132,153 sequences) with our previously identified EST sequences (341,984 sequences, unpublished data), and the ESTs available from GenBank (12,371 sequences), we built the first Alfalfa Gene Index (MSGI 1.0). MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1, 294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Transcript profiling of stem internodes of genotypes 708 and 773 was conducted by quantifying the number of Illumina EST reads that were mapped to sequences in MSGI 1.0. We identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index (MSGI 1.0) assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a cellulosic feedstock. Examination of 2 different tissue types at different developmental stages (Elongating vs. post-elongation stem internodes) in two alfalfa genotypes (708 and 773) with divergent cell wall composition in stems.
2011-04-21 | E-GEOD-26757 | biostudies-arrayexpress
Project description:Alfalfa is the most produced perennial forage crop in Canada. Drought stress is a major form of abiotic stress, affecting its productivity and annual yield. A small RNA, miR156, plays a major role in drought tolerance by downregulating downstream SPL genes, but its effects at the proteome level are unknown. In this study, the protein level perturbations of miR156 overexpression (A8) and empty vector (EV) control genotypes were compared under drought stress. Using label-free quantification, 3,000 protein groups were identified, of which 68 were upregulated in A8 and 84 were downregulated relative to EV under control conditions. Conversely, under drought stress, 610 proteins were upregulated and only 52 proteins were downregulated in A8 relative to EV. Functional analysis using PlantRegMap showed that the enriched proteins are likely involved in biological and molecular processes including antioxidant response, response to stress, signal transduction and biosynthesis of secondary metabolites. These proteins/pathways might be involved in the enhancement of drought stress tolerance mediated by miR156. Protein groups related to signaling, such as MAP kinase, calcium-dependent protein kinase, protein phosphatase 2C, and transcriptional regulators including bZIP and zinc finger proteins were found to be differentially expressed when a search was conducted against a drought stress gene database. The proteomic dataset was validated by immunoblotting of selected proteins. The results of this study provide a better understanding and insight into the role of miR156 in drought stress tolerance in alfalfa at the proteomic level.
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Project description:Drought often compromises yield in non-irrigated crops such as rainfed rice, imperiling the communities that depend upon it as a primary food source. In this study, two cultivated species (Oryza sativa cv. Nipponbare and Oryza glaberrima cv. CG14) and an endemic, perennial Australian wild species (Oryza australiensis) were grown in soil at 40% field capacity for 7-d (drought). The hypothesis was that the natural tolerance of O. australiensis to erratic water supply would be reflected in a unique proteomic profile. Leaves from droughted plants and well-watered controls were harvested for label-free quantitative shotgun proteomics. Physiological and gene ontology analysis confirmed that O. australiensis is responded uniquely to drought, with superior leaf water status and enhanced levels of photosynthetic proteins. Moreover, distinctive patterns of expression of proteins in drought were observed across the entire O. australiensis proteome. An intermediate impact of drought on photosynthetic and stress-response proteins is reported in O. glaberrima relative to O. sativa but the drought response was most striking in O. australiensis. For example, photosynthetic proteins decreased when O. sativa after drought, while a narrower range of stress-responsive proteins was up-regulated. Distinctive proteomic profiles and the expression levels of individual proteins with specific functions in response to drought in O. australiensis indicate the importance of this species as a source of stress tolerance genes.
