Dataset Information

Identification of novel protein-coding sequences in Eucalyptus grandis plants by high-resolution mass spectrometry

ABSTRACT: Eucalyptus species are widely used in the forestry industry, and a significant increase in the number of sequences available in database repositories has been observed for these species. In proteomics, a protein is identified by correlating the theoretical fragmentation spectrum derived from genomic/transcriptomic data against the experimental fragmentation mass spectrum acquired from large-scale analysis of protein mixtures. Proteogenomics is an alternative approach that can identify novel proteins encoded by regions previously considered as non-coding. This study aimed to confidently identify and confirm the existence of previously unknown protein-coding sequences in the Eucalyptus grandis genome.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Eucalyptus Grandis

TISSUE(S): Plant Cell, Stem

SUBMITTER: Gabriel Jorge

LAB HEAD: Gabriel Lemes Jorge

PROVIDER: PXD022110 | Pride | 2021-01-07

REPOSITORIES: Pride

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Publications

Identification of novel protein-coding sequences in Eucalyptus grandis plants by high-resolution mass spectrometry.

Jorge Gabriel Lemes GL Balbuena Tiago Santana TS

Biochimica et biophysica acta. Proteins and proteomics 20201229 3

Eucalyptus species are widely used in the forestry industry, and a significant increase in the number of sequences available in database repositories has been observed for these species. In proteomics, a protein is identified by correlating the theoretical fragmentation spectrum derived from genomic/transcriptomic data against the experimental fragmentation mass spectrum acquired from large-scale analysis of protein mixtures. Proteogenomics is an alternative approach that can identify novel prot ...[more]

PMID: 33385527

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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:Purpose: Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection Methods: We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Reads mapping to predicted transcripts were analysed with edgeR pacakge to study differntial expression of genes under control and water-stress conditions. SNPs within the genes were obtained using pileup file generated with samtools and the SNP frequencies were analysed with VarScan package. SNPs were further characterised as sysnonymous and non-synonymous using popoolation2 pacakge. Results: Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with total gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. Conclusions: Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study. Leaf mRNA of 12 samples were used to study the gene expression under control and water-stress conditions. Three samples under water-stress conditions and three samples under control conditions were used as biological replicates for differenctial gene expression between the treatments. High-throughput sequence reads from mRNA samples were generated by deep sequencing with Illumina GAIIx.

Dataset Information

Identification of novel protein-coding sequences in Eucalyptus grandis plants by high-resolution mass spectrometry

Publications

Identification of novel protein-coding sequences in Eucalyptus grandis plants by high-resolution mass spectrometry.

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