Elisabeth Jamet

Cell wall proteins: a new insight through proteomics

Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translational modifications have been characterized in cell wall proteins to date? The purpose of this review is to discuss the experimental results obtained to date using proteomics, as well as some of the new questions challenging future research.

Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

BackgroundThe ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure, (ii) polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50%) of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins.ResultsThe rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. Critical steps were revealed: (i) homogenization in low ionic strength acid buffer to retain CWP, (ii) purification through increasing density cushions, (iii) extensive washes with a low ionic strength acid buffer to retain CWP while removing as many cytosolic proteins as possible, and (iv) absence of detergents. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%), belonging to the same functional classes as proteins identified using previously described protocols. Finally, removal of intracellular proteins was obtained using detergents, but their amount represented less than 3% in mass of the total protein extract, based on protein quantification.ConclusionThe new cell wall preparation described in this paper gives the lowest proportion of proteins predicted to be intracellular when compared to available protocols. The application of its principles should lead to a more realistic view of the cell wall proteome, at least for the weakly bound CWP extractable by salts. In addition, it offers a clean cell wall preparation for subsequent extraction of strongly bound CWP.

Bioinformatics as a Tool for Assessing the Quality of Sub-Cellular Proteomic Strategies and Inferring Functions of Proteins: Plant Cell Wall Proteomics as a Test Case

Bioinformatics is used at three different steps of proteomic studies of sub-cellular compartments. First one is protein identification from mass spectrometry data. Second one is prediction of sub-cellular localization, and third one is the search of functional domains to predict the function of identified proteins in order to answer biological questions. The aim of the work was to get a new tool for improving the quality of proteomics of sub-cellular compartments. Starting from the analysis of problems found in databases, we designed a new Arabidopsis database named ProtAnnDB ( http://www.polebio.scsv.ups-tlse.fr/ProtAnnDB/ ). It collects in one page predictions of sub-cellular localization and of functional domains made by available software. Using this database allows not only improvement of interpretation of proteomic data (top-down analysis), but also of procedures to isolate sub-cellular compartments (bottom-up quality control).

Isolation of Plant Cell Wall Proteins

The quality of a proteomic analysis of a cell compartment strongly depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific drawbacks: (1) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure; (2) polysaccharide networks of cellulose, hemicelluloses, and pectins form potential traps for contaminants such as intracellular proteins; (3) the presence of proteins interacting in many different ways with the polysaccharide matrix require different procedures to elute them from the cell wall. Three categories of CWP are distinguished: labile proteins that have little or no interactions with cell wall components, weakly bound proteins extractable with salts, and strongly bound proteins. Two alternative protocols are decribed for cell wall proteomics: (1) nondestructive techniques allowing the extraction of labile or weakly bound CWP without damaging the plasma membrane; (2) destructive techniques to isolate cell walls from which weakly or strongly bound CWP can be extracted. These protocols give very low levels of contamination by intracellular proteins. Their application should lead to a realistic view of the cell wall proteome at least for labile and weakly bound CWP extractable by salts.

Cell Wall Proteome

In this chapter, we will focus on the contribution of proteomics to the identification and determination of the structure and function of CWPs as well as discussing new perspectives in this area. The great variety of proteins found in the plant cell wall is described. Some families, such as glycoside hydrolases, proteases, lectins, and inhibitors of cell wall modifying enzymes, are discussed in detail. Examples of the use of proteomic techniques to elucidate the structure of various cell wall proteins, especially with post-translational modifications such as Nglycosylations, proline hydroxylation and O-glycosylations, addition of GPI anchors, and phosphorylation, are given. Finally, the emerging understanding of the functions of cell wall proteins is discussed, as well as proposals for future research.

Understanding the Remodelling of Cell Walls during Brachypodium distachyon Grain Development through a Sub-Cellular Quantitative Proteomic Approach

Brachypodium distachyon is a suitable plant model for studying temperate cereal crops, such as wheat, barley or rice, and helpful in the study of the grain cell wall. Indeed, the most abundant hemicelluloses that are in the B. distachyon cell wall of grain are (1-3)(1-4)-β-glucans and arabinoxylans, in a ratio similar to those of cereals such as barley or oat. Conversely, these cell walls contain few pectins and xyloglucans. Cell walls play an important role in grain physiology. The modifications of cell wall polysaccharides that occur during grain development and filling are key in the determination of the size and weight of the cereal grains. The mechanisms required for cell wall assembly and remodelling are poorly understood, especially in cereals. To provide a better understanding of these processes, we purified the cell wall at three developmental stages of the B. distachyon grain. The proteins were then extracted, and a quantitative and comparative LC-MS/MS analysis was performed to investigate the protein profile changes during grain development. Over 466 cell wall proteins (CWPs) were identified and classified according to their predicted functions. This work highlights the different proteome profiles that we could relate to the main phases of grain development and to the reorganization of cell wall polysaccharides that occurs during these different developmental stages. These results provide a good springboard to pursue functional validation to better understand the role of CWPs in the assembly and remodelling of the grain cell wall of cereals.

