Transcriptome reorganization during seasonal wood development in Pinus radiata
ABSTRACT: Seasonal wood development results in two distinct wood types: earlywood (EW) and latewood (LW), which is the major cause of wood qaulity variation. We investigate transcriptome reorganization during seasonal wood development in radiata pine using a newly developed 18k cDNA microarrays. Three sampling trees each at juvenile (5 yrs), transition (9 yrs) and mature (14 yrs) ages (based on the wood rings at breast height) were selected from a plantation forest of radiata pine at Bondo, NSW , Australia (35º 16' 44.04 S, 148º 26' 54.66 E). The sampling trees at juvenile and mature ages were grown within 50 m distance and under similar environment. Two sampling trees at rotation age (30 yrs) were chosen at Yarralumla, ACT, Australia (35° 18' 27'' S, 149° 7' 27.9'' E).
Project description:Wood maturation produces two distinct wood tissues: juvenile wood (JW) and mature wood (LW), which are the major cause of wood qaulity variation within a tree. We investigate transcriptome reorganization during wood maturation process in radiata pine using a newly developed 18k cDNA microarrays. Developing xylem tissues from nine sampled trees at 5- and 13-year-old each were randomly divided into three groups with three trees each. Total RNA samples extracted from three trees within a group were pooled at equal amount before using for microarray experiments. Using this pooling strategy three biological replicates were formed for each microarray experiment. Dye swap was applied in each biological replicate. Comparisons between JW and MW in spring (EW) and autumn (LW) were arranged in two separate microarray experiments: juvenile earlywood (JE) vs. mature earlywood (ME), juvenile latewood (JL) vs. mature latewood (ML)
Project description:Wood stiffness is the most important wood quality trait of forest trees for structural timber production. We investigated genes differentially transcribed in radiate pine trees with distinct wood stiffness using bulked segregant analysis (BSA) and cDNA microarrays. Transcript accumulation in earlywood (EW) and latewood (LW) of high (HS) and low stiffness (LS) trees in two progeny trials was compared. Radiata pine trees used for microarray experiment were selected from two progeny trials planted at Flynn and Kromelite, Australia. Based on the IML-based MOE measurement, five families with highest and lowest MOE each were selected from each trial, which represented two segregant populations with contrasting wood stiffness. Two individuals from each selected family were further sampled. Developing xylem tissues of selected trees in Flynn trial were sampled in spring (October) and autumn (April), representing earlywood (EW) and latewood (LW) of juvenile aged trees, respectively. Collection of xylem tissues from Kromelite trial was arranged in summer (late November) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. In Flynn trial EW and LW tissues were collected from the same sampled trees on opposite sides of the trunk. Transcript accumulation was compared in trees with highest (HS) and lowest stiffness (LS) using xylem samples from Flynn collected in spring (EW) and autumn (LW), as well as Kromelite in summer (LW), respectively. Bulked segregant analysis (BSA) was used for the experiment design. Total RNA samples extracted from the five trees with HS were pooled at equal amount, and compared to the bulked five individuals with LS. This pooling strategy can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.
Project description:Wood density is a foundamental quality trait for structural timber, bioenergy and pulp industries. We investigated genes differentially transcribed in radiate pine juvneile trees with distinct wood density using cDNA microarrays. Radiata pine trees were selected from a progeny trial planted at Flynn, Australia. Based on the gravitical measurement of wood cores, 12 families with highest and lowest density each were selected, representing two groups of trees with contrasting wood density. One individual with higher or lower density were further sampled in each selected family. Developing xylem tissues of selected trees were sampled in autumn (April) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. Wood cores of the sampled trees were further measured using SilviScan 2. Total RNA extracted from ten developing xylem tissues with confirmed distinct density in each tree group were pooled into two bulks (five trees each), and the two bulks of HD were compared with two LD bulks in the microarray experiment (named the bulk experiment). Six developing xylem tissues with the most distinct density from each tree group were further chosen. Six xylem tissues with HD were individually compared with bulked six xylem tissues with LD in the second microarray experiment (named individual experiment). These two different pooling strategies can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.
Project description:Compression (CW) and opposite wood (OW) are formed in the uniderside and upperside of conifer branches respectively in response to gravity stress. We investigated genes differentially transcribed between the underside and upside of radiate pine branches using cDNA microarrays with a view to plant gravitropism. Six trees with well-developed branches were selected from a radiata pine commercial plantation (aged 13 years) located at Bondo, NSW, Australia (35º 16' 44.04 S, 148º 26' 54.66 E). The largest branch from each tree was further selected for sampling, including three branches sampled in April 2007 (autumn in Bondo) and three sampled in October 2007 (spring). Bark was removed from the base part (about 10 cm in length) of each branch. Developing xylem tissues were scraped from the exposed upperside and underside surface respectively with a sharp chisel. Samples were immediately placed into 50 ml BD FalconTM tubes filled with liquid nitrogen. Gene expression in the underside and upperside of branches was compared using radiata pine cDNA microarrays.
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:Seasonal wood development results in two distinct wood types: earlywood (EW) and latewood (LW), which is the major cause of wood qaulity variation. We investigate transcriptome reorganization during seasonal wood development in radiata pine using a newly developed 18k cDNA microarrays. Overall design: Three sampling trees each at juvenile (5 yrs), transition (9 yrs) and mature (14 yrs) ages (based on the wood rings at breast height) were selected from a plantation forest of radiata pine at Bondo, NSW , Australia (35º 16' 44.04 S, 148º 26' 54.66 E). The sampling trees at juvenile and mature ages were grown within 50 m distance and under similar environment. Two sampling trees at rotation age (30 yrs) were chosen at Yarralumla, ACT, Australia (35° 18' 27'' S, 149° 7' 27.9'' E).
Project description:microRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression by targeting specific mRNAs. Altered expression of circulating miRNAs have been associated with age-related diseases including cancer and cardiovascular disease. Although we and others have found an age-dependent decrease in miRNA expression in peripheral blood mononuclear cells (PBMCs), little is known about the role of circulating miRNAs in human aging. Here, we examined miRNA expression in human serum from young (mean age 30 years) and old (mean age 64 years) individuals using next generation sequencing technology and real-time quantitative PCR. Of the miRNAs that we found to be present in serum, three were significantly decreased in 20 older individuals compared to 20 younger individuals: miR-151a-5p, miR-181a-5p and miR-1248. Consistent with our data in humans, these miRNAs are also present at lower levels in the serum of elderly rhesus monkeys. In humans, miR-1248 was found to regulate the expression of mRNAs involved in inflammatory pathways and miR-181a was found to correlate negatively with the pro-inflammatory cytokines IL-6 and TNFa and to correlate positively with the anti-inflammatory cytokines TGFb and IL-10. These results suggest that circulating miRNAs may be a biological marker of aging and could also be important for regulating longevity. Identification of stable miRNA biomarkers in serum could have great potential as a noninvasive diagnostic tool as well as enhance our understanding of physiological changes that occur with age. Examination of microRNAs isolated from human serum from 11 young (mean age 30 yrs) and 11 old (mean age 64 yrs) individuals and from peripheral blood mononuclear cells from one young (30 yr) and one old (64 yr) individual.
Project description:Identification of miRNAs in the xylem of Eucalyptus globublus. Samples were taken from different seasons and at different time points from the tension and opposition wood of bent branches.
Project description:Following spinal cord injury, skeletal muscle loss is rapid. This severe atrophy is attributed to declines in protein synthesis and increases in protein breakdown. However, the signaling mechanisms controlling these changes are not well understood. Nine male patients and one female patient with spinal cord injury (SCI) (Mean ± SEM = 43.9 ± 6.7 yrs) were recruited for this study. Six patients were quadriplegics and four patients were paraplegics. Inclusion criteria were as follows: patients above the age of 18 yrs, absence of severe brain injury (Glasgow Coma Scale > 13), absence of muscle-crush injury or compartment syndrome, absence of all of the following conditions: hypoxic injury, systemic sepsis, systemic inflammatory or autoimmune disease, and malignancy. Muscle biopsies were obtained from the vastus lateralis muscles of the SCI patients two days and five days post-SCI. Biopsies collected two days post-SCI were included in the current analysis. Expression changes were measured by microarray and gene clustering; identification of enriched functions and canonical pathways were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). Functional analysis found that 48h following SCI expression, gene expression changes were related to decreases in metabolic functions such as the tricarboxylic acid cycle and oxidative phosphorylation as well as increases in functions associated with protein degradation such as proteasome activity and ubiquitination. Furthermore, increases in expression of metallothioneins were found to be the most over-represented functional group in the DAVID analysis. Results from this study showed that functional categories of gene expression changes in human skeletal muscle are consistent with previous findings in animals. Muscle biopsies were obtained from the vastus lateralis muscles of the SCI patients two days and five days post-SCI. (N = 10). A 5mm Berstrom biopsy needle was used. Biopsy samples were immediately snap-frozen in liquid nitrogen upon excision. All samples were stored at -80° C until analysis.
Project description:Histone modifications play an integral role in plant development, but have been poorly studied in woody plants. Investigating chromatin organization in wood-forming tissue and its role in regulating gene expression allows us to understand the mechanisms underlying cellular differentiation during xylogenesis (wood formation) and identify novel functional regions in plant genomes. However, woody tissue poses unique challenges for using high-throughput chromatin immunoprecipitation (ChIP) techniques for studying genome-wide histone modifications in vivo. We investigated the role of the modified histone H3K4me3 (trimethylated lysine 4 of histone H3) in gene expression during the early stages of wood formation using ChIP-seq in Eucalyptus grandis, a woody biomass model. Plant chromatin fixation and isolation protocols were optimized for developing xylem tissue collected from field-grown E. grandis trees. A “nano-ChIP-seq” procedure was employed for ChIP DNA amplification. Over 9 million H3K4me3 ChIP-seq and 18 million control paired-end reads were mapped to the E. grandis reference genome for peak-calling using Model-based Analysis of ChIP-Seq. The 12,177 significant H3K4me3 peaks identified covered ~1.5% of the genome and overlapped some 9,623 protein-coding genes and 38 noncoding RNAs. H3K4me3 library coverage, peaking ~600 - 700 bp downstream of the transcription start site, was highly correlated with gene expression levels measured with RNA-seq. Overall, H3K4me3-enriched genes tended to be less tissue-specific than unenriched genes and were overrepresented for general cellular metabolism and development gene ontology terms. Relative expression of H3K4me3-enriched genes in developing secondary xylem was higher than unenriched genes, however, and highly expressed secondary cell wall-related genes were enriched for H3K4me3 as validated using ChIP-qPCR. In this first genome-wide analysis of a modified histone in a woody tissue, we developed optimized a ChIP-seq procedure suitable for field-collected samples. In developing E. grandis xylem, H3K4me3 enrichment is an indicator of active transcription, consistent with its known role in sustaining pre-initiation complex formation in yeast. The H3K4me3 ChIP-seq data from this study paves the way to understanding the chromatin landscape and epigenomic architecture of xylogenesis in plants, and complements RNA-seq evidence of gene expression for the future improvement of the E. grandis genome annotation. Examination of H3K4me3 in developing secondary xylem tissue from two clonal individuals of E. grandis growing in the field