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.

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Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions.

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amyloselike polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with ␣-amylases. Exactly how this enzyme catalyzes the formation of ␣-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg 415 , subsite ؉4) and the correct positioning (Asp 394 and Arg 446 , subsite ؉1) of acceptor molecules. On the other hand, Arg 226 (subsites ؉2/؉3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.Amylosucrase (EC 2.4.1.4) is a glucansucrase belonging to glycoside-hydrolase (GH) 1 family 13 (1, 2). 2 This transglucosidase catalyzes the synthesis of an insoluble amylose-like polymer from sucrose (3), a cheap and easily available agroresource. This is in contrast to starch or glycogen synthases (4), which require nucleotide-activated sugar as a donor. Amylosucrase is thus attractive for the industrial synthesis of amyloselike polymers and for the modification of glucans (in particular to form nondigestible glucans) (5). Remarkably, amylosucrase is the only member of GH family 13 displaying polymerase activity and is clearly unique in this family that mainly contains starch-degrading enzymes. Amylosucrase was first isolated in the culture supernatant of Neisseria perflava (3) and later identified in various Neisseria strains (6, 7). Recently, data mining has revealed the presence of genes encoding putative amylosucrases in the genome of many other organisms such as Deinococcus radiodurans (8), Caulobacter crescentus (9), Xanthomonas campestris, Xanthomonas axonopodis (10), and Pirellula sp. (11). Recombinant amylosucrase from Neisseria polysaccharea (AS) has been the most extensively studied amylosucrase. The gene encoding AS (1) has been cloned, and its product has been purified to homogeneity. Characterization of the reaction products synthesized from sucrose substrate showed that sucrose isomers (turanose and trehalulose), glucose, maltose, and maltotriose were also produced besides the insoluble...

The recombinant amylosucrase from Neisseria polysaccharea was used to synthesize in vitro amylose from sucrose as unique substrate. The morphology and structure of the insoluble residue were shown to depend only on the initial sucrose concentration (100, 300, or 600 mM), which controlled both the chain length and concentration at the precipitation stage. The average degree of polymerization (DP) in the precipitated product varied from 58 for the lowest initial sucrose concentration (100 mM) to 45 and 35 for higher sucrose concentrations (300 and 600 mM, respectively). The shorter chains (DP 35 and 45), produced in high yields (54 and 24 g/L respectively), precipitated as polycrystalline aggregates with exceptional crystallinity, without optimization of the reaction medium for crystallization. The longer chains (DP 58), produced in lower amount (2.9 g/L), formed networks similar to those observed for amylose gels. All synthesized products displayed a B-type crystal structure. Their melting behavior was also studied, the thermostability being higher for the precipitate containing the longer chains. Further thermal treatments were shown to still improve the crystallinity and yield substrates usable as new standards for the determination of the relative crystallinity of starchy products. The kinetics of chain elongation and aggregation were thoroughly investigated in order to explain how the action of amylosucrase resulted in such different amylose structures. These results emphasize the potentiality of amylosucrase in the design of amylodextrins with controlled morphology, structure, and physicochemical properties.

The structure of amylosucrase from Neisseria polysaccharea in complex with beta-D-glucose has been determined by X-ray crystallography at a resolution of 1.66 A. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0 A. The D-glucose complex shows two well-defined D-glucose molecules, one that binds very strongly in the bottom of a pocket that contains the proposed catalytic residues (at the subsite -1), in a nonstrained (4)C(1) conformation, and one that binds in the packing interface to a symmetry-related molecule. A third weaker D-glucose-binding site is located at the surface near the active site pocket entrance. The orientation of the D-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrose complex shows one molecule bound in the active site, where the glucosyl moiety is located at the alpha-amylase -1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible access channel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtained through enzyme interactions with the glucosyl ring and that an important part of the enzyme function is a precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328. The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, and Arg509. Both Asp394 and Arg446 are located in an insert connecting beta-strand 7 and alpha-helix 7 that is much longer in amylosucrase compared to other enzymes from the alpha-amylase family (family 13 of the glycoside hydrolases).

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.

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.