Plant Glycobiology—a diverse world of lectins, glycoproteins, glycolipids and glycans

Glycosylation is essential for the growth, development or survival of every organism (Varki and Lowe, 2009). Defects in glycan signaling often lead to abnormal development and severe diseases. Glycosylation is ubiquitous and the tremendous structural complexity of glycans makes it quite impossible to predict the biological importance of individual structures. Nowadays, glycans are no longer regarded solely as an energy reservoir, but are associated with storage and transfer of biological information as part of a highly complicated multidimensional coding system (Rudiger and Gabius, 2009; Gabius et al., 2011; Solis et al., 2014). Plants synthesize a wide variety of unique glycan structures and glycan-binding proteins which play pivotal roles during their life cycle. The increasing number of excellent publications, both in primary and applied plant glycobiology research, demonstrates the great promise and importance of this area for current and future plant science. With 13 original contributions, this Research Topic is a nice compilation of Mini Reviews and Reviews, an Original research paper, and an Opinion Article, highlighting important aspects of plant glycobiology. In plant glycobiology, N-glycans constitute core structures which are grafted on polypeptide backbones. Complex N-glycans are ubiquitously present in plants (Wilson et al., 2001), yet their biological function is virtually unknown. Nguema-Ona et al. (2014) provide an overview of the biosynthesis of N-glycans. Maeda and Kimura nicely review the group of free N-glycans that are released from misfolded proteins or originate from fully processed and secreted proteins by the action of the N-glycan releasing enzymes ENGase and PNGase. They discuss the impact of these plant complex N-glycans in terms of plant development and fruit ripening (Maeda and Kimura, 2014). The paper from Strasser continues this discussion and focuses on recent developments with respect to N-glycan signaling in transgenic A. thaliana and rice plants with disabled N-glycan processing, which ultimately could lead to the development of some new glyco-engineering tools (Strasser, 2014). Next to N-glycans, photosynthesis-derived small sugars such as sucrose, fructose, glucose, trehalose, and derived oligosaccharides, which are generally accepted to be involved in plant energy metabolism and plant growth, have very recently been suggested to act as signal molecules in important plant developmental programs (Ruan, 2014; Smeekens and Hellmann, 2014). In his Opinion Article, Van den Ende (2014) focuses on this intimate communication between plant hormones and small sugars, better-known as the sugar sensing mechanism, and the putative role of small sugars in apical dominance. Plant cell walls are formed of complex interlaced networks of polysaccharides (cellulose, hemicelluose and pectins) and hydroxyproline-rich O-glycoproteins (HRGPs) which are considered as structural proteins (Carpita and Gibeaut, 1993). However, the way these macromolecules are arranged in supramolecular scaffolds is still poorly understood. Knoch et al. (2014) focus on the recent discoveries of carbohydrate active enzymes (CAZy) (Lombard et al., 2014) that are involved in the synthesis as well as in the degradation of arabinogalactan proteins (AGPs), i.e., a highly diverse class of cell surface HRGPs found in most plant species. They discuss the role of these enzymes in plant development. Nguema-Ona et al. (2014) and Hijazi et al. (2014) broaden this discussion and present an overview of the enzymes not only involved in the synthesis of AGPs, but also of extensins, another type of HRGPs, and discuss the importance of both AGPs and extensins for proper cell wall development and morphology as well as their role in biotic stress responses. Hijazi et al. (2014) propose a new model to explain how all types of HRGPs could contribute to a continuous glyco-network with their respective partners including polysaccharides to form a complex architecture in plant cell walls. In the case of secondary cell walls, lignin, and different types of hemicelluloses are found. Hao et al. (2014) present an Original Research paper in which they identified a galacturonosyltransferase (GAUT12) from A. thaliana as a new glycosyltransferase possibly contributing to the synthesis of a polysaccharidic structure including pectins allowing the deposition of xylan and lignin. Plant cell walls not only have a structural function, but also play a critical role in the perception of invading pathogens and the activation of specific plant defense responses, as discussed by Lannoo and Van Damme (2014). This review elaborates how plants can recognize plant pathogens or predators upon perception of characteristic epitopes or damage-associated patterns, using protein-protein interactions as well as protein-glycan interactions mediated by lectins. In addition, they highlight that protein-glycan interactions mediated by different types of nucleocytoplasmic lectins are part of signaling pathways implicated in plant defense responses. Plant lectins not only attracted a lot of attention due to their phytoprotective properties, they are also of interest for medical applications and use in biomedical diagnosis. They can be purified from natural resources, but with the increasing demand for biopharmaceuticals, different expression platforms are being exploited for their recombinant production. Oliveira et al. (2014) describe how they can produce recombinant frutalin, a lectin from Artocarpus incisa (breadfruit) which possesses immuno-modulatory, anti-tumor, and tumor biomarker properties, in distinct microbial systems. Since the presence and quality of glycosylation plays a crucial role for the pharmacological properties of the therapeutic protein, also plants have received growing attention for molecular farming. In this Research Topic, several papers review the humanization of the plant glycosylation pathway allowing the production of human proteins with optimized glycosylation profiles in eukaryotic microalgae (Mathieu-Rivet et al., 2014), lower plants (mosses) (Decker et al., 2014) and in higher plants (De Meyer and Depicker, 2014; Loos and Steinkellner, 2014). The major aim of this Research Topic was to provide the reader an overview of the latest progress in plant glycobiology research. All contributions demonstrate recent and exciting breakthroughs and present the intrinsic capacity of this particular scientific research area for further improvement of plant biotechnology. We hope that this e-book can provide useful information to readers and stimulate future research in the dynamic plant glycobiology community.