Dynamics of Protein Synthesis and Degradation in FLG22-Stimulated Arabidopsis
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Date
2017
Publication Type
Doctoral Thesis
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Abstract
Gene expression is a multi-step process that shapes the most central aspects of a cell including its
physiology and metabolism. As such, it is governed by precise regulatory mechanisms so that the
protein levels can respond swiftly and precisely to stimuli. The regulatory cascade includes
transcription, translation and protein degradation, yet the relative contributions of these regulation
levels are not well studied on a systems-wide level, especially for plants.
In this study, I quantified four aspects of gene expression on a time-resolved, genome-wide scale
using RNA sequencing (RNA-Seq, transcription), ribosome footprint profiling (Ribo-Seq, translation),
quantitative mass spectrometry with spike-in stable isotopic labelling with amino acids in cell
culture (SILAC MS, protein abundance) and dynamic SILAC MS (protein degradation). All datasets
were obtained from the same Arabidopsis suspension cell cultures after stimulation with the
pathogen-associated molecular pattern (PAMP) flg22, the elicitor-active epitope of bacterial
flagellin, or under control conditions.
In the process of obtaining these datasets, I developed an improved SILAC method for plant cell
cultures, created an optimized Ribo-Seq protocol for plant samples, identified gene-specific
differences in translational efficiency, showed that protein turnover is modelled more accurately
with a logistic decay model compared to an exponential decay model, and determined the baseline
half-lives for over 2500 Arabidopsis proteins. This is the largest plant protein turnover dataset
obtained so far and the first one acquired with dynamic SILAC.
Statistical analyses of the data revealed that transcription showed the largest response to flg22
stimulation both in size of the fold-changes and the number of regulated genes. The translational
response reflected the transcriptional response. In fact, I found only few examples with some
regulation of translational efficiency upon flg22 treatment. Analysis of the quantitative proteomics
data revealed that some protein abundances did not follow the transcriptional and translational
changes. For the proteins that maintained constant protein levels despite transcriptional and
translational upregulation, I could show that the protein degradation rates increased
correspondingly, counteracting the raised protein synthesis. Downregulation of gene expression is
determined by a more gradual reduction in transcription, which slowly affects the protein
abundance. The speed in which the protein level responds to the transcriptional downregulation
presumably depends on the turnover rate of the individual protein.
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Examiner : Gruissem, Wilhelm
Examiner : Bärenfaller, Katja
Examiner : Robinson, Mark
Examiner : Krijgsveld, J.
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ETH Zurich
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Subject
Systems Biology; Arabidopsis; Gene Expression Regulation; Translation; Plants; Proteomics; Protein Degradation
Organisational unit
03554 - Gruissem, Wilhelm (emeritus) / Gruissem, Wilhelm (emeritus)