Plant phosphoproteomics: An update

Abstract

Phosphoproteomics involves identification of phosphoproteins, precise mapping, and quantification of phosphorylation sites, and eventually, revealing their biological function. In plants, several systematic phosphoproteomic analyses have recently been performed to optimize in vitro and in vivo technologies to reveal components of the phosphoproteome. The discovery of novel substrates for specific protein kinases is also an important issue. Development of a new tool has enabled rapid identification of potential kinase substrates such as kinase assays using plant protein microarrays. Progress has also been made in quantitative and dynamic analysis of mapped phosphorylation sites. Increased quantity of experimentally verified phosphorylation sites in plants has prompted the creation of dedicated web-resources for plant-specific phosphoproteomics data. This resulted in development of computational prediction methods yielding significantly improved sensitivity and specificity for the detection of phosphorylation sites in plants when compared to methods trained on less plant-specific data. In this review, we present an update on phosphoproteomic studies in plants and summarize the recent progress in the computational prediction of plant phosphorylation sites. The application of the experimental and computed results in understanding the phosphoproteomic networks of cellular and metabolic processes in plants is discussed. This is a continuation of our comprehensive review series on plant phosphoproteomics.

Response to biotic and oxidative stress in Arabidopsis thaliana: Analysis of variably phosphorylated proteins

“…Reversible protein phosphorylation is a pivotal regulatory mechanism in eukaryotic cells and increasing evidence indicates that it plays a fundamental role also in plant response to biotic and abiotic stresses [8,31]. In this study, we have described the quantitative changes of phosphoproteins present in A. thaliana leaves after challenge with elicitors of plant basal defense (chitosan and benzothiadiazole), an inducer of oxidative stress (methyl viologen) as well as mechanical injury (wounding).…”

“…In this context, protein phosphorylation is a crucial event for regulation of many processes essential for plant biochemistry and physiology. Increasing evidence indicates that this post-translational modification plays a pivotal role also at different stages of plant response to pathogens, such as signal transduction events, control of the cellular redox status, adjustments to metabolism and accumulation of defense molecules [8]. However, systematic knowledge about in vivo plant protein phosphorylation in response to pathogen stimuli is lacking.…”

“…Identified protein species, together with their quantitative variations as result of the different treatments, are reported in Table 1. All proteins, except three, have been previously described in recent large-scale phosphoproteome studies as being phosphorylated [8,[24][25][26][27][28][29][30][31]. Information on protein phosphorylation was deduced by searching Arabidopsis phosphoproteome RIPP-DB, PhosPhAt and P3DB database, available on the WEB at the following addresses: https://phosphoproteome.psc.database.…”

Plant Protein Analysis

“…Many tags are currently used in plant protein purification including green fluorescent protein (Peckham et al 2006), gluthatione S-transferase (Sridhar et al2006), hexahistidine (Koroleva et al 2009), maltose binding protein (Koroleva et al 2009 To improve the purification of plant protein complexes new protein tags (TAP tags) based on Biotin carboxyl carrier domain have been developed (Qi & Katagiri 2009). One important application of these techniques has been the investigation of post-translational modifications (PTMs) in plant proteins, for example protein phosphorylation is one of the most extensively studied PTMs in plants where -immobilized metal affinity chromatography (Fe-IMAC) is widely used to enrich phosphopeptides from complex peptide mixtures (Kersten et al 2009). …”

Revealing plant defense signaling

“…The sequential application of IMAC and TiO 2 helps capture most phosphopeptides since IMAC appears to have a stronger selectivity for multiply phosphorylated peptides, whereas TiO 2 seems to have a bias toward monophosphorylated peptides. 5 Recently, in comparative analyses of rice and Arabidopsis phosphoproteomes, different phosphopeptide enrichment methods were applied, i.e., lactic acid-modified titania and β-hydroxypropanoic acid modified zirconia utilized hydroxy acid-modified metal oxide chromatography (Ti-HAMMOC and Zr-HAMMOC) and Fe-IMAC coupled to liquid chromatography-mass spectrometry (LC-MS). 6,7 A large scale data set of rice phosphorylation sites was collected from nonstimulated suspension-cultured rice cells.…”