RNA-seq transcriptome profiling in the Fagus sylvatica trunk
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ABSTRACT: In a field study, trees from two sites in Lower Saxony, Germany, were compared. RNA-seq (performed on an Illumina HiSeq 2000) was conducted on RNA from developing xylem tissue from 4 different harvests throughout the growth season to analyze transcriptional changes related to variations in wood formation and development. A transcriptome contig database was created from the combined raw reads using ABySS. Mapping of reads from distinct samples to the contig database was performed using Bowtie.
Project description:Comparison of transcriptomes from bark, developing xylem and xylem of P. radiata saplings exposed to 0 or 1mg of Ethephon in lanolin for 1 or 8 weeks We developed an oligonucleotide microarray using sequences (mostly from Pinus taeda) from public sequence databases. These sequences were reconstituted into a non-redundant database by CAP3 assembly and used as templates for automated design of 60-mer oligonucleotide probes through eArray, AgilentM-bM-^@M-^Ys online facility. The microarray slides, manufactured by Agilent, were used to monitor gene expression in an Ethephon-induction experiment. Ethephon was dispersed in lanolin paste and applied in a 3 cm band near the base of the stem of 2-year old Pinus radiata saplings. RNA was extracted from bark, cambial region, also known as M-bM-^@M-^\developing xylemM-bM-^@M-^], and xylem tissues exposed for 1 or 8 weeks to Ethephon. The transcriptomes from these extracts were compared by hybridization onto the All-Pinus microarray slides. Statistically significant differentially expressed genes identified by limma (Linear Models for Microarray Data) were subsequently analysed by singular enrichment analysis through the Database for Annotation, Visualization and Integrated Discovery (DAVID) portal. Results revealed that bark, cambial region and xylem generate mostly mutually exclusive cohorts of genes and Gene Ontology (GO) classes. Ethephon induction led to the upregulation of xylem genes related to the metabolism of phenylpropanoids and flavonoids and to defence responses, specifically, fungal/insect attack and oxidative stress. Independent validation of the microarray data for five genes was obtained by quantitative RT-PCR. The results are also interpreted in reference to gross and microscopic morphological changes. These results confirm the utility of the All-Pinus microarray for transcriptomic research in P. radiata. Series of 2-color, 2 condition experiments in 12 180k arrays. Main comparison is within tissues exposed to 0 [control] or 1 mg Ethephon. 2nd level of comparison is between tissues [bark, xylem scraping, xylem]. Third level of comparison is between time [1 or 8 week exposure]. One slide is hybridized with cRNA generated from control and treated tissues with the same duration of exposure to Ethephon. Two biological replicates [each biological rep is a 2 y old cutting propagated clone] for treated plants whilst control consists of RNA pooled, in equal proportions [estimated by UV absorbance], from 2 untreated biological replicates.]. Dyes used for each sample are indicated in sample description.
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:P. maximowiczii × P. trichocarpa and P. nigra × P. maximowiczii plants were grown in the field, each pure and mixed with Robinia pseudoacacia, at two locations differing in soil nutrient levels. After 3 years of growing, samples of developing xylem were harvested and analyzed by RNA-seq.
Project description:In order to further determine whether genes related to xylem development have changed at the protein level, we conducted proteomic analysis of stem xylem at different stages.
Project description:Woody plant material represents a renewable resource that has the potential to produce biofuels and/or novel materials with greatly reduced CO2 emissions. The study of viral infection in plants has largely focussed on detrimental symptoms, such as leaf yellowing or cell death that result in reduced crop yields. Apple rubbery wood (ARW) disease is the result of a viral infection that causes woody stems to exhibit increased flexibility. Biochemical and histochemical studies suggest the phenotype is a result of reduced lignification, specifically within the fibre cells of woody xylem. Expression analysis and proteomic data suggests that the downregulation of phenylalanine ammonia lyase (PAL) is responsible for decreased lignification. PAL is required for the first committed step in the phenylpropanoid pathway that leads to lignin biosynthesis. This is consistent with a large increase in soluble phenolics, including the lignin precursor phenylalanine, in symptomatic xylem. Downregulation of PAL appears to result from a widespread siRNA induction by the infected host, triggered by the virus. Symptomatic wood exhibited increased digestibility comparable to those seen in genetically engineered plants that alter lignin biosynthesis. To our knowledge this is the first example of a virus that alters lignin metabolism and offers a unique route to address the problem of the recalcitrant nature of plant biomass and a possible route to generating wood with altered mechanical properties.
Project description:Woody plant material represents a renewable resource that has the potential to produce biofuels and/or novel materials with greatly reduced CO2 emissions. The study of viral infection in plants has largely focussed on detrimental symptoms, such as leaf yellowing or cell death that result in reduced crop yields. Apple rubbery wood (ARW) disease is the result of a viral infection that causes woody stems to exhibit increased flexibility. Biochemical and histochemical studies suggest the phenotype is a result of reduced lignification, specifically within the fibre cells of woody xylem. Expression analysis and proteomic data suggests that the downregulation of phenylalanine ammonia lyase (PAL) is responsible for decreased lignification. PAL is required for the first committed step in the phenylpropanoid pathway that leads to lignin biosynthesis. This is consistent with a large increase in soluble phenolics, including the lignin precursor phenylalanine, in symptomatic xylem. Downregulation of PAL appears to result from a widespread siRNA induction by the infected host, triggered by the virus. Symptomatic wood exhibited increased digestibility comparable to those seen in genetically engineered plants that alter lignin biosynthesis. To our knowledge this is the first example of a virus that alters lignin metabolism and offers a unique route to address the problem of the recalcitrant nature of plant biomass and a possible route to generating wood with altered mechanical properties.
