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Background: The N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles., Results: We analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer., Conclusions: This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.

Staining of two-dimensional gel constitutes a crucial step in comparative proteome analysis with respect to the number of proteins analyzed, the accuracy of spot quantification, and compatibility with mass spectrometry. The present chapter describes procedures for several visible and fluorescent dyes compatible with mass spectrometry: colloidal Coomassie blue, silver nitrate, Sypro Ruby, Deep Purple, and 5-hexadecanoylamino-fluorescein.

Two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting were used to investigate the natural variation in the proteome among 8 Arabidopsis thaliana ecotypes, of which 3 were previously shown to display atypical responses to environmental stress. Comparison of 2-D maps demonstrated that only one-quarter of spots was shared by all accessions. On the other hand, only 15% of the 25 majors spots accounting for half the total protein amount could be classified as major spots in all ecotypes. Identification of these major spots demonstrated large differences between the major functions detected. Accordingly, the proteomes appeared to reveal important variations in terms of function between ecotypes. Hierarchical clustering of proteomes according to either the amount of all anonymous spots, that of the 25 major spots or the functions of these major spots identified the same classes of ecotypes, and grouped the three atypical ecotypes. It is proposed that proteome comparison has the capacity to evidence differences in the physiological status of ecotypes. Results are discussed with respect to the possibility to infer such differences from limited comparisons of major proteins. It is concluded that classical proteomics could constitute a powerful tool to mine the biodiversity between ecotypes of a single plant species.

Ethylene regulates fruit ripening and several plant functions (germination, plant growth, plant-microbe interactions). Protein quantification of ethylene receptors (ETRs) is essential to study their functions, but is impaired by low resolution tools such as antibodies that are mostly nonspecific, or the lack of sensitivity of shotgun proteomic approaches. We developed a targeted proteomic method, to quantify low-abundance proteins such as ETRs, and coupled this to mRNAs analyses, in two tomato lines: Wild Type (WT) and Never-Ripe (NR) which is insensitive to ethylene because of a gain-of-function mutation in ETR3. We obtained mRNA and protein abundance profiles for each ETR over the fruit development period. Despite a limiting number of replicates, we propose Pearson correlations between mRNA and protein profiles as interesting indicators to discriminate the two genotypes: such correlations are mostly positive in the WT and are affected by the NR mutation. The influence of putative post-transcriptional and post-translational changes are discussed. In NR fruits, the observed accumulation of the mutated ETR3 protein between ripening stages (Mature Green and Breaker + 8 days) may be a cause of NR tomatoes to stay orange. The label-free quantitative proteomics analysis of membrane proteins, concomitant to Parallel Reaction Monitoring analysis, may be a resource to study changes over tomato fruit development. These results could lead to studies about ETR subfunctions and interconnections over fruit development. Variations of RNA-protein correlations may open new fields of research in ETR regulation. Finally, similar approaches may be developed to study ETRs in whole plant development and plant-microorganism interactions. [ABSTRACT FROM AUTHOR]

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress. [ABSTRACT FROM AUTHOR]

Staining of two-dimensional gel constitutes a crucial step in comparative proteome analysis with respect to both the number of proteins analysed, the accuracy of spot quantification and reproducibility. In this work, we compared the efficiency of recent fluorophores to stain Arabidopsis total protein extract: Sypro Ruby® (SR), Deep Purple® (DP) and 5-hexadecanoylamino-fluorescein (C16-F). In addition, classical visible dyes, colloidal Coomassie blue (CCB) and silver nitrate (SN), were also included. High quality images were obtained for the three fluorescent dyes, DP giving the cleaner background, whereas spikes were observed with SR and a rough background with C16-F. On the other hand, saturation occurred for abundant spots with SR and DP. For a same protein load the number of detected spots ranged between 250 for CCB and 800 for SR in the sequence SR > DP ≈ SN > C16-F > CCB. These differences were shown to rely mainly on the sensitivity between dyes leading to the detection of additional spots belonging to classes of lower abundance. Analysis of the distribution of variation coefficients for spots from replicates showed differences in the staining reproducibility between dyes that ranged in the order SR > C16-F > DP > SN > CCB. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects. [Copyright &y& Elsevier]

Iron-sulfur (Fe-S) proteins play critical functions in plants. Most Fe-S proteins are synthetized in the cytosol as apo-proteins and the subsequent Fe-S cluster incorporation relies on specific protein assembly machineries. They are notably formed by a scaffold complex, which serves for the de novo Fe-S cluster synthesis, and by transfer proteins that insure cluster delivery to apo-targets. However, scarce information is available about the maturation pathways of most plastidial Fe-S proteins and their specificities towards transfer proteins of the associated SUF machinery. To gain more insights into these steps, the expression and protein localization of the NFU1, NFU2, and NFU3 transfer proteins were analyzed in various Arabidopsis thaliana organs and tissues showing quite similar expression patterns. In addition, quantitative proteomic analysis of an nfu3 loss-of-function mutant allowed to propose novel potential client proteins for NFU3 and to show that the protein accumulation profiles and thus metabolic adjustments differ substantially from those established in the nfu2 mutant. By clarifying the respective roles of the three plastidial NFU paralogs, these data allow better delineating the maturation process of plastidial Fe-S proteins. [ABSTRACT FROM AUTHOR]