Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome

Abstract

Cereals contain proteinaceous inhibitors of endo-β-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography (CEC) followed by affinity chromatography (AC), were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI-and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity.Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.2

The three classes of wheat xylanase-inhibiting proteins accumulate in an analogous way during wheat ear development and germination

“…This assumption is supported by the fact that XIs comprise large gene families (Croes et al, 2008;Takahashi-Ando et al, 2007), of which some, but not all members are inducible by pathogen attack and/or stress-related conditions (Igawa et al, 2004(Igawa et al, , 2005Takahashi-Ando et al, 2007). In fact, divergent evolutionary forces, brought about by plant-pathogen interactions, presumably caused the three types of XIs to coevolve in order to better withstand attack by exogenous xylanases (Igawa et al, 2004).…”

“…Claire). Comparison of 2D-immunoblot patterns of XIs in crude wheat extracts with 2D-fingerprints of their affinity-purified counterparts indicated that the PAbs were reactive against the different XI forms (Croes et al, 2008).…”

“…Extracted wheat grain albumins/globulins were separated by 2D-gel electrophoresis as described previously (Croes et al, 2008). In brief, protein pellets (see above) were fully solubilized in 2D-lysis buffer (2.0 M thio-urea, 7.0 M urea, 4.0% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 20 mM dithiothreitol, 0.5% Immobiline pH gradient (IPG) buffer pH 6-11, traces of bromophenol blue).…”

“…Wheat grain extracts for 2D-gel electrophoresis were prepared as described previously (Croes et al, 2008). Briefly, samples were crushed in liquid nitrogen and 250 mg was suspended in 1.0 mL Tris-HCl buffer (50.0 mM, pH 7.8).…”

Kinetics and substrate selectivity of a Triticum aestivum xylanase inhibitor (TAXI) resistant D11F/R122D variant of Bacillus subtilis XynA xylanase

“…By now, three distinct types of such xylanase inhibitors have been identified in cereals: TAXI (Triticum aestivum xylanase inhibitor), XIP (xylanase-inhibiting protein) and TL-XI (thaumatin-like xylanase inhibitor) (Croes et al, 2008). TAXI-type inhibitors are proteins of ∼40 kDa that specifically inhibit bacterial and fungal GH11 xylanases (Gebruers et al, 2001).…”