Quantitative Phosphoproteomics after Auxin-stimulated Lateral Root Induction Identifies an SNX1 Protein Phosphorylation Site Required for Growth

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 over-

ARF-Aux/IAA interactions through domain III/IV are not strictly required for auxin-responsive gene expression

“…Auxin might play a direct or indirect role in altering the state of chromatin and thus influence the binding of ARFs to auxin response promoters. Phosphorylation of ARF proteins (e.g., ARF2) might also influence their DNA-binding activity and phosphorylation of ARF2 may be an auxin-dependent process 24 , 25 . The study reported here along with the chromatin and phosphorylation studies summarized above suggest that it would be worthwhile to launch investigations into mechanisms involved in auxin-responsive gene expression that explore areas beyond ARF-Aux/IAA CTD interactions on promoters of auxin response genes.…”

The Vacuolar Manganese Transporter MTP8 Determines Tolerance to Iron Deficiency-Induced Chlorosis in Arabidopsis

“…However, the way by which FRO2 is regulated is not yet clear. FRO2 is modified by Ser phosphorylation at three sites (Zhang et al, 2013) and by Met oxidation at two sites (Engelsberger and Schulze, 2012). It remains to be determined if any of those modifications have a regulatory function that is sensitive to Mn.…”

Parallel Proteomic and Phosphoproteomic Analyses of Successive Stages of Maize Leaf Development

“…Moreover, none of these resources provide simultaneous quantification of unphosphorylated and phosphorylated forms of a protein. Such parallel comparisons are essential to distinguish changes in the phosphorylation state of a particular protein from changes in the overall abundance of that protein, as illustrated by studies in plants (Reiland et al, 2011;Zhang et al, 2013) and other systems (Rigbolt et al, 2011). Until very recently, comprehensive coverage of the proteome was not possible; therefore, the likelihood of identifying the same protein in both a phosphoproteomic and proteomic analysis was low, making such parallel comparisons difficult.…”