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Project description:The invasive marine mussel Mytilus galloprovincialis has displaced the native congener Mytilus trossulus from central and southern California, but the native species remains dominant at more northerly sites that have high levels of freshwater input. To determine the extent to which interspecific differences in physiological tolerance to low salinity might explain limits to the invasive species’ biogeography, we used an oligonucleotide microarray to compare the transcriptional responses of these two species to an acute decrease in salinity. Among 6,777 genes on the microarray, 117 genes showed significant changes that were similar between species, and 12 genes showed significant species-specific responses to salinity stress. Osmoregulation and cell cycle control were important aspects of the shared transcriptomic response to salinity stress, whereas the genes with species-specific expression patterns were involved in mRNA splicing, polyamine synthesis, exocytosis, translation, cell adhesion, and cell signaling. Forty-five genes that changed expression significantly during salinity stress also changed expression during heat stress, but the direction of change in expression was typically opposite for the two forms of stress. These results (i) provide insights into the role of changes in gene expression in establishing physiological tolerance to acute decreases in salinity, and (ii) indicate that transcriptomic differences between M. galloprovincialis and M. trossulus in response to salinity stress are subtle and involve only a minor fraction of the overall suite of gene regulatory responses. Two species (Mytilus galloprovincialis, Mytilus trossulus), hypo-osmotic shock for four hours (850 mOs/kg), one control group (1000 mOs/kg) sampled at the end of the treatment exposure (850 mOs/kg), one control group (1000 mOs/kg) sampled at the beginning. Biological replicates: 6 in each treatment group, 6 in each control group. Heterologous and homologous hybridization to a microarray constructed from Mytilus californianus and Mytilus galloprovincialis sequences. A reference design that used separate pools of reference RNA for each species was employed. Reference amplified RNA (aRNA) was created for each species by pooling RNA before and after amplification. The reference pool was made up of RNA from six different samples: two base-line control samples from the beginning of the experiment, two treatment samples from the end of the four-hour hypo-osmotic exposure, and two time-control samples from the end of the four-hour exposure time. To accurately compare the transcriptomes of Mytilus galloprovincialis and M. trossulus, we chose to develop a common microarray format that could be used for both species. This microarray design consisted of probe sequences generated from the out-group species, M. californianus. M. trossulus and M. galloprovincialis are approximately 7.5 million years divergent from M. californianus, yet only 3.5 million years divergent from each other (Seed, 1992). Therefore, heterologous hybridization to the microarray allowed us to compare transcriptional responses of M. galloprovincialis and M. trossulus without the inherent sequence biases that would result from a microarray that was designed from sequences of either M. galloprovincialis or M. trossulus. A limited number of sequences (556) from ESTs from M. galloprovincialis that matched M. californianus ESTs were included on the microarray to test for the effects of sequence mismatches. Only probes that performed well for both M. galloprovincialis and M. trossulus were used in our analyses. In order to determine significant changes in expression, we conducted a two-way ANOVA, in which salinity and species were modeled as fixed effects, and focused on genes that were significant for the salinity effect or the species x temperature interaction. We ignored the species term from the ANOVA as this effect could have highlighted genes that differentially bound to probes on the microarray due to differences in sequence homology, thus not reflecting true differences in gene expression. In accordance with statistical convention, all genes with a significant species x temperature interaction were deemed not to have significant temperature effects, even if the temperature term from the ANOVA had a low P-value. All genes with FDR-corrected (Benjamini and Hochberg, 1995) P-values less than 0.05 were considered significant. Analyses were conducted in the R statistical programming environment (R Development Core Team, 2009).
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Project description:Gene expression profiling of leaf tissue in transgenic alfalfa expressing Legume Anthocyanin Production 1.
2009-10-13 | E-MEXP-1855 | biostudies-arrayexpress
Project description:Two alfalfa varieties contrasting in heat tolerance, MS30 and MS37, were used for this experiment, and their seeds were provided by Sichuan Academy of Grassland Sciences. After two months of growth, both alfalfa varieties grow into adult-plants. Select 10-15 healthy plants with consistent growth to remain in each nutritional bowl. All materials are transferred to artificial climate boxes for high temperature stress treatment. The light cycle is still the day/night cycle:16/8h. According to the results of a prior experiment and based on the relevant report, the treatment temperature was set to 20℃, 25℃, 30℃, 35℃, 40℃ and 4℃, which is a gradient upward trend. Among them, alfalfa growing at 20°C were used as the control (non-HT stress). All alfalfa varieties were treated at each temperature gradient for 7 days and the leaves was collected for the determination of physiological indicators. Moreover, the physiological conditions of the plants and the humidity of the climate box were observed at any time. water the plants properly once a day, and the alfalfa special fertilizer was applied once in each cycle of temperature stress treatment to ensure the water and nutrition demand of the plant. Based on the results of the above experiment, some physiological responses demonstrated that most alfalfa varieties showed the most significant variation at 20℃, 35℃ and 43℃. Therefore, leaf samples collected from plants after exposure to the rising temperature 7 days were harvested for further TMT quantitative proteomic analysis.
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