Project description:In order to pinpoint the most differentially expressed genes between Eucalyptus grandis leaf blades and vascular (xylem) tissues as well as between E. grandis and Eucalyptus globulus xylem tissues, a total number of nine 50mer-oligoprobes covering the length of each one of 21,432 unique sequences derived from the Genolyptus EST dataset were synthesized “on-chip” in duplicate, randomly distributed in two blocks of each slide. Probes were also synthesized from ten cDNA sequences encoding known human proteins as negative controls, totaling 21,442 sequences. Leaves and xylem samples were taken from two E. grandis clonal trees, i.e., both derived from the same matrix tree and harboring the same genotype. Two additional xylem samples were collected from two other E. grandis clonal trees of a different genotype, as well as from two E. globulus clonal trees. Therefore, ten cDNA samples and ten identical chips were produced at Roche NimbleGen for the microarray assays, with a total number of 385,956 features per slide. Besides the discovery of differentially expressed genes between leaf and xylem, we wanted to test the validity of the assumed “technical” and “biological duplicates” since all trees were field-grown and four years-old in age. A ten chip study using total RNA recovered from mature leaf and vascular (xylem) tissues of Eucalyptus grandis and xylem from Eucalyptus globulus trees. Two clonal trees of E. grandis (E.grandis_Clone A_Ramet 1 and E.grandis_Clone A_Ramet 2), derived from a single matrix tree and therefore genomically identical, were the source of two samples of leaf RNA and two samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Two other clonal trees of E. grandis (E.grandis_Clone B_Ramet 1 and E.grandis_Clone B_Ramet 2), derived from a different matrix tree, were the source of two additional samples of xylem RNA individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Likewise, two pairs of clonal trees of E. globulus (E.globulus_Clone A_Ramet 1 and E.globulus_Clone A_Ramet 2/ E.globulus_Clone B_Ramet 1 and E.globulus_Clone B_Ramet 2), derived from two distinct matrix trees, were the source of four additional samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Each chip measures the expression level of 21,432 genes from Eucalyptus sp. and ten human genes (negative controls) with nine 50-mer probe pairs (PM/MM) per gene in two separate blocks per chip (technical duplicate), totalizing 18 hybridization signal values per gene per chip.
Project description:* In response to gravitational stresses, angiosperm trees form tension wood in the upper sides of branches and leaning stems in which cellulose content is higher, microfibrils are typically aligned closely with the fibre axis and the fibres often have a thick inner gelatinous cell wall layer (G-layer). * Gene expression was studied in Eucalyptus nitens branches oriented at 45° using microarrays containing 4 900 xylem cDNAs, and wood fibre characteristics revealed by X-ray diffraction, chemical and histochemical methods. * Xylem fibres in tension wood (upper branch) had a low microfibril angle, contained few fibres with G-layers and had higher cellulose and decreased Klason lignin compared to lower branch wood. Expression of two closely related fasciclin-like arabinogalactan proteins and a B-tubulin was inversely correlated with microfibril angle in upper and lower xylem from branches. * Structural and chemical modifications throughout the secondary cell walls of fibres sufficient to resist tension forces in branches can occur in the absence of G-layer enriched fibres and some important genes involved in responses to gravitational stress in eucalypt xylem are identified. Keywords: tissue comparison Two nine-year-old Eucalyptus nitens trees were used as a source of biological material. RNA was isolated from xylem from the vertical main stem and from the upper and lower quarter of branches oriented at approximately 45° from vertical. For each tree, slides were hybridized with probes synthesized from vertical xylem and one or other of upper or lower branch xylem.
Project description:A microarray analysis of whole-genome gene expression in roots was carried out in a (Populus trichocarpa X Populus deltoides) X Populus deltoides pseudo-backcross pedigree. Genetic variation in gene expression was quantified for 55,793 predicted gene models based on a single probe per gene. Resultant data contributed to the analysis of the genetic architecture of gene expression in roots of Populus. Data include one biological replicate of 163 individuals segregating from a pseudo-backcross pedigree of (Populus trichocarpa X Populus deltoides) X Populus deltoides analyzed for gene expression (GE) in roots using one probe per gene for 55793 independent gene models (probes E_POPLARSxxxxxPxxxxx) and single feature sequence polymorphism (SFP) using one probe per gene for 12084 independent gene models (probes G_POPLARSxxxxxPxxxxx). GE and SFP probes were selected from 6-7 probes per gene previously tested in a pilot study of the two parent trees of the cross (Populus deltoides X Populus trichocarpa)
Project description:A microarray analysis of whole-genome gene expression in leaves was carried out in a (Populus trichocarpa X Populus deltoides) X Populus deltoides pseudo-backcross pedigree. Genetic variation in gene expression was quantified for 55,793 predicted gene models based on a single probe per gene. Resultant data contributed to the analysis of the genetic architecture of gene expression in leaves of Populus. Data include one biological replicate of 183 individuals segregating from a pseudo-backcross pedigree of (Populus trichocarpa X Populus deltoides) X Populus deltoides analyzed for gene expression (GE) in roots using one probe per gene for 55793 independent gene models (probes E_POPLARSxxxxxPxxxxx) and single feature sequence polymorphism (SFP) using one probe per gene for 12084 independent gene models (probes G_POPLARSxxxxxPxxxxx). GE and SFP probes were selected from 6-7 probes per gene previously tested in a pilot study of the two parent trees of the cross (Populus deltoides X Populus trichocarpa)