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RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of the plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs.

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid-ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO3(2-)) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys(21) and Cys(22) as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO3(2-) to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Reactive oxygen species fulfill key roles in development and signaling, but lead at high concentration to damage in macromolecules. In proteins, methionine (Met) is particularly prone to oxidative modification and can be oxidized into Met sulfoxide (MetO). MetO reduction is catalyzed by specialized enzymes, termed methionine sulfoxide reductases (MSRs), involved in senescence and protection against diseases and environmental constraints. The precise physiological functions of MSRs remain often elusive because of very poor knowledge of their substrates. In this study, affinity chromatography was used to isolate partners of Arabidopsis thaliana plastidial methionine sulfoxide reductase B1 (MSRB1). Twenty-four proteins involved in photosynthesis, translation, and protection against oxidative stress, as well as in metabolism of sugars and amino acids, were identified. Statistical analysis shows that the abundance of MSRB1 partners in chromatography affinity samples is proportional to Met content. All proteins, for which structural modeling was feasible, display surface-exposed Met and are thus potentially susceptible to oxidation. Biochemical analyses demonstrated that H(2)O(2) treatment actually converts several MSRB1-interacting proteins into MSRB substrates. In consequence, we propose that affinity chromatography constitutes an efficient tool to isolate physiological targets of MSRs.

Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as "guards". The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity.

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker alpha-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion constructs.