Intercultivar, spatio-temporal and pathogen induced variation of polymorphic families of three types of xylanase inhibitors in wheat grains: a proteomic approach

Since the discovery of proteinaceous xylanase inhibitors in 1997 in whea t, three classes of such naturally occurring xylanase inhibiting protein s (XIs) were identified and purified from wheat, i.e. Triticum aestivum xylanase inhibitor (TAXI), xylanase inhibiting protein (XIP) and thaumat in-like xylanase inhibitor (TLXI). The initial finding that XIs only inh ibit endo-1,4-ß-D-xylanases (xylanases) of microbial origin, wherea s they do not interact with plant endogenous xylanases has led to the hy pothesis that XIs play a role in plant defence. They may limit pathogen invasion either by reducing the hydrolytic activity of the secreted xyla nases or by ensuring the generation of elicitor-active (arabino)xylo-oli gosaccharide fragments that might have been extensively degraded in the absence of the inhibitors. Elicitors in turn trigger subsequent plant de fence responses. In addition, XIs which make up a considerable part of the albumin/globul in fraction in wheat grains have a large impact on cereal-based applicat ions in which inhibitor-sensitive xylanases are used. Xylanases affect t he functionality of arabinoxylans by altering their physico-chemical pro perties and are applied as technical aids in cereal-based processes, as they can improve processing and product end-quality. The current study aimed to unravel the intercultivar, spatio-temporal an d pathogen induced distribution of the three known classes of polymorphi c XIs in the wheat proteome. A comprehensive view on the variability in TAXI, XIP and TLXI (iso)forms contributes to a more profound insight in their presumed role in plant protection and provides a stronger scientif ic basis to understand their impact on biotechnological applications. Using an antibody-based technique TAXI, XIP and TLXI protein levels were measured in different wheat cultivars and milling fractions. Although n o intercultivar correlation was discovered between the levels of the thr ee classes of XIs (showing two- to three-fold variations), their spatial distribution within the wheat grain was comparable. The highest TAXI, X IP and TLXI concentrations were found in the aleurone fraction. Immunobl otting and densitometric analysis also revealed that the three classes o f XIs accumulate in an analogous way during grain development and germin ation. The largest increases in TAXI, XIP and TLXI levels were observed from the milky to the soft dough stage of grain development, after which high levels were maintained until grain maturity. They further accumula ted in the developing seedlings up till 5 to 9 days post-imbibition. Whi le TLXI was found to be restricted to wheat grain tissues, TAXI and XIP also occurred in roots and shoots of the young seedlings. No detectable amounts of the three types of XIs were present in roots, leaves or stems at later stages of ear development. A proteomic approach, using 2D gel electrophoresis (2-DE) and mass spect rometry (MS) revealed the existence of polymorphic families of XIs in wh eat grains, comprising different genetic variants as well as post-transl ationally modified forms. Thereby, a link was provided between data on t he genomic and proteomic levels. Some XI isoforms corresponding to putat ive genes, for which it was not yet known if they are actually expressed in wheat grains, were identified. Other genetic variants were not detec ted and may only be transcribed in other wheat plant parts or under othe r (stress) conditions. The intercultivar, temporal and pathogen induced variability of these po lymorphic XI families was assessed in detail by the combined use of 2-DE /MS and difference in-gel electrophoresis (DIGE) technology. Differences in XI levels between wheat cultivars could be attributed to various ind ividual differences in the multiple (iso)forms, in which they occur. The similarity in abundance between some XI (iso)forms and several chitinas es may well point to co-regulation of their expression. Early in grain d evelopment the XI polymorphism was found to be less pronounced than at t he harvest ripe stage. Fusarium graminearum ∆Tri5 infection o f wheat grains at anthesis resulted in enhanced, reduced as well as unch anged levels of specific XI forms. This is in line with earlier results on the transcriptomic level. The disease effect was mostly visible soon after inoculation (5 days post-anthesis). XI levels at the harvest ripe stage are possibly not affected. In general, the results obtained in this doctoral dissertation provide a dditional evidence for the presumed plant protective role of the three c lasses of XIs. The accumulation of XIs at later stages of grain developm ent and their persistence throughout the early stages of germination are consistent with a defensive function, in particular for the young wheat seedling, which is highly susceptible to pathogen attack. Their locatio n within the wheat kernel, showing high concentrations in aleurone and p eripheral layers, contributes to this hypothesis. Besides their spatio-t emporal distribution, also the large polymorphism of XIs fits well with their suggested role as plant defence-related proteins. A XI population showing distinct recognition specificities towards xylanases might be th e result of plant evolution to counteract the many xylanases secreted by bacteria and fungi, analogous to polygalacturonase-inhibiting proteins in dicots. Finally, the abundance of some XI forms was found to be induc ed in response to F. graminearum ∆Tri5 infection, while that of others was repressed or remained constant. It can be assumed that som e XI genes are constitutively expressed while others are inducible by bi otic or abiotic stresses. The basal level of the various XI (iso)forms i s probably cultivar-, tissue- and time-specific. However, the exact quan titative and qualititative composition of the heterogenous XI population under certain circumstances additionally depends upon the pathogen or s tress encountered, the site of infestation and the signaling pathway inv olved.

Publication year:2009

Accessibility:Closed