Abstract
Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. The implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.
Highlights
Plant cell wall recalcitrance is one of the major hurdles for the development of economically viable biomass-based biofuels (Himmel et al, 2007; Dashtban et al, 2009; Ding et al, 2012; Inoue et al, 2014)
Our microscopic observation of late growth stage Arabidopsis stems shows that a significant proportion of plant cells and their organelles were broken, resulting in segments and debris of plasma membranes and organelles in the stems at the late growth stage (Figures 1A–D)
The image shows the broken cell membrane, and highlights the in vivo cell walls at the late growth stage are already mixed with intracellular components and protein
Summary
Plant cell wall recalcitrance (caused by cell wall integrity and strength) is one of the major hurdles for the development of economically viable biomass-based biofuels (Himmel et al, 2007; Dashtban et al, 2009; Ding et al, 2012; Inoue et al, 2014). Plant cell walls are predominantly composed of cellulose, hemicellulose, and lignin. They contain both enzymes and structural proteins. Cell wall structural proteins usually account for 1–5% (mass)of cell wall, and can be characterized into three major classes: extensins, hydroxyproline/prolinerich proteins, and glycine rich proteins (Carpita et al, 2001). The other group of cell wall proteins is non-structural, and includes enzymes that contribute a range of functions during growth and development including stress response, oxidoreductase activities, and hydrolytic activities (Carpita et al, 2001; Minic et al, 2007; Jamet et al, 2008). For the purpose of exploring potential routes for engineering bioenergy crops, in this study we focus on the nonstructural cell wall proteins
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