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Wheat grains contain three classes of xylanase inhibitors (XIs), i.e. TAXI (Triticum aestivum xylanase inhibitor), XIP (xylanase inhibiting protein) and TLXI (thaumatin-like xylanase inhibitor). These proteins are involved in plant defence and strongly affect cereal-based processes in which inhibitor-sensitive xylanases are used. This paper reports on the successful use of 2D-DIGE and tandem MS to discriminate XI (iso)forms and measures their qualitative and quantitative variation in six different wheat cultivars. In total, 18 TAXI-, 27 XIP- and 3 TLXI-type XI spots were identified. The multiple members of the large TAXI-gene family make a considerable contribution to the total TAXI population. For XIP-type XIs, XIP-I is expressed as the predominant form, albeit under variable degrees of PTMs. Only one TLXI genetic variant was identified, showing different degrees of glycosylation. Multiple comparison analysis revealed up to 5-fold intercultivar differences in protein level of XI (iso)forms. Evaluation of abundance patterns using multivariate statistical tools revealed highly distinctive as well as correlated levels of different XI forms among the six cultivars. As the constitutive (and induced) levels of the different XI (iso)forms, which are differentially regulated in response to various forms of stress in other wheat plant parts, considerably vary between the cultivars, it can be assumed that their degree of resistance against pathogenic attack differs. Similarities in abundance profiles between XI (iso)forms and pathogenesis-related chitinases are also in line with a role in plant defence.

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

In wheat ( Triticum aestivum ) grains, TAXI- (T. aestivum xylanase inhibitor), XIP- (xylanase inhibiting protein), and TLXI-type (thaumatin-like xylanase inhibitor) xylanase inhibitors (XIs) are expressed in considerable levels and under different forms. As these proteins have a significant impact on microbial xylanases frequently used in cereal-based biotechnological processes, knowledge of their quantitative and qualitative variability in wheat is of great interest. This paper reports the successful use of immunoquantification by Western blotting to determine the intercultivar variation in the three structurally different classes of XIs, as well as their distribution among various industrial milling fractions. TAXI and XIP protein levels in eight wheat cultivars ranged from 81 to 190 ppm and from 156 to 371 ppm, with average values of 133 and 235 ppm, respectively. Using immunoblotting, TLXI protein levels could be measured directly for the first time. They ranged from 51 to 150 ppm and amounted to 112 ppm on average. The three classes of XIs were distributed among different wheat milling fractions in a similar way, with 4 and 10 times higher concentrations in the aleurone-enriched fraction than in white flour and pericarp fractions, respectively. Immunoblot patterns suggested that the observed intercultivar and spatial variabilities within the wheat grain are not due to the presence or absence of specific members of the large polymorphic XI families but to differences in the overall level and/or proportions of the specific members.