The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK.
Proteomics (Impact Factor: 3.81). 05/2009; 9(9):2355-72. DOI: 10.1002/pmic.200800721
Source: PubMed


The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion-exchange chromatography, could be determined accurately since, xylem-specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.

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    • "occurring primarily in Fabaceae that associate with nitrogen-fixing bacteria have been used in proteomics studies. Finally, extracellular fluids such as root exudates [142], xylem and phloem sap [80] [143] [144], and intercellular washing (apoplast) fluid [145] [146] have been analyzed at the protein level. "
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    ABSTRACT: The topic of plant proteomics is reviewed based on related papers published in the journal "Proteomics" since publication of the first issue in 2001. In total, around 300 original papers and 41 reviews published in "Proteomics" between 2000 and 2014 have been surveyed. Our main objective for this review is to help bridge the gap between plant biologists and proteomics technologists, two often very separate groups. Over the past years a number of reviews on plant proteomics have been published [1-7]. To avoid repetition we have focused on more recent literature published after 2010, and have chosen to rather make continuous reference to older publications. The use of the latest proteomics techniques, its integration with other approaches in the Systems Biology direction are discussed more in detail. Finally we comment on the recent history, state of the art, and future directions of plant proteomics, using publications in "Proteomics" to illustrate the progress in the field. The review is organized into two major blocks, the first devoted to provide an overview of experimental systems (plants, plant organs, biological processes) and the second one to the methodology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Proteomics 03/2015; 15(5-6). DOI:10.1002/pmic.201400349 · 3.81 Impact Factor
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    • "Plant cell walls contain diverse enzymes that can hydrolyse or re-model xyloglucan (Millar et al., 2009; Franková and Fry, 2013). "
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    ABSTRACT: Xyloglucan plays an important structural role in primary cell walls, possibly tethering adjacent microfibrils and restraining cell expansion. There is therefore considerable interest in understanding the role of xyloglucan endotransglucosylase/hydrolases (XTHs), which are encoded in Arabidopsis by a 33-member gene family. We compared the key catalytic properties of two very different Arabidopsis XTHs (heterologously produced in Pichia), both of which are aluminium-repressed. Reductively tritiated oligosaccharides of xyloglucan were used as model acceptor substrates. Untransformed Pichia produced no xyloglucan-acting enzymes; therefore purification of the XTHs was unnecessary. XTH15, a classical group-I/II XTH, had high XET and undetectable XEH activity in vitro; its XET Km values were 31 μM XXXGol (acceptor substrate) and 2.9 mg/ml xyloglucan (donor substrate). In contrast, XTH31, a group-III-A XTH, showed predominant XEH activity and only slight XET activity in vitro; its XET Km was 86 μM XXXGol (acceptor), indicating a low affinity of this predominantly hydrolytic protein for a transglycosylation acceptor substrate. The Km of XTH31’s XEH activity was 1.6 mg/ml xyloglucan. For both proteins, the preferred XET acceptor substrate, among five cellotetraitol-based oligosaccharides tested, was XXXGol. XTH31’s XET activity was strongly compromised when the second Xyl residue was galactosylated. XTH15’s XET activity, in contrast, tolerated substitution at the second Xyl residue. The two enzymes also showed different pH preferences, XTH31 exhibiting an unusually low pH optimum and XTH15 an unusually broad optimum. XTH31’s hydrolase activity increased almost linearly with decreasing pH in the apoplastic range, 6.2-4.5, consistent with a possible role in ‘acid growth’. In conclusion, these two Al3+-repressed XTHs differ, in several important enzymic features, from other members of the Arabidopsis XTH family.
    Phytochemistry 10/2014; 112(1). DOI:10.1016/j.phytochem.2014.09.020 · 2.55 Impact Factor
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    • "Due to these possible interactions, many technical difficulties are encountered during extraction and analysis of cell wall proteins. Moreover, accessibility and availability of CWPs from specific tissues in a living plant are not always straightforward, making the analysis experimentally limited [11]. Hence, tissue culture systems provide a convenient and attractive source of cell wall proteins. "
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    ABSTRACT: Secreted class III peroxidases (EC are implicated in a broad range of physiological processes throughout the plant life cycle. However, the unambiguous determination of the precise biological role of an individual class III peroxidase isoenzyme is still a difficult task due to genetic redundancy and broad substrate specificity in vitro. In addition, many difficulties are encountered during extraction and analysis of cell wall proteins. Since class III peroxidases are also secreted into the apoplast, the use of suspension cell cultures can facilitate isolation and functional characterization of individual isoforms. Here, we report on the characterization of class III peroxidases secreted in the spent medium of sugarcane suspension cell cultures. After treatment with specific inducers of cell wall lignification, peroxidases were isolated and activities assayed with guaiacol, syringaldazine and coniferyl alcohol. Enzymatic activity was not significantly different after treatments, regardless of the substrate, with the exception of methyl-jasmonate treatment, which led to a decreased guaiacol peroxidase activity. Remarkably, peroxidases isolated from the medium were capable of oxidizing syringaldazine, an analog to sinapyl alcohol, suggesting that sugarcane cultures can produce peroxidases putatively correlated to lignification. A proteomic approach using activity staining of 2-DE gels revealed a complex isoperoxidase profile, composed predominantly of cationic isoforms. Individual spots were excised and analyzed by LC-ESI-Q-TOF and homology-based search against the Sugarcane EST Database resulted in the identification of several proteins. Spatio-temporal expression pattern of selected genes was determined for validation of identified class III peroxidases that were preferentially expressed during sugarcane stem development.
    Plant Physiology and Biochemistry 11/2012; 62C:1-10. DOI:10.1016/j.plaphy.2012.10.015 · 2.76 Impact Factor
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