Transcription profiling of alfalfa trichomes and stems from potato leaf hopper resistant and susceptible varieties
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ABSTRACT: Two varieties of Alfalfa plants (potato leaf hopper resistance line and sensitive line, provided by Forage Genetics International, West Salem, WI, USA) were selected to compare their transcriptomics in order to discover the mechanism of potato leaf hopper resistance in the resistant line.
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.
Project description:Two alfalfa varieties, 'Chilean' (M. sativa ssp. sativa var. Chilean, drought sensitive) and 'Wisfal' (M. sativa ssp. falcata var. Wisfal, drought tolerant), with contrasting water use efficiency were subjected to water withholding for 11 days followed by re-watering. Samples were taken for well-watered plants and plants after five, eight, eleven days of drought stress as well as plants after recovery for one day following drought stress. Roots and shoots were sampled and analyzed separately by expression profiling using Affymetrix Medicago GeneChip.
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.
Project description:Time series response of potato cv. Désirée, which is tolerant to PVY infection, was analysed in both inoculated as well as upper non-inoculated leaves. Additionally, transgenic plants deficient in accumulation of salicylic acid (NahG- Désirée) were studied in the same setting.
Project description:Identifying the intracellular and cell wall-ionically bound glycoside hydrolases (GHs) carbohydrate esterases (CEs) at the late growth stage of Arabidopsis stems is important for understanding the mechanisms regulating the cell wall integrity. The fact that whole plant stems are used as biomass feedstocks and the mixing of intracellular proteins with the cell wall proteome caused by an increasing proportion of broken cells of stems at the late growth stage pose a challenge in identifying these GHs and CEs. Here, we used a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by protein solution method for direct identification of stem proteins, and by SDS-PAGE separation and protein gel band method for improving the identification of stem proteins, using Nano-LC-MS/MS analysis. Totally, 75 and 236 stem proteins were identified by using these two methods, respectively, confirming the later method (i.e. protein gel method) identified three time more stem proteins. Among these proteins, based on cell wall protein databases and data mining analyses, 6 and 22 proteins were identified as cell wall proteins from the late growth stage Arabidopsis stems, by using these two methods respectively.
Project description:This work aimed to characterize the molecular adaptations occurring in cork oak (Quercus suber) stems in adaptation to drought, and identify key genetic pathways regulating phellem development. One-year-old cork oak plants were grown for additional 6 months under well-watered (WW) or water-deficit (WD) conditions and main stems were targeted for transcriptomic analysis. WD had a negative impact on secondary growth, decreasing the activity of the vascular cambium and phellogen. Following a tissue-specific approach, we analyzed the transcriptional changes imposed by WD in phellem (outer bark), inner bark, and xylem, and found a global downregulation of genes related to cell division, cell wall biogenesis, lignin and/or suberin biosynthesis. Phellem and phloem showed a concerted upregulation of photosynthesis-related genes, suggesting a determinant role of stem photosynthesis in the adaptation of young plants to long-term drought. The data gathered will be important to further harness the diverse genetic background of this species for the development of optimized management practices.
Project description:Three 2cm segments were excised from different parts (TOP, MID, BOT) along the vertical axis of a 4 week old (25cm) stem of flax (L. usitatissimum) were compared using a cDNA amplicon array. Each segment represented a different developmental stage, especially in relation to bast fibre differentiation (i.e. TOP= elongation, MID=transition, BOT= thickening). Only the cDNAs that showed the highest differential expression were sequenced.