Abstract
Protein phosphorylation is instrumental to early signaling events. Studying system-wide phosphorylation in relation to processes under investigation requires a quantitative proteomics approach. In Arabidopsis, auxin application can induce pericycle cell divisions and lateral root formation. Initiation of lateral root formation requires transcriptional reprogramming following auxin-mediated degradation of transcriptional repressors. The immediate early signaling events prior to this derepression are virtually uncharacterized. To identify the signal molecules responding to auxin application, we used a lateral root-inducible system that was previously developed to trigger synchronous division of pericycle cells. To identify and quantify the early signaling events following this induction, we combined (15)N-based metabolic labeling and phosphopeptide enrichment and applied a mass spectrometry-based approach. In total, 3068 phosphopeptides were identified from auxin-treated root tissue. This root proteome dataset contains largely phosphopeptides not previously reported and represents one of the largest quantitative phosphoprotein datasets from Arabidopsis to date. Key proteins responding to auxin treatment included the multidrug resistance-like and PIN2 auxin carriers, auxin response factor2 (ARF2), suppressor of auxin resistance 3 (SAR3), and sorting nexin1 (SNX1). Mutational analysis of serine 16 of SNX1 showed that overexpression of the mutated forms of SNX1 led to retarded growth and reduction of lateral root formation due to the reduced outgrowth of the primordium, showing proof of principle for our approach.
Highlights
In recent years, immense progress has been made in the global analysis of cellular protein phosphorylation events [1,2,3]
A modest number of the reported plant phosphoproteomic datasets contain quantitative data; the majority of these datasets were obtained with cultured cells (9 –11)
In Arabidopsis, lateral roots are initiated by the local activation of pericycle cells at the xylem poles [27] by auxin-triggered degradation of the transcriptional repressor SOLITARY ROOT (SLR/IAA14) and subsequent derepression of auxin response factors ARF7 and ARF19 [28]
Summary
Immense progress has been made in the global analysis of cellular protein phosphorylation events [1,2,3]. Auxin-induced Protein Phosphorylation in Arabidopsis Root be grown on media containing 15N as the sole nitrogen source [13]. We deployed 15N-labeled Arabidopsis seeds germinated and grown on solid 15N medium, allowing us to investigate auxin-induced changes in protein phosphorylation using LRIS.
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