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Identification of novel peptide forms associated with seasonality and water restriction imposition in Eucalyptus grandis leaf proteome

ABSTRACT: Climate change is escalating the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding how plants cope with all those abiotic factors are indeed crucial. Here, we investigate how Eucalyptus grandis, a plant genus with several industry applications, cope with seasonal variation and water restriction imposition at their proteomic level under field conditions. For that, we attempted to confidently identify known proteins and novel peptides associated with the effect of seasonality and water restriction impositions, as well as to provide insights into how novel peptides behave under such conditions. Leaf proteome of E. grandis plants was studied under both a conventional proteomic workflow and a dedicated proteogenomics approach. The highest proteomic variability was identified in the summer season and the most abundant known proteins associated with seasonal variation were related to the photosynthesis process. Post-translational modifications, protein turnovers, and chaperones were the main functional classifications identified among biological treatments. Furthermore, we were able to identify 144 novel peptides by both spectral correlations against modified databases (43) and by a de novo peptide sequencing approach (101) not predicted by current proteomics pipelines. We believe that most single amino acid substituted (SAS) peptides, mainly associated with the photosynthesis process, decreased the protein stability by altering the quantitative change upon ΔΔG values and non-covalent interactions. MRM validation assays were performed for selected novel peptide identifications to provide an extra level of evidence showing to be a very robust mass-spectrometry method of validation.

INSTRUMENT(S): TSQ Quantiva, Q Exactive

ORGANISM(S): Eucalyptus Grandis

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Gabriel Jorge

LAB HEAD: Tiago Santana Balbuena

PROVIDER: PXD031100 | Pride | 2024-03-25

REPOSITORIES: Pride

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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

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Identification of novel peptide forms associated with seasonality and water restriction imposition in Eucalyptus grandis leaf proteome

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