Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase

Abstract

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (K(d)) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.

Current potential and limitations of immunolabeling in cereal grain research

“…Consequently, LM11 has proven useful in localizing AX in different cereal grains Wilson, et al, 2006). Localization of poorly substituted AX has also been achieved by means of anti-(14)--unsubstituted xylan antiserum (Beaugrand, et al, 2005;Beaugrand, Reis, et al, 2004). Using immunolabeling, AX has been detected in the outer layers, aleurone, starchy endosperm, and transfer cells of different cereals (Table 1).…”

“…However, the outer layers (bran without aleurone), transfer cells, and the embryo (germ) remain less well characterized (Beaugrand, et al, 2005;Chen, et al, 2012;Jerkovic, et al, 2010;Robert, et al, 2011). Table 1.…”

“…Outer layers / Bran without aleurone Arabinoxylan Wheat (Beaugrand et al, 2005;Beaugrand, Reis, et al, 2004;Philippe et al, 2006;Philippe et al, 2007); oat, rye, barley (13),(14)--D-glucan Wheat (Philippe et al, 2006) Callose Wheat (Philippe et al, 2006) Ferulic acid Wheat (Philippe et al, 2007) Xylanase Wheat (Beaugrand, Cronier, et al, 2004;Beaugrand et al, 2005;Beaugrand, Reis, et al, 2004) Oxalate oxidase Wheat (Jerkovic et al, 2010) Xylanase inhibitor I protein Wheat (Jerkovic et al, 2010) Wheatwin1 Wheat (Jerkovic et al, 2010) …”

“…Maize (Galuszka et al, 2005) Aleurone Arabinoxylan Wheat (Beaugrand et al, 2005;Beaugrand, Reis, et al, 2004;Guillon et al, 2004;Lovegrove et al, 2013;Philippe et al, 2006;Philippe et al, 2007;Robert et al, 2011;Suliman et al, 2013); oat, rye ; barley Xylan Wheat (Lovegrove et al, 2013) Xyloglucan Barley (13),(14)--D-glucan Wheat Philippe et al, 2006;Robert et al, 2011;; barley Callose Wheat (Philippe et al, 2006); barley (Wilson et al, 2012) Mannan Barley Ferulic acid Wheat (Philippe et al, 2007;Robert et al, 2011) p-Coumaric acid Wheat Tranquet et al, 2009) Xylanase Wheat (Beaugrand, Cronier, et al, 2004;Beaugrand et al, 2005;Beaugrand, Reis, et al, 2004) Glycosyltransferase Wheat (Suliman et al, 2013) Oxalate oxidase Wheat ( Wheat (Van Herpen et al, 2008;Wiley et al, 2007) Oleosin / Caleosin Rice (Chen et al, 2012); oat (Heneen et al, 2008) Puroindolines Wheat (Capparelli et al, 2005) Transfer cells Arabinoxylan Wheat Lovegrove et al, 2013;Robert et al, 2011); oat, rye, barley …”

“…Silver enhancement is also necessary when the gold particles bound to the secondary antibody are too small to be detected even by TEM (Lesage, et al, 2011). Observation of highly contrasted labeling by light microscopy is also possible using an epipolarization system in which gold particles are visualized as bright spots (Beaugrand, et al, 2005).…”