Cécile Albenne

Recent advances in plant cell wall proteomics

The plant extracellular matrix contains typical polysaccharides such as cellulose, hemicelluloses, and pectins that interact to form dense interwoven networks. Plant cell walls play crucial roles during development and constitute the first barrier of defense against invading pathogens. Cell wall proteomics has greatly contributed to the description of the protein content of a compartment specific to plants. Around 400 cell wall proteins (CWPs) of Arabidopsis, representing about one fourth of its estimated cell wall proteome, have been described. The main points to note are that: (i) the diversity of enzymes acting on polysaccharides suggests a great plasticity of cell walls; (ii) CWPs such as proteases, polysaccharide hydrolytic enzymes, and lipases may contribute to the generation of signals; (iii) proteins of unknown functions were identified, suggesting new roles for cell walls. Recently, the characterization of PTMs such as N‐ and O‐glycosylations improved our knowledge of CWP structure. The presence of many glycoside hydrolases and proteases suggests a complex regulation of CWPs involving various types of post‐translational events. The first 3‐D structures to be resolved gave clues about the interactions between CWPs, or between CWPs and polysaccharides. Future work should include: extracting and identifying CWPs still recalcitrant to proteomics, describing the cell wall interactome, improving quantification, and unraveling the roles of each of the CWPs.

Combining various strategies to increase the coverage of the plant cell wall glycoproteome

Glycoproteomics recently became a very active field, mostly in mammals. The first part of this paper consists of a mini-review on the strategies used in glycoproteomics, namely methods for enrichment in glycoproteins and mass spectrometry (MS) techniques currently used. In a second part, these strategies are applied to the cell wall glycoproteome of etiolated hypocotyls of Arabidopsis thaliana, showing their complementarity. Several sub-glycoproteomes were obtained by: (i) affinity chromatography on concanavaline A (ConA) and analysis of glycoproteins by MALDI-TOF MS; (ii) multidimensional lectin chromatography (using AIL, PNA, ConA and WGA lectins) and subsequent identification of glycoproteins by MALDI-TOF MS and LC-MS/MS; (iii) boronic acid chromatography followed by identification of glycoproteins by MALDI-TOF MS. Altogether, 127 glycoproteins were identified. Most glycoproteins were found to be putative N-glycoproteins and N-glycopeptides were predicted from MS data using the ProTerNyc bioinformatics software.

Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins

Xylem plays a major role in plant development and is considered part of the apoplast. Here, we studied the proteome of Brassica oleracea cv Bartolo and compared it to the plant cell wall proteome of another Brassicaceae, the model plant Arabidopsis thaliana. B. oleracea was chosen because it is technically difficult to harvest enough A. thaliana xylem sap for proteomic analysis. We studied the whole proteome and an N‐glycoproteome obtained after Concanavalin A affinity chromatography. Altogether, 189 proteins were identified by LC‐MS/MS using Brassica EST and cDNA sequences. A predicted signal peptide was found in 164 proteins suggesting that most proteins of the xylem sap are secreted. Eighty‐one proteins were identified in the N‐glycoproteome, with 25 of them specific of this fraction, suggesting that they were concentrated during the chromatography step. All the protein families identified in this study were found in the cell wall proteomes. However, proteases and oxido‐reductases were more numerous in the xylem sap proteome, whereas enzyme inhibitors were rare. The origin of xylem sap proteins is discussed. All the experimental data including the MS/MS data were made available in the WallProtDB cell wall proteomic database.

