Fusarium elicitor (400 |lg/mL), 24 h
Germination in presence or not of aminooxyacetic acid (AOA, 250 |xM, ACC synthase inhibitor) followed by treatment or not with ACC (100 nM) Salicylic acid (SA) Cells grown in presence of 0, 0.01, 0.1, and 1 mM SA
Seed germination in presence of SA, 500 |XM, 1 day
4-day-old seedlings (Columbia)
Seeds after 1 day of imbibition
Root, specific target enzyme families
Cellular and specific target enzyme families
2-D DIGE + MALDI Apoplast alkalinization 132
accumulation, real-time PCR
2-DE + MALDI Root hair formation, 133
2-DE + LC-MS/MS Western-blot, Glutathione 124
affinity chromatography, Real-time PCR
2-DE + MALDI Influence of SA, 67
sulfo-SA, and acetyl-SA on germination in optimal conditions or under salt stress, Detection of oxidized proteins and metabolic labeling processing, occur after protein synthesis. They modulate enzymatic activity, binding ability, subcellular localization, how long proteins remain active, and so on. Protein modification by reversible phosphorylation is the most extensively studied PTM, and it is involved in almost all signaling pathways in plants . The regulation of such an extensive and dynamic phosphorylation status mobilizes more than 5% of the Arabidopsis genome (approximately 1100 protein kinases and between 100 and 200 protein phosphatases) [81, 82], allowing a large degree of combinatorial regulation at the post-translational level (see PlantsP, Table 10.1). Phosphorylated proteins have been characterized using classical tools such as incorporation of radioactive orthophosphate by cells submitted to bacterial or fungal elicitors, followed by 2-DGE separation and MS identification of labeled proteins [83, 84]. Phosphoaffinity column chromatography combined with the use of phosphatase treatment and phosphospecific staining has allowed the characterization of a set of phosphorylated late embryogenesis abundant (LEA) proteins in mature Arabidopsis seed . To decipher the phospho-proteome, sophisticated approaches using IMAC charged with transition metals (Fe3+ or Ga3+) were developed in order to selectively purify phosphopeptides from complex mixtures. Using this approach, the evolution of the phosphorylation status of different photosynthetic proteins under several physiological conditions were analyzed [86-88]. Phosphorylation sites of proteins involved in RNA metabolim were also determined . The first successful large-scale IMAC study published led to the identification of more than 300 phosphorylation sites in Arabidopsis PM proteins [89, 90] (Table 10.2). A potential downfall of IMAC technology is that it may also bind peptides containing acidic residues, and methylation of these acidic residues can help to improve the specific binding of phosphopeptides . Recently, Wolschin and Weckwerth  have developed a promising procedure for the enrichment of phosphorylated proteins and peptides from complex mixtures based on the specific interaction of the phosphate group with metal oxide/hydroxide affinity chromatography (MOAC). A growing number of observations show that protein phosphorylation occurs at different sites in a single protein [92-94]. Moreover, the modifications of multiple sites constitute an additional layer of molecular information beyond the amino acid sequence. These modifications are coordinated, reversible, and kinetically regulated, and they work in tandem to control innumerable cellular functions. Specific methods are required to analyze the dynamics of these events in a cellular context and have been developed to quantify them. To this end, Hegeman et al.  have developed a method of determining phosphorylation stoichiometries by using methyl-esterification with an isotopic labeling reagent (methyl alcohol-^4). Using the increasing technological capacities of MS technology, Glinski and Weckwerth  have developed a highly selective LC-MS/MS-based method using high-resolution multiple reaction monitoring on a triple quadrupole mass spectrometer, to eliminate the need for SIL. The strategy combines enrichment of phosphoproteins using the MOAC technique and selective ion trap MS, providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.
With the objective of revealing novel post-translational regulations of plant proteins, several groups have recently engaged a thorough analysis of the modification status in various Arabidopsis tissues, cells or organelles. Santoni et al.,  have shown that the PIP aquaporins of the PM are methylated. PM proteins are also modified by GPI. The GPI-anchored proteins (GAPs) are targeted to the plant cell surface and are likely to be involved in extracellular matrix remodeling, cell adhesion, differentiation, and host-pathogen interactions. Proteomics works have allowed the identification of several GAPs [27, 97, 98]. PTM by N -myristoylation has also been studied and the putative Arabidopsis "N-myristoylome" consists of 437 proteins, accounting for 1.7% of the complete proteome . A growing body of evidence suggests that protein S -nitrosylation, the covalent attachment of nitric oxide (NO) to the thiol group of Cys residues, plays a regulatory role in animals but also in plants. Lindermayr et al. [100, 101] have identified more than 100 proteins as S -nitrosylation targets by the in vitro biotin switch method suggesting that a number of activities are regulated by S -nitrosylation. Reversible modification of Cys residues by S-glutathionylation was also recently studied in Arabidopsis and could play a role in redox regulation of protein activities .
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