ABSTRACT: Comparison of wild type Populus to transgenics expressing either a miRNA-resistant Populus ortholog of ATHB15/CORONA or miRNA-resistant Populus ortholog of REVOLUTA RNA was extracted from stem tissue of four biological replicates each for two mutants and wild type controls. Mutants were lines HBMut2-4-1 (Populus ATHB15/CAN ortholog) and HBMut3-8-1 (Populus REVOLUTA ortholog).
Project description:Comparison of wild type Populus to transgenics expressing either a miRNA-resistant Populus ortholog of ATHB15/CORONA or miRNA-resistant Populus ortholog of REVOLUTA
Project description:Illumina HiSeq technology was used to generate mRNA profiles of bark from MIR15 compared to wildtype plants. Wild type (WT) and transgenic poplars (Populus tremula x P. alba, clone INRA 717-1B4) were grown aseptically on Woody Plant Medium. Total RNA was extracted using Tri-Reagent according to the manufacturer’s instructions. Reads of 2X100bp were generated and aligned to Populus trichocarpa v3.0 reference transcripts (http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Ptrichocarpa; Ptrichocarpa_210_transcript_primaryTranscriptOnly) using CLC Genomics Workbench 7. mRNA profiles of bark from MIR15 compared to wildtype plants were generated by paired-end (2x100bp) Illumina HiSeq2000 sequencing. Two biological replicates were sequenced for MIR15 and WT samples.
Project description:Oligoarray analysis was used to identify genes differentially expressed in 2 mm sections of the basal stem region of wild type T89, PtAIL1 over-expressing or PtAIL1 RNAi lines of P. tremula x Populus tremuloides 24h and 72h after adventitious rooting induction. The Populus trichocarpa custom-exon expression array (12x135K) manufactured by Roche NimbleGen Systems Limited (Madison, WI) (http://www.nimblegen.com/products/exp/index.html) contained three independent, nonidentical, 60-mer probes per gene model coding sequence. Included in the oligoarray were 43,929 annotated gene models (Populus trichocarpa genome v2 ; Phytozome 5.0), 5,859 random 60-mer control probes and labelling controls. We performed 27 hybridizations from wildtype, PtAIL1 over-expressing or PtAIL1 RNAi lines of P. tremula x Populus tremuloides at time 0, 24h and 72h after adventitious rooting induction. For each timepoint we performed three biological replicates.
Project description:The Poplar transcriptome was analyzed in Populus tremulaxPopulus alba clone 717-1B4 control roots and in two poplar lines overexpressing MiSSP7. We performed 9 hybridizations (NimbleGen) with samples derived from Populus tremulaxPopulus alba clone 717-1B4 control roots, as well as from roots of LINE1 and LINE2 MiSSP7 overexpressor poplars (3 biological replicates each). All samples were labeled with Cy3.
Project description:Secretory Carrier-Associated Membrane Proteins (SCAMPs) are highly conserved 32–38 kDa proteins that are involved in membrane trafficking. A proteomics approach was taken to elucidate function of the SCAMPs in wood formation of transgenic Populus trees carrying an RNAi construct for Populus tremula x tremuloides SCAMP3 (PttSCAMP3; Potri.019G104000). Secondary xylem tissues were run on a SynaptTM G2 HDMS mass spectrometer equipped with a nanoflow electrospray ionization interface. Multivariate OnPLS (orthogonal projections to latent structures) modeling was applied to identify consistent changes in the proteomes of the transgenic lines compared to the wild type trees. The woody tissues of the transgenic trees displayed increased amounts of both polysaccharides and lignin oligomers, indicating increased deposition of both the carbohydrate and lignin components of the secondary cell walls. This coincided with slightly increased wood density as well as significantly increased thickness of the suberized cork in the transgenic lines. The OnPLS model identified a rather large number of proteins that were more abundant in the transgenic lines than in the wild type. Several of these were related to secretion and/or endocytosis as well as both primary and secondary cell wall biosynthesis, suggesting function of the Populus SCAMP proteins in membrane trafficking to fine-tune the abundance of cell wall precursors and/or proteins involved in cell wall biosynthesis and transport. The data provides a multi-level source of information for future studies on the function of the SCAMP proteins in plant stem tissues.
Project description:The brown rot fungus, Fomitopsis pinicola strain FP-58527, was cultivated in media containing ground Populus tremuloides, Pinus taeda or Picea glauca wood as sole carbon source. Mass spectrometry analyses identified proteins likely involved in the degradation of lignocellulose. Patterns of enzymes detected varied with substrate.
Project description:The brown rot fungus, Fomitopsis pinicola strain FP-58527, was cultivated in media containing ground Populus tremuloides, Pinus taeda or Picea glauca wood as sole carbon source. Mass spectrometry analyses identified proteins likely involved in the degradation of lignocellulose. Patterns of enzymes detected varied with substrate.
Project description:In this study we report on transgenic hybrid aspen (Populus tremula x P. tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after five years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula x tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 (Potri.019G116400) that was selected from a gene-mining program for novel regulators of wood formation. A proteomic analysis was performed to characterize the overall effect of the overexpression of PttVAP27-17 on plant metabolic pathways. 20 mg of xylem sample for the selected wild type (WT) and three transgenic lines (lines 1,2 and 3) was collected in the following manner from two-months-old, greenhouse grown trees: The frozen bottom-part of the stem (10-17 cm portion from the base of the stem) was peeled, and the surface of the secondary xylem consisting of living vessels and fibers (into the depth of approximately one mm from the surface) was scraped. The xylem scrapings were ground to fine powder in liquid nitrogen and stored at -80oC. The analyses included 3-5 biological replicates for each of the transgenic lines, and seven replicates for the wild type.
Project description:Trichomes are specialised epidermal cells that generally play a role in reducing transpiration and act as a deterrent to herbivory. In a screen of activation tagged Populus tremula x P. alba 717-1B4 trees, we identified a mutant line, fuzzy, with increased foliar trichome density. This mutant also had a 35% increase in growth rate and a 200% increase in the rate of photosynthesis as compared to wild-type poplar. The fuzzy mutant had significant resistance to feeding by larvae of the white spotted tussock moth (Orgyia leucostigma), a generalist insect pest of poplar trees. The fuzzy phenotype is attributable to activation tagging and increased expression of the gene encoding PtaMYB186, which is related to Arabidopsis thaliana MYB106, a known regulator of trichome initiation. The fuzzy phenotype can be recapitulated by overexpressing PtaMYB186 in poplar. PtaMYB186 overexpression results in reconfiguration of the poplar transcriptome, with changes in the transcript abundance of suites of genes that are related to trichome differentiation. It is notable that this gene responsible for trichome development also altered traits related to growth rate and pest resistance, suggesting that non-intuitive facets of plant development might be useful targets for plant improvement. 6 arrays total. 2 genotypes – WT, FUZZY – PtaMYB186 OE
Project description:RNA was extracted from xylem tissue of the first 5cm of the lower stem of four replicates of three overexpression lines and wild-type. Samples were collected from 3-month-old Populus from growth-chamber experiments at 25oC and 35oC. 2 treatments (growth at 25C and 35C temperature), 4 genotypes (wildtype and 3 35S::EVE1 independent lines) and 4 biological reps per genotype