Plant Cell Wall Proteomics： Mass Spectrometry Data, a Trove for Research on Protein Structure/Function Relationships

Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organelles. In particular, mass spectrometry (MS) analysis of gel-separated proteins produces data not only for protein identification, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics of Arabidopsis thaliana. These analyses show that highly homologous members of multigene families can be differentiated. Two lectins presenting 93% amino acid identity were identified using peptide mass fingerprinting. Although the identification of structural proteins such as extensins or hydroxyproline/proline-rich proteins (H/PRPs) is arduous, different types of MS spectra were exploited to identify and characterize an H/PRP. Maturation events in a couple of cell wall proteins (CWPs) were analyzed using site mapping. N-glycosylation of CWPs as well as the hydroxylation or oxidation of amino acids were also explored, adding information to improve our understanding of CWP structure/function relationships. A bioinformatic tool was developed to locate by means of MS the N-terminus of mature secreted proteins and N-glycosylation.

Arabinogalactan protein 31 (AGP31), a putative network-forming protein in Arabidopsis thaliana cell walls?

BACKGROUND AND AIMS Arabinogalactan protein 31 (AGP31) is a remarkable plant cell-wall protein displaying a multi-domain organization unique in Arabidopsis thaliana: it comprises a predicted signal peptide (SP), a short AGP domain of seven amino acids, a His-stretch, a Pro-rich domain and a PAC (PRP-AGP containing Cys) domain. AGP31 displays different O-glycosylation patterns with arabinogalactans on the AGP domain and Hyp-O-Gal/Ara-rich motifs on the Pro-rich domain. AGP31 has been identified as an abundant protein in cell walls of etiolated hypocotyls, but its function has not been investigated thus far. Literature data suggest that AGP31 may interact with cell-wall components. The purpose of the present study was to identify AGP31 partners to gain new insight into its function in cell walls. METHODS Nitrocellulose membranes were prepared by spotting different polysaccharides, which were either obtained commercially or extracted from cell walls of Arabidopsis thaliana and Brachypodium distachyon. After validation of the arrays, in vitro interaction assays were carried out by probing the membranes with purified native AGP31 or recombinant PAC-V5-6xHis. In addition, dynamic light scattering (DLS) analyses were carried out on an AGP31 purified fraction. KEY RESULTS It was demonstrated that AGP31 interacts through its PAC domain with galactans that are branches of rhamnogalacturonan I. This is the first experimental evidence that a PAC domain, also found as an entire protein or a domain of AGP31 homologues, can bind carbohydrates. AGP31 was also found to bind methylesterified polygalacturonic acid, possibly through its His-stretch. Finally, AGP31 was able to interact with itself in vitro through its PAC domain. DLS data showed that AGP31 forms aggregates in solution, corroborating the hypothesis of an auto-assembly. CONCLUSIONS These results allow the proposal of a model of interactions of AGP31 with different cell-wall components, in which AGP31 participates in complex supra-molecular scaffolds. Such scaffolds could contribute to the strengthening of cell walls of quickly growing organs such as etiolated hypocotyls.

The highly conserved spermatophyte cell wall DUF642 protein family: Phylogeny and first evidence of interaction with cell wall polysaccharides in vitro

The evolution of spermatophyte plants involved fundamental changes in cell wall structure and function which resulted from diversification of carbohydrates and proteins. Cell wall proteomic analyses identified a novel family of proteins of yet unknown function, the DUF642 (Domain of Unknown Function 642) proteins. To investigate the evolution of the DUF642 gene family, 154 gene sequences from 24 plant species were analyzed, and phylogenetic inferences were conducted using the Maximum Likelihood and Bayesian Inference methods. Orthologous genes were detected in spermatophyte species and absent in non-seed known plant genomes. Protein sequences shared conserved motifs that defined the signature of the family. Distribution of conserved motifs indicated an ancestral intragenic duplication event. Gene phylogeny documented paleoduplication events originating three or four clades, depending on root position. When based on mid-point rooting, it retrieved four monophyletic clades: A, B, C, and D. A glycosylphosphatidylinositol (GPI)-anchor site and one or two galactose-binding domains-like (GBDLs) could be predicted for some DUF642 proteins. The B, C, and D clades grouped the predicted GPI-anchored proteins. First evidence of in vitro interaction of a DUF642 protein with a cell wall polysaccharide fraction is provided. A competition assay with cellulose prevented this interaction. The degree of diversification and the conservation of the family suggested that DUF642 proteins are key components in seed plant evolution.

