Project description:Woody plant material represents a vast renewable resource that has the potential to produce biofuels and other bio-based products with favourable net CO2 emissions1,2. Its potential has been demonstrated in a recent study that generated novel structural materials from flexible mouldable wood3. 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 flexibility4. While ARW disease is associated with the presence of an RNA virus5 (ARWV), the mechanism underlying the development of its unique symptoms is not known. We demonstrate that ARWV symptoms arise from reduced lignification within the secondary cell wall of xylem fibres, while the mid-lamellae region and xylem ray cells are largely unaffected, and results in increased wood digestibility. Gene expression and proteomic data from symptomatic xylem clearly show the downregulation of phenylalanine ammonia lyase (PAL), the enzyme catalysing the first committed step in the phenylpropanoid pathway leading 2 to lignin biosynthesis. PAL downregulation is the likely cause of the decreased lignification, which is consistent with a large increase in soluble phenolics, including the lignin precursor phenylalanine, seen in symptomatic xylem. ARWV-infection results in the accumulation of many host-derived viral activated small interfering RNAs (vasiRNAs). PAL-derived vasiRNAs are among the most abundant vasiRNAs in symptomatic xylem and are likely the cause of reduced PAL activity. Apparently, the mechanism used by the virus to alter lignin exhibits similarities to the RNAi strategy that has been used to alter lignin in genetically modified trees to generate comparable improvements in wood properties6–8. CRISPR technology was recently used to increase yields in maize by introducing the naturally occurring waxy mutation into maize9. A similar biomimetic approach for biomass improvement based upon our understanding of ARWV symptoms offers a promising route to improve wood properties and provides important context for the genetic manipulation of trees.

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

Project description:The within-tree variation in wood properties constitutes an exceptional model to study the mechanisms that adjust the different biosynthetic pathways providing substrates with the massive and variable demands of different biosynthetic reactions of cell wall polymers. Although a few genes have been reported as differentially expressed in differentiating compression wood compared to normal or opposite wood, the expression of a larger set of genes is expected to change due the broad range of features that distinguish this reaction wood. By combining the construction of different cDNA libraries with microarray analyses, using samples from different Pinus pinaster provenances collected in different years and geographic locations, we have identified a total of 496 genes that change their expression during differentiation of compression wood (331 up-regulated and 165 down-regulated compared to opposite wood). Consistent with the well-known structural and chemical characteristics of compression wood, a large number of genes involved in the biosynthesis of cell wall components were shown to be up-regulated during compression wood differentiation, including genes involved in synthesis of cellulose, hemicellulose, lignin and lignans. In particular, further analysis of a set of these genes involved in providing S-adenosylmethionine, ammonium recycling, lignin and lignans biosynthesis showed parallel expression profiles to levels of lignin accumulation in cells undergoing xylogenesis in vivo and in vitro. The comparative transcriptomic analysis of compression and opposite wood formation in this work have revealed a broad spectrum of coordinated transcriptional modulation of biosynthetic reactions for different cell wall polymers associated to within-tree variations in softwood structure and composition. In particular, it suggest the occurrence of a mechanism that modulates at transcriptional level genes encoding enzymes involved in S-adenosylmethionine synthesis and ammonium assimilation with coniferyl alcohol demand for lignin and lignan synthesis, as a key metabolic requirement in cells undergoing lignification. Two-condition experiment including dye-swap experiments, Compression Differentiating Xylem vs. Opposite Differentiating Xylem. Biological replicates: 4 compression xylem, 4 opposite xylew, harvested from four different individual pine trees. Two replicates per array.

Dataset Information

Flexible wood caused by altered lignin, following viral-induced suppression of phenylalanine ammonia lyase (PAL) activity. sRNA-seq

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