Arabidopsis thaliana root cell wall proteomics: Increasing the proteome coverage using a combinatorial peptide ligand library and description of unexpected Hyp in peroxidase amino acid sequences.

Plant cell walls (CWs) contain a large proportion of polysaccharides (90–95% of CW mass) and proteins (5–10%) that play major roles in CW plasticity during development and in response to environmental cues. Here, we present CW proteomics data of Arabidopsis thaliana roots. Plants were cultivated in hydroponic conditions. CW protein (CWP) extracts were prepared and analyzed in two different ways in order to enlarge the coverage of the root CW proteome: proteins were analyzed either directly or following an affinity chromatography on a combinatorial peptide ligand library (CPLL) to reduce the concentration dynamic range. Proteins were identified by LC‐MS/MS and bioinformatics. Altogether, 424 proteins having predicted signal peptides have been identified (CWPs). CPLL permitted to identify low‐abundant CWPs never described before, thus enlarging the coverage of the root CW proteome. The number of oxidoreductases is particularly high and includes a large collection of class III peroxidases (CIII Prxs; 38 out of the 73 A. thaliana CIII Prxs). For the first time, hydroxyproline residues were localized at conserved positions in CIII Prx amino acid sequences.

Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins.

Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.

Job contenders: roles of the β-barrel assembly machinery and the translocation and assembly module in autotransporter secretion: Mechanisms of autotransporter biogenesis

In Gram‐negative bacteria, autotransporters secrete effector protein domains that are linked to virulence. Although they were once thought to be simple and autonomous secretion machines, mounting evidence reveals that multiple factors of the bacterial envelope are necessary for autotransporter assembly. Secretion across the outer membrane of their soluble effector “passenger domain” is promoted by the assembly of an outer membrane‐spanning “β‐barrel domain”. Both reactions require BamA, an essential component of the β‐barrel assembly machinery (BAM complex) that catalyzes the final reaction step by which outer membrane proteins are integrated into the lipid bilayer. A large amount of data generated in the last decade has shed key insights onto the mechanistic coordination of autotransporter β‐barrel domain assembly and passenger domain secretion. These results, together with the recently solved structures of the BAM complex, offer an unprecedented opportunity to discuss a detailed model of autotransporter assembly. Importantly, some autotransporters benefit from the presence of an additional machinery, the translocation and assembly module (TAM), a two‐membrane spanning complex, which contains a BamA‐homologous subunit. Although it remains unclear how the BAM complex and the TAM cooperate, it is evident that multiple preparatory steps are necessary for efficient autotransporter biogenesis.

An enlarged cell wall proteome of Arabidopsis thaliana rosettes

Plant cells are surrounded by cell walls playing many roles during development and in response to environmental constraints. Cell walls are mainly composed of polysaccharides (cellulose, hemicelluloses and pectins), but they also contain proteins which are critical players in cell wall remodeling processes. Today, the cell wall proteome of Arabidopsis thaliana, a major dicot model plant, comprises more than 700 proteins predicted to be secreted (cell wall proteins—CWPs) identified in different organs or in cell suspension cultures. However, the cell wall proteome of rosettes is poorly represented with only 148 CWPs identified after extraction by vacuum infiltration. This new study allows enlarging its coverage. A destructive method starting with the purification of cell walls has been performed and two experiments have been compared. They differ by the presence/absence of protein separation by a short 1D‐electrophoresis run prior to tryptic digestion and different gradient programs for peptide separation before mass spectrometry analysis. Altogether, the rosette cell wall proteome has been significantly enlarged to 361 CWPs, among which 213 newly identified in rosettes and 57 newly described. The identified CWPs fall in four major functional classes: 26.1% proteins acting on polysaccharides, 11.1% oxido‐reductases, 14.7% proteases and 11.7% proteins possibly related to lipid